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RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. C-ERB B ACTIVATION AND AVIAN LEUKOSIS VIRUS INDUCED ERYTHROBLASTOSIS by Maribeth Anne Raines A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1987 ABSTRACT C-ERB B ACTIVATION AND AVIAN LEUKOSIS VIRUS INDUCED ERYTHROBLASTOSIS by Maribeth Anne Raines Both qualitative and quantitative alterations in cellular gene expression have been associated with oncogenesis. Molecular characterization of avian leukosis virus (ALV) induced erythroblastosis indicates that there is an absolute correlation between alteration of the proto-oncogene c-erb B and erythroblastosis induction. The c-erb B gene is closely related to the human epidermal growth factor receptor (hEGF—R). ALV can activate the oncogenic potential of c-erb B by two distinct mechanisms: insertional activation (IA c-erb B) and transduction of c-erb B. In the former, the majority of integrated proviruses are intact and are situated in the same transcriptional direction. C-erb B transcription initiates in the 5' LTR of the provirus and continues into erb B to generate a truncated c-erb B RNA. Alternatively, a portion of the c—erb B gene can be transduced into ALV to become new viruses capable of inducing rapid erythroblastosis. These viruses encode c-erb B products identical to the IA c-erb B products; both products lack sequences corresponding to the extracellular ligand-binding domain of hEGF-R. It is the elevated and perhaps inappropriate expression of a truncated growth factor receptor which causes the uncontrolled proliferation and differentiation of erythroblasts. The identification of two transduced c-erb B virus Maribeth Anne Raines mutants suggest that the c-terminal portion of the c-erb B protein is also important in determining the oncogenic potential of c-erb B. ACKNOWLEDGEMENTS Although there are many people who should be acknowledged for contributing in some way to this dissertation only a few are listed here. I am especially grateful to Lyman Crittenden and his colleagues at the Regional Poultry Research Lab for not only providing various chicken and virus strains but also for teaching me the basics of ‘chickenology'. None of this work would have been possible without their help and guidance. I would like to thank Carlo Moscovici for taking over some of the in vivo studies (described in Chapters 4 and 5) and thereby enabling me to stay in the lab and out of the chicken barns. I also thank my friends and colleagues at both Michigan State and Case Western (especially Wynn Lewis, Karen Frederici, Paul Bates, Jerrleodgson, Sue Conrad, N. Maihle, Bob Radinsky, and Tom Flickinger) for their stimulating dissussions, advice, and constant support; they have helped improve both the quality of my science and my life. A special note of thanks goes to those few people who actually helped put this work together into a formal dissertation - Nita Maihle, Tom Flickinger, Rich Schneeberger, and Susan and Mark Gorman. Last but not least, I would like to thank Hsing-Jien Kung for giving me the opportunity to work in his lab and on a truly exciting research project as well as the freedom to pursue my own scientific interests. iv TABLE OF CONTENTS List of Tables List of Figures Introduction and Literature Review Chapter Chapter Chapter Chapter Chapter Chapter Summary A. Hematopoiesis B. Retroviruses and Cancer C. Retrovirus Life Cycle D. Oncogenesis by Nonacute Retroviruses E. Oncogenesis by Acute Transforming Viruses F. Structure and Function of Viral erb B Nonacute Retrovirus Induction of Avian Erythroblastosis in Different Strains of Chickens Structure and Expression of the Chicken c-erb B Gene Transcription from the 3' Long Terminal Repeat of Integrated ALV Proviruses On the Mechanism of c-erb B Transduction: Newly Released Transducing Viruses Retain Poly A tracts of erb B Transcripts and Encode C-terminally Intact erb B Proteins Identification of New c-erb B Transducing Viruses: Deletions in the C-terminal Domain Activate Sarcomagenic Potential Evidence for Differentiation of ALV Transformed Erythroblasts 1g Vivg vii viii l3 16 22 34 40 53 73 93 107 132 160 179 Appendix A: Appendix B: Appendix C: Appendix D: References vi C-erb B Activation in Avian Leukosis Virus Induced Erythroblastosis: Clustered Integration Sites and the Arrangement of ALV Provirus in the c-erb B Alleles. (1985) M. A. Raines, W. G. Lewis, L. B. Crittenden, and H. J. Kung, Proc. Natl. Acad. Sci. USA 82:2287-2291. C-erb B Activation in ALV-Induced Erythroblastosis: Novel RNA Processing and Promoter Insertion Result in Expression of an Amino-Truncated EGF Receptor. (1985) T. W. Nilsen, P. A. Maroney, R. G. Goodwin, F. M. Rottman, L. B. Crittenden, M. A. Raines, and H. J. Kung, Cell 41:719-726. RAV-l Induced Erythroleukemic Cells Exhibit a Weakly Transformed Phenotype In Vitro and Release c-erb B-Containing Retroviruses Unable to Transform Fibroblasts. (1986) H. Beug, M. S. Hayman, M. A. Raines, H. J. Kung, and B. Vennstrom, J. of Virology 57:1127-1138. Materials and Methods 183 188 196 208 215 LIST OF TABLES Chapter 1 1. Incidence of erythroblastosis in different 54 chicken lines Chapter 5 2. Induction of erythroblastosis from leukemia 133 samples containing transduced c-erb B viruses 3. Disease Potential of clone purified virus 150 stocks vii LIST OF FIGURES Chapter 1 1. Gross Lesions of ALV induced erythroblastosis 2. Histological examination of ALV induced erythroblastosis 3. Development of erythroblastosis Chapter 2 4. Molecular cloning of the alpha and beta- alleles of c-erb B 5. Northern blot analysis of c-erb B related RNAs in preleukemic and uninfected liver tissue 6. Northern blot analysis of normal c-erb B RNAs 7. Selective cDNA cloning of c-erb B sequences located 5' of VBl 8. Southern Blot analysis using cDNA clone 167 Chapter 3 9. Detection of novel c-erb B related RNAs in leukemic samples containing insertionally activated c-erb B genes. 10. 81 nuclease analysis of erb B related RNAs in erythroleukemic and normal tissues Chapter 4 11. The presence of transduced erb B proviruses in ALV induced erythroblastosis 12. The expression of erb B transducing viral RNA in ALV induced erythroblastosis and secondary leukemias viii 59 62 65 75 81 84 88 9O 95 99 109 112 13. 14. 15. Chapter 5 16. 17. 18. 19. 20. 21. Chapter 6 22. 23. 24. 25. 26. ix The 5' and 3' junctions of erb B transducing viruses The structure of c-erb B transduced proviral clones 9141, 9134E1, and 913453 Nucleotide sequence of the 5' and 3' viral-erb B junctions in 9134El and 913483 Purification scheme for three different c-erb B transducing viruses in erythroleukemic chicken 9134 Northern analysis of erb B related RNAs in sarcomas induced by 9134 viral extracts Sl nuclease analysis of transduced erb B sequences in RNA from newly isolated sarcomas and transformed fibroblasts Immunoprecipitation of erb B related proteins in cell lines infected by different c-erb B containing viruses Structure and sequence of the 913483 provirus Schematic comparison of the erb proteins displaying different oncogenic potentials Blood smears from preleukemic and leukemic chickens Appearance of circulating polychromatic erythrocytes prior to erythroleukemia developmant define a preleukemic phase Comparison of bone marrow smears from birds in preleukemic and leukemic phases Structural alterations in the c-erb B locus of preleukemic and leukemic DNA samples Northern blot analysis of leukemic and preleukemic tissue samples 116 119 123 136 138 141 146 148 155 162 165 168 171 174 INTRODUCTION AND LITERATURE REVIEW AW Leukemia is a disruption of hematopoiesis. In order to understand the role of oncogenes and retroviruses in leukemogenesis, it seems essential that the basic principles of hematopoiesis be discussed. The main focus of this discussion is concerned with avian hematopoiesis with special emphasis on erythropoiesis. The murine system is also described since most of the pioneering work has been done in this system. Hematopoiesis involves the production and maintenance of blood cells. The site of blood formation in the chicken, like other animals, is developmentally regulated. Hematopoiesis begins in the chick embryo in the blood islands of the yolk sac (Romannoff, 1960). This remains the major site of hematopoiesis during embryogenesis. Limited hematopoietic activity has also been detected in the liver and spleen between days 7 and 9 of development. Bone marrow hematopoiesis begins as early as days 8 and 9 and becomes the major source for blood cell formation after hatching. Hematopoiesis outside of the bone marrow, known as extramedullary hematopoiesis, rarely if ever occurs in the hatched chicken. This is in contrast to mammals where the liver and spleen play an important role in embryonic hematopoieisis and during periods of hematopoietic stress in the adult animal (Rifkind et a1., 1980). The hematopoietic system is composed of blood cells which are capable of proliferating, differentiating, and maturing into several cell types. The mature cells include erythrocytes, granulocytes, macrophages, eosinophils, megakaryocytes, and B- or T-lymphocytes. These cells are morphologically distinct and each performs a different function necessary for survival. Their lifespans are relatively short and therefore, they must be continuously replaced. The mature cells, although routinely observed in the bloodstream, are thought to originate in the bone marrow from a common pluripotent stem cell. This stem cell has the ability to proliferate (or self-renew) and differentiate into the various cell types. Under normal physiologic conditions, the stem cell is quiescent, but is capable of rapid proliferation if necessary. The most convincing evidence in support of a pluripotent stem cell was first provided by reconstitution experiments (Till et a1., 1961; McCulloch et a1., 1965). Recipient mice were made hematopoiesis deficient by irradiation and were injected with bone marrow cells from a donor containing a chromosome marker. Distinct hematopoietic colonies of donor origin appeared in the spleens of the recipients. Examination of individual colonies revealed a heterogeneous population of hematopoietic cells containing either erythroid or myeloid cells, or both. Each colony was shown to arise from a single cell and maintained the ability to reconstitute other irradiated mice. Based on these observations a pluripotent stem cell, sometimes referred to as CFU-S or colony forming unit-spleen, was proposed. Later experiments extended the pluripotency of the stem cell to lymphoid cells (Abramson et a1., 1977). Similar types of reconstitution experiments have been performed in the chicken. In this case, colonies appear in the bone marrow rather than the spleen and are called CFU-M for colony forming unit- marrow. This observation is consistent with the idea that the liver and spleen of the chicken do not provide a suitable environment for extramedullary hematopoiesis. As the stem cell differentiates a hierarchy of cell types and cell lineages appear. This heirarchy has been defined based on functional as well as morphological properties. Progenitor cells, the immediate progeny of stem cells, were first identified using in vigrg colony assays. Colonies containing distinct progenitor forms were observed after seeding bone marrow cells in semisolid media. The in_!1§;g colony assay is now routinely used to identify distinct progenitor cells (Pluznik et a1., 1964; Bradley et a1., 1966; Metcalf et a1., 1979). Unlike stem cells, the progenitor cells are "committed” to differentiate along a particular cell lineage and display only limited proliferative capacity. Two of the best characterized progenitor cells are those of the erythroid and granulomyelocytic lineages. The granulomyelocytic progenitors represent a distinct subset of precursor cells which differentiate into granulocytes or macrophages 13_yi§rg and v vo. Colonies containing both granulocytes and macrophages originate from these progenitors and are called CFU-GM for colony forming unit-granulocyte/macrophage. When specific in vitro culture conditions were used, two types of erythroid colonies were identified (Stephenson et a1., 1971; McCleod et a1., 1974; Samarut et a1., 1980). One is a more mature erythroid colony, termed the colony forming unit-erythroid (CFU-E). It is characterized by small diffuse colonies of 20 to 60 cells in methylcellulose or plasma clot cultures. These cells, when removed from semisolid media and suspended in culture medium, differentiate into hemaglobinized cells (mature erythrocytes) within 3 days. Another colony resembling a more primitive erythroid precursor is also observed. These colonies are called the burst forming unit-erythroid (BFU-E) since they are composed of tightly packed cells and contain as many as a thousand cells per colony. The BFU-E derived cells are also capable of differentiating into hemoglobinized cells in liquid culture, but require a longer incubation period, usually between 7 and 10 days. Thus the BFU-E and CFU-E are functionally distinct. In the chicken, the CPU-E and BFU-E can be characterized further based on the presence of specific antigenic markers (Samarut et a1., 1979). BFU-E and CFU-M preferentially express a brain related antigen (Br-antigen) whereas CPU-E displays an antigen characteristic of immature red cells (Im- antigen). Antibody mediated cytolysis of cells containing the Im- antigen does not inhibit BFU-E formation (Samarut et a1., 1980). Thus the BFU-E is considered an intermediate cell type between the pluripotent CPU-M (CFU-S in the murine system) and CFU-E and has the potential for limited self-renewal. Several other erythroid cell types have been distinguished based on cell morphology and histochemical staining. .These include the erythroblast, polychromatic erythrocyte, and the mature erythrocyte. This is the terminology used by avian hematologist and is somewhat different than the conventional terminology which uses normoblast as the form for most erythroid intermediates (Lucas et a1., 1961; Beck, 1977). Where these cell types fit into the cell lineages defined in tissue culture is unclear. The most immature erythroid cell that can be identified morphologically is the erythroblast (or pronormoblast). Unlike the other erythroid intermediates the erythroblast expresses little if any hemoglobin, stains benzidine negative, and reacts with anti-Im. This erythroid cell type is rarely seen in the bloodstream and is observed at very low numbers in the bone marrow of the chicken (less than 0.1%) (Nelson et a1., 1980). An increased number of erythroblasts has been observed in anemic birds. The erythroblast does appear to be capable of cell division since mitotic figures have been observed. These properties taken collectively suggest that the erythroblast is probably identical to the CFU-E. Thus erythropoiesis traverses the following pathway. Stem cell > BFU-E > CFU-E/erythroblast > polychromatic erythrocytes > mature erythrocytes. The identification of several intermediates in the erythroid lineage illustrates the prevailing features of hematopoietic differentiation. That is, once a stem cell “commits" itself to a particular cell lineage, further proliferation and differentiation occur almost simultaneously. A more mature phenotype is accompanied by a progressive loss of proliferative potential. The end result is a cell with no proliferative ability but serving a very specific function to the organism. In the case of erythrocytes, it is to maintain tissue oxygen levels by transporting oxygen. The presence of committed, but intermediate cell types which can readily differentiate provides a means for rapid response to hematopoietic stress such as bleeding or infection. As expected for any developmental system, hematopoiesis is very tightly regulated and its regulation appears to be influenced by its microenvironment. The bone marrow is composed of a complex stromal cell network consisting of several cell types (Dexter 1982; Sorrell et a1., 1980). The sites of erythropoiesis and granulopoiesis are localized to two distinct compartments in the chick bone marrrow. Erythropoiesis occurs intravascularly with the more immature erythroid cells being associated with endothelial cells which line the vascular region of the marrow. Granulopoiesis, on the otherhand, occurs extravascularly, and is often associated with reticulum cells. The reticular cells are fibroblastic cells which form a loose cellular network in the marrow. Adipocytes are also found associated with macrophages and together with the reticular cells, they fill the perisinusoids of the marrow. The role of the bone marrow microenvironment in hematopoiesis is still unclear. This microenvironment can be reconstituted in vitro by establishing long term bone marrow cultures consisting of an adherent stromal cell layer (Dexter et a1., 1974; Dexter et a1., 1976). These cultures, known as Dexter cultures, support the proliferation and differentiation of both stem cells and their maturing progeny. Although the original Dexter cultures selectively supported only myeloid and erythroid cultures, modifications of this culture system have been used to support lymphoid cells (Whitlock et a1., 1982). The adherent multilayer of stromal cells is a key element in these cultures. It has been speculated that these cells function as a matrix for stem cell attachment and that specific cell-cell interactions affect stem cell proliferation. The particular localization in the bone marrow may determine which lineage will ultimately develop, while the stromal cells serve to maintain the stem cell population in general. It is apparent from the in vitro colony assays that the proliferation and development of the committed progenitor cells do not require bone marrow derived cultures. A feeder cell layer or conditioned media, however, is usually necessary for successful colony formation (Dexter 1984). The importance of conditioned media in colony formation supported the idea that humoral factors were important in regulating hematopoiesis and has become an area of intensive research in the last decade. Several hematopoietic growth factors have been purified to homogeneity and molecularly cloned (Cough et a1., 1984; FUng et a1., 1984 ; Kawaski et a1., 1985; Wong et a1., 1985; Lin et a1., 1986; McDonald et a1., 1986; Shoemaker et a1., 1986). These growth factors can be divided into two types based on their colony stimulating activities in;gi§;g. Some are multipotent, like interleukin-3 (IL-3) and granulocyte/macrophage-colony stimulating factor (GM-CSF). IL-3 stimulates the proliferation and development of all myeloid and erythroid precursor cells and can facilitate the self- renewal of CFU-S (Ihle et a1., 1982). GM-CSF appears to be more restricted than IL-3 in its activity and promotes growth of progenitor cells of the granulocyte and macrophage lineages (Burgess, et a1., 1980). The formation of erythroid bursts can be stimulated by GM-CSF but only in the presence of another growth factor, erythropoietin (Sieff et a1., 1985). Erythropoietin is one of many hematopoietic growth factors whose activity is confined to a specific cell lineage. Erythropoietin acts exclusively in the development of erythroid progenitor cells (Marks et a1., 1978). Other unipotent growth factors include G-CSF (granulocyte-colony stimulating factor) and CSF-l (colony stimulating factor-1) which affects growth of granulocytes and macrophages, respectively. All of the above mentioned growth factors are low molecular weight glycoproteins. Most of them have been purified from the media of cultured cells with the exception of erythropoietin. Erythropoietin is synthesized exclusively in the liver and kidney and has been purified from urine and serum (Miyake et a1., 1977). The other CSFs are synthesized by multiple cell types (Metcalf 1985). These cells are distributed throughout most tissues and include fibroblasts, activated T-lymphocytes, and endothelial cells (Cline et a1., 1979; Whetton et a1., 1986). CSF-l and GM-CSF have also been detected in the urine and serum of animals but at lower levels (Burgess et a1., 1977; Stanley 1985). IL-3 was purified from a leukemic cell line and has yet to be detected in 2132. Continuous exposure to the growth factor appears to be required for proliferation and survival of the appropriate target cells, since without them the cells deteriorate. This poses somewhat of a problem since the bone marrow cells themselves do not secrete growth factors. G-CSF is one of the few growth factors known to be synthesized by bone marrow cells. The function of the ultrastructure of the bone marrow may be to act as a surface on which hematopoietic growth factors can be concentrated. In this way growth factors synthesized in other tissues can reach the appropriate hematopoietic cell. This interaction could be achieved via the extracellular matrix and membrane associated glycoconjugates characteristic of stromal cells. Recent evidence supports this view (Hunt et a1., 1987). 'Although the hematopoietic growth factors are widely distributed, they act on very specific cell types. Their action has been shown to be mediated by receptor molecules present on the cell surface. The characterization of specific hematopoietic growth factor receptors has been limited by the low number of progenitor cells and the heterogeneous nature of the bone marrow. Radio-iodonated growth factors have been used to identify specific growth factor receptors on the surface of bone marrow cells. Each growth factor binds to a receptor of different apparent molecular weight suggesting that each growth factor has its own receptor (Whetton et a1., 1986). Growth 10 factor receptors are coexpressed on cells at different stages of hematopOietic development (Metcalf, 1985). There appears to be no direct competition among growth factors. That is, different growth factors do not bind to a common receptor molecule. Regulation of growth factor receptors does, however, appear to be cooordinately regulated (Walker et a1., 1985; Lotem et a1., 1986). This regulation, termed transmodulation, involves the down regulation or removal of receptor molecules from the cell surface. The particular growth factors which are down-regulated coincide with the hierarchy of hematopoietic cell lineages and the multipotency of the growth factors. For example, binding of bone marrow cells with IL-3 down regulates not only its own receptor but also receptors for GM-CSF, CSF-l, and G-CSF. Similarly, GM-CSF down modulates GM-CSF, CSF-l, and G-CSF receptors. CSF-l and G-CSF only down regulate their own receptors. Down regulation of these receptors appears to reduce the number of avalaible receptors and not the affinity of the receptor for its ligand. It is not clear how a down-regulated receptor leads to an active state of differentiation and proliferation, especially since very low levels of receptor occupancy (less than 10%) are necessary to achieve the biological effects associated with erythropoietin, GM-CSF, and G-CSF (Metcalf, 1985). Down-modulation may serve a more instructive role; where a cell once down-regulated by a particular growth factor is destined to proceed down a particular differentiation pathway. The presence of specific cell surface receptors on hematopoietic cells raises the question of what is the signal that is transduced and 11 what are the immediate effects on the cell? Does ligand binding signal proliferation, then differentiation or both? Studies with erythropoietin (epo) suggest that the principal biological effect of epo is not differentiation but proliferation of the target precursor cells (Marks et a1., 1978). Differentiation to the mature erythrocyte appears to be a property inherent in an epo-responsive cell. If differentiation is the only consequence of epo action, one would expect to see large numbers of erythroid precursors in the bone marrow. The use of a mitogenic signal as an initial response provides an efficient way of rapidly recovering from hematopoietic stress. How a growth factor receptor signals mitogenesis and how this signal is coupled to a specific differentiation program awaits further elucidation of the specific growth factor receptors and their signal transduction pathways. The majority of work done on hematopoietic growth factors and their receptors has been done in the human and murine systems. Similar activities have been identified in the avian system, but none have been purified to homogeneity (Samarut et a1., 1976,1978). Some hematopoietic growth factors do cross react between species, albeit with diminished binding and biological effects in the heterologous system. Erythropoietin appears to be especially species specific, since murine erythropoietin is not active on rat erythroid cells and displays only limited sequence homology (McDonald et a1., 1986). The establishment of long-term bone marrow cultures and the crude purification of hematopoietic growth factor-like activity from the 12 avian system suggests that hematopoiesis in the chicken is similar to that of humans and mice. That is, there is a hierarchy of hematopoietic cells which are regulated by humoral factors. The cells and molecules which mediate these responses may prove to be physically distinct, but the general principles of hematopoietic development appear to be similar. The above discussion illustrates the complex nature of blood cell development. It involves the multiplication of stem cells and their progressive differentiation into mature cells of various types, each having an important function. This program of events is exquisitely regulated by a variety of growth factors which can act alone or in synergy to produce a specific cell type. Leukemia results from an imbalance in the normal hematopoietic program. It is typically manifested as the accumulation of blast-like cells in the bone marrow and bloodstream of the affected animal. The leukemic blast cells are usually of a specific hematopoietic cell lineage and are analagous to the committed progenitor cells. Unlike other progenitor cells, leukemic cells have increased proliferative ability and in many cases become "immortalized" so that they can be continuously passaged in tissue cell culture. Some leukemic cells can be induced to differentiate after treatment with agents like phorbol esters and dimethylsulfoxide, but are resistant to the effects of the appropriate hematopoietic growth factors. Leukemic cells display features suggesting that the proliferative and differentiation signals have been uncoupled or perhaps one signal, i.e., the proliferative signal, is l3 potentiated. Characterization of leukemias and the oncogenes involved should provide useful information regarding what the proliferation and differentiation signals are, and also how that signal may be transduced to complete the differentiation program. B. e v e and Can er Unlike the study of avian hematopoiesis, the avian system has been at the forefront of retrovirus research. Indeed it has only been in the last decade that a human retrovirus has been found associated with leukemia. It seems ironic that in 1987 one of the major threats to the human population is a retrovirus when just 10 to 20 years ago retroviruses were thought to be obscure agents restricted to the avian system and not mainstream to cancer biology. The following discussion focuses primarily on avian retroviruses, their oncogenes, and how they interact within the cell. Retroviruses were first identified as the causative agents of avian leukemias and tumors and are commonly referred to as RNA tumor viruses. In 1911, Peyton Rous demonstrated that chicken sarcomas could be transplanted using a cell free filtrate. It is the virus from this sarcoma now known as the Rous Sarcoma Virus (RSV), which has been instrumental in establishing a large number of concepts central to retrovirology as well as cancer biology. Direct inoculation of RSV onto chorioallantoic membranes resulted in small tumors and supported the idea that a retrovirus could "transform" a normal cell into a tumor cell (Keogh, 1939). In 1958, Temin and Rubin further described a 14 similar ”transformation" in vitro using monolayers of chicken embryo fibroblasts. RSV transformed cells stood out as distinct "foci" on the cell monolayer and the number of foci correlated with the amount of virus applied to the cells. The development of, conditional and transformation defective mutants of RSV suggested that RSV contained a specific gene responsible for the tumorous phenotype (Martin et a1. , 1972). This transforming gene, now known as v-src, was shown to originate from cellular sequences and was highly conserved among metazoans (Stehelin et a1. , 1976; Spector et a1, 1978). RSV is just one of many avian retroviruses. The avian retroviruses are divided into two classes based on their pathogenic properties and genetic content (Teich, 1982). The slow transforming Viruses or non-acute retroviruses, as the name implies, induce neoplastic disease after a long latency period of several months. They are capable of inducing a wide spectrum of diseases and are often found associated with naturally occurring tumors. The non-acute class of retroviruses is replication competent and can be routinely found in infectious stocks of defective viruses. For this reason, they are also known as ”associated" or "helper" viruses. These viruses do not transform cells in vitro and do not contain oncogenes. Avian leukosis Virus (ALV) and reticulo-endotheliosis virus (REV) are two distinct subclasses of non-acute retroviruses. The avian leukosis virus group is further subdivided based on the expression of specific envelope coat proteins. The molecular basis of oncogenesis by ALV and REV will be discussed in detail later. 15 The other class of retroviruses is the acute transforming viruses. These viruses induce acute and fatal neoplastic disease within a few weeks or even within days. They usually induce one predominant neoplasm. Based on the predominant neoplasm which they induce, the acute transforming viruses are subdivided into either the sarcoma viruses or the leukemia viruses (Teich, 1984b). RSV is an example of the former subclass. Most of the acute transforming viruses are defective for replication. That is, they lack one or more of the three essential genes for virus replication (see next section). Virus production can occur if the defective genes are supplied in trans by a replication competent virus, i.e. , the non-acute retrovirus. Unlike the non-acute viruses, the acute transforming viruses transform cells in vitro and contain additional oncogenic sequences. At least 12 different types of oncogenic sequences, referred to as viral oncogenes, have been found associated with avian retroviruses (Bishop et a1. , 1985). Like the v-src gene of RSV, normal chicken cells'contain genes which are homologous with each of these viral oncogenes. These cellular sequences are called proto-oncogenes or cellular oncogenes. Acute transforming viruses presumably arose by recombination between Proto-oncogenes and a replication competent, non-acute virus. The Process by which these cellular oncogenes are incorporated into a retrovirus is known as transduction and will be discussed in detail later. Although cellular oncogenes were originally defined as a result of retroviral transduction, chromosomal translocation, gene amplification, and other relevant mutations of these same proto- oncogenes have been associated with cancers in other species including 16 humans (Weinberg, 1982, Varmus, 1984, Alitalo, 1985). It is in this respect that avian retrovirology has had its most profound effect on cancer biology today. C. ov s i e C cle The unique way in which retroviruses interact with their hosts make them potent cancer causing agents. The following discussion focuses on aspects of the retrovirus life cycle which are relevant to oncogenesis (for a more complete review see Varmus, 1983, 1984). Retroviruses are single-stranded RNA viruses. Their genomes are diploid, consisting of two identical single- stranded RNA molecules. All the genetic information necessary for virus replication is contained within a single RNA molecule. The retrovirus genome consists of three structural genes; gag encodes the group associated antigens which comprise the core structure of the virus; 22; encodes reverse transcriptase, an RNA directed DNA polymerase; and egg is responsible for the synthesis of the envelope glycoproteins. These three genes are arranged 5'gag-pgl-ggx3' in the genome. In general viruses which do not contain any one of these three genes are defective for replication. Successful infection and replication of defective viruses can occur if complemented with a replication competent, or so-called helper virus. The retroviral genome closely resembles a eukaryotic mRNA. Not only is it a plus strand virus, but it also contains a 5' cap site, internally methylated adenosines, and a poly (A) tract. In addition, 17 the genome contains several noncoding regions most of which are situated at the termini of the virus. These noncoding sequences possess a variety of functions most of which are essential for a complete virus life cycle. Important noncoding.sequences include: 1) the R region, repeated sequences present at both ends of the viral RNA, 2) U5 sequences, sequences unique to the 5' end of the viral genome and represented twice in the integrated viral DNA, 3) the primer binding site (PBS), the site where the tRNA primer binds to initiate synthesis of the first DNA strand, 4) the leader sequence, sequences preceding the gag gene important in packaging of viral RNA and in some cases containing the splice donor site required for generation of subgenomic RNAs, 5) 3' noncoding region (NT), sequences between 2g! and the beginning of U3 which contain a purine-rich tract capable of priming plus strand DNA synthesis, and 6) U3 sequences, sequences unique to the 3' end of the viral genome and present twice in proviral DNA as a component of the LTR. In addition U3 contains a consensus polyadenylation signal, and promoter and enhancer sequences important in the regulation of viral RNA synthesis. The virus life cycle begins with entry into the cell. Although retroviruses adsorb rather nonspecifically to the cell, internalization is mediated through an interaction between the envelope glycoproteins and specific cell surface receptors. In the case of Avian Leukosis Viruses (ALVs), there are five envelope subgroups, A through E, which have been designated based on their host range, cross interference of receptors, and neutralization of antibodies (Payne, 1985). Four 18 autosomal loci govern susceptibility to the viral subgroups. They are called tx- a, tg-b, 52:9, and tv-e, £2 standing for tumor virus. No locus corresponding to subgroup D has been identified. Subgroup B and subgroup D viruses, however, appear to use the same set of receptors. In each case the susceptibility allele is dominant. Once the virus has bound to its receptor, it is not clear how the virion is internalized. Internalization could occur through fusion with the plasma membrane or by receptor-mediated endocytosis. It should be noted that mammalian cells do not express any of the t1 loci, yet can be infected by ALVs. This process occurs with very low efficiency, and entry presumably occurs through nonspecific adsorption to the plasma membrane. Once the cell has been infected, viral DNA is synthesized and integrated into the host genome. Successful DNA synthesis requires not only the appropriate subtrates, primers, and enzymes, but also an active cellular environment. The final product of DNA synthesis is a linear duplex DNA which contains duplicated copies of U3 and US at its termini. The duplicated sequence is referred to as the long terminal repeat (LTR). These sequences are necessary for integration into the host genome and subsequent expression of viral gene products. Reverse transcriptase is responsible for synthesis of both the plus and minus strands of DNA. The minus strand is synthesized first. It is initiated by binding of a host tRNA to the primer binding site situated at the 5' end of the viral RNA. The polymerase synthesizes only 100 to 180 nucleotides before reaching the end of the template. This short minus strand DNA, called strong stop DNA, contains R 19 sequences complementary to the sequences present at the 3' end of the viral genome. Hybridization to complementary DNA sequences situated at the 3' end of the viral genome serves as primer for the synthesis of the entire minus strand. Plus strand synthesis procedes similar to minus strand synthesis. It begins in the 3' noncoding region of the virus and is quickly halted due to termination of the template. Synthesis terminates at sequences complementary to the primer binding site of the strong stop DNA. This duplication enables the second ‘jump' of the polymerase and completes plus strand synthesis. The final product is a linear duplex DNA with two copies of U3-R-U5 at each end. Errors in reverse transcription may account for the relatively high frequency (10'3 to 10'4) at which viral mutants are generated during viral passage. Deletion mutants may be generated by inappropriate transfer of nascent DNA strands from one template to the other (Coffin, 1979). Reverse transcriptase also shows a high rate of misincorporation in vitro (Gopinathan et a1., 1979) and may account for viral mutants containing point mutations. The infidelity of reverse transcriptase may in part be due to the lack of an associated editing function. Errors introduced into the viral genome during reverse transcription are essential to the transduction of host sequences since most viral oncogenes contain multiple mutations relative to their cellular counterparts. After the linear duplex DNA is synthesized it is transported to the nucleus where a portion of it becomes circularized. The circular 20 DNA is specifically cleaved by a virus associated endonuclease. As a result it is always inserted into the host chromosome colinear with the viral genome. Insertion appears to be random and there is no obvious sequence specificity at the integration sites. The completion of the virus life cycle depends on the transcription of the provirus into progeny genomes and mRNAs. The structure of the integrated provirus is similar to that of the virus except for the presence of the LTRs at each end. The LTR provides most of the regulatory sequences necessary for transcription. The cap site is located at the 5' boundary of R. 25-30 nucleotides upstream of the initiation site are sequences resembling the so-called "TATAA box". Also present are CCAAAT sequences 70-85 nucleotides upstream of the cap site. Both these sequences have been implicated in determining the transcriptional start site of most eukaryotic genes. A polyadenylation signal, AATAAA, is located 20 nucleotides from the U3-R boundary. In addition to these sequences, the LTR also contains sequences, called enhancer sequences, which can increase the activity of a nearby heterologous promoter (Banerji et a1., 1981; Khoury et a1., 1983). These sequences are situated in the 5' two-thirds of U3, and appear to operate in a position and orientation independent manner. It is not clear whether these enhancer sequences in any way influence the promoter activity of the LTR. Transcription is catalyzed by RNA polymerase II. Capping and other processing events are presumably performed by additional host enzymes. Transcription is initiated at the U3/R boundary of the 5' LTR and 21 terminates at the R/US boundary of the 3' LTR. Two polyadenylated RNAs are generated. One encodes a full-length genomic mRNA which can be packaged into virions or serve as mRNA for the gag and 221 genes. The other RNA is generated by splicing from the leader sequence of the virus into any. This subgenomic RNA serves as template for the gag polyprotein. ALV is unique in that its splice donor dite is located within the gag coding sequence. As a result, six amino acids of the gag gene are present at the amino-terminus of gay. The presence of a translational start site before the splice donor site is of special significance in activating the proto-oncogene c-erb B. The primary translation products of gag, 221, and gay are precursor polyproteins which are post-translationally modified to generate mature virion proteins. Expression of the gag, 221, and gay genes is required for productive infection. The majority of RNA packaged into virions is the genomic viral RNA, although some nonspecific packaging has also been reported (Boccara et a1., 1982; Linial, 1987). This selection is presumbably due to the recognition of specific viral sequences. Sequences present in the leader region of the virus have been shown to contain sequences important in packaging (Shank et a1., 1980; Koyama, 1984). These sequences, however, are present in both the subgenomic and genomic RNAs. Sequences present in the gag gene, or the ”intron" region of ALV, have been implicated in viral packaging (Pugatsch et a1., 1984). The latter case offers an explanation for preferential packaging of the genomic RNA since these sequences are spliced out in the subgenomic RNA. Copackaging of 22 genomic RNA and cellular RNA can facilitate viral recombination and the generation of defective viruses which contain oncogenes. D. Qngggeaaaia by Nonacate Regrgvirases The complex nature of retrovirus replication and its intimate interaction with the host cell suggests several ways in which non-acute retroviruses can mediate pathogenesis. Expression of the viral structural genes themselves may directly or indirectly affect the growth of the host cell. For example, the gay gene product is expressed on the cell surface and can serve as antigen to activate the immune system (Teich, 1982). Continuous expression could lead to immunosuppression thereby allowing tumor cells to escape immune surveillance. The gay gene product itself has been implicated in murine leukemogenesis. In this case the normal gag gene is altered as a result of recombination between the infecting virus and endogeneous viral sequences. The recombinant virus, called the spleen focus forming virus (SFFV), induces rapid tumor formation (Teich, 1985). It is as yet unclear how the structurally altered gay gene mediates leukemogenesis in the murine system. It seems most likely that expression of the recombinant gay gene product mimics some other molecule (perhaps a growth factor receptor) which is instrumental in regulating hematopoiesis. More recently it has been demonstrated that certain viral genes of the human T-cell leukemia viruses (HTLV-l and HTLV-Z) can act 1a_§;ana thereby affecting transcription and translation of other viral gene products. The tat-1 and tat-2 genes are known to affect expression of cellular genes, namely interleukin-2 23 and the interleukino2 receptor (Greene et a1., 1986). This illustrates yet another way in which virus infection can disrupt normal cell function and perhaps lead to oncogenesis. Retroviruses can also act as mutagens since their DNA form, the provirus, integrates randomly into cellular DNA. Insertion of a provirus into a cellular gene can disrupt its normal expression. Inactivation of the gene by proviral insertion may not be readily detected since the host genome is diploid and only one allele would be disrupted. A number of genetic markers such as coat color and developmental disorders have been identified which are associated with retroviruses inserted within a few specific loci (Kozak, 1985; King et a1., 1985). These are usually examples of recessive mutations and have only been identified through classical genetics. Tumor formation may result from a dominant mutation and would therefore select for a different type of mutation rather than gene inactivation. The provirus carries with it strong transcriptional regulatory elements at its termini. The introduction of these regulatory elements into the host genome by retroviral insertion has the potential to produce a dominant phenotype. A retrovirus in this context could induce tumor formation or leukemia production by placing the cellular gene under the regulation of viral sequences. This results in increased and perhaps inappropriate expression of the cellular gene. This type of alteration is often referred to as insertional activation because of the associated increase in transcription. Insertional activation of certain cellular genes, namely proto-oncogenes, appears 24 to be the predominant mechanism by which most non—acute retroviruses induce neoplasia. A mechanism for pathogenesis by non-acute retroviruses was first provided by studies B-cell lymphomas. The majority of lymphomas analyzed contain a provirus inserted in the c-myc locus. C-myc is the cellular counterpart of the viral oncogene, v-myc, the transforming gene found in the avian retrovirus, MC-29. All tumors showed marked increases in the relative levels of c-myc mRNA (30-100 fold; Hayward et a1., 1981). Molecular analysis of B-cell lymphomas induced by avian leukosis virus (ALV) established that there are at least two distinct mechanisms by which non-acute retroviruses can activate transcription of adjacent cellular sequences. The chicken c-myc gene contains three exons, the first of which is noncoding (Watson et a1., 1983; Shih et a1., 1984). Integrated proviral sequences were detected upstream of the first coding exon (exon 2) in over 80% of the B-cell lymphomas analyzed (Hayward et a1, 1981; Shih et a1., 1984; Fung et a1., 1982b; Payne et a1., 1982). Most of the insertion sites were located within the first intron, but others were observed as far as 5 kb upstream. Proviruses were usually situated in the same transcriptional orientation. Elevated levels of c-myc were detected in tumor samples containing this type of viral-c- myc arrangement. The elevated c-myc transcripts comigrated with novel viral related RNAs. These virally linked transcripts exclusively hybridized to US sequences of the LTR. Based on these observations, it was proposed that c-myc transcription is initiated in the 3' LTR and 25 that transcription reads through into the c-myc gene (Hayward et a1., 1981). In this situation, the 3' LTR of the provirus acts as a promoter for the adjacent c-myc gene. This was somewhat unusual since viral transcription normally utilizes the promoter within the 5' LTR. The deletion of additional ALV sequences within or near the 5' LTR in most, if not all, of the tumors analyzed suggested that their removal may potentiate the use of the 3' LTR as a promoter (Neel et a1, 1981; Payne et al, 1981; Fung et a1., 1981). This particular mechanism of activation is known as promoter insertion. C-myc expression is presumably deregulated by putting it under the activity of the viral promoter. In a few other cases the proviral DNA was found either a) inserted downstream of c-myc and in the same transcriptional orientation, or b) upstream of c-myc but in the opposite orientation (Payne et a1., 1982). The former case may be an exceptional one since it was observed in only one tumor sample. This particular sample contained elevated c-myc RNA levels and the c-myc related transcripts were found to terminate prematurely due to the insertion of viral sequences at the 3' end of the gene. In the latter case, elevated levels of c-myc expression were also observed but no ALV sequences were found associated with the c-myc RNAs. The positioning of an ALV provirus in the opposite transcriptional orientation would not allow the viral promoter to be used for initiating c-myc transcription. In these cases other viral sequences are thought to be responsible for elevated c-myc expression. These sequences, now known as enhancer elements, reside in the U3 26 region of the viral LTR, and act independent of position and orientation to enhance the transcriptional efficiency of the normal cellular promoter. This mechanism of c-myc activation, referred to as enhancer insertion, is different from promoter insertion in that transcription is not initiated from viral sequences but uses cellular promoters. The cellular promoters utilized may not be the normal cellular promoters since several tumor samples appear to utilize cryptic promoters. Both mechanisms affect the level of c-myc transcription. The highest levels are usually associated with tumors containing promoter insertion arrangements. Thus promoter insertion may prove to be a more efficient mechanism for activating the c-myc gene in B-cell lymphomas since most of the tumors analyzed display this type of activation mechanism. In both promoter insertion and enhancer insertion, the 5' untranslated region of the normal c-myc mRNA is removed due to integration within the first intron. This loss of untranslated sequence may further affect c-myc expression. It has been proposed that the noncoding sequences of c-myc RNA may form a potential hairpin or rho-like structure which would subsequently inhibit efficient translation of c-myc RNA (Saito et a1., 1983, and Nottenburg et a1., 1986). Removal of the untranslated region as a result of proviral insertion may enhance translation as well as transcription. The relative level of the c-myc protein produced may actually exceed the 30 to 100 fold estimates made based on transcription. 27 The truncated c-myc RNA should encode a protein virtually identical to that of the normal c-myc protein. In only one case, however, has the structure of the c-myc protein from a tumor sample been determined. When the nucleotide sequence of the activated c-myc gene was compared to that of normal c-myc, several point mutations were found (Westaway et a1., 1984). Some of the differences did result in amino acid changes. It is not clear whether these structural changes in the activated c-myc gene product contribute in any way to the development of B-cell lymphomas. The prevailing theme of oncogenesis by nonacute retroviruses is one that implies an alteration in gene regulation. This observation raises the question of whether qualitative changes as well as quantitative changes in gene expression may be necessary for tumor formation. The B-cell lymphoma studies described above are correlative and do not demonstrate that c-myc is responsible for lymphomagenesis. Experiments to directly address the disease potential of c-myc have been limited to studies using acute leukemia viruses which carry v-myc sequences. There are several strains of v-myc containing viruses. These include MC-29, MH-2, and OK-II. Like the activated c-myc gene, the v-myc genes of these viruses contain several nonconservative point mutations (Bister et a1., 1986). These point mutations do not, however, affect common amino acids. Most of the v-myc proteins are synthesized as viral fusion proteins and therefore are not identical to the activated c-myc protein. Interestingly, these viruses have a different oncogenic spectrum and do not induce B-cell lymphoma. 28 Myelocytomatosis, the predominant disease associated with v-myc containing viruses, is quite distinct from B-cell lymphomas and affects cells of the early myeloid lineage (Graf et a1., 1978). Closer examination of MC-29 infected birds has revealed small B-cell lymphomas. These are not the predominant lesion and therefore may have been missed in earlier studies (Hayward et a1., 1983). A newly isolated variant of MC-29, HB-l, induces short latency lymphomas of B- and/or T-cell origin (Enrietto et a1., 1983). The difference in the pathogenicity of the HB-l virus is thought to be due to recombination between v-myc and c-myc sequences. This hypothesis needs to be tested more rigorously since the recombinant virus has also undergone additional recombination events in the helper virus sequences. Inconsistencies in other experimental parameters such as the age and strain of the chickens used, as well as the route of injection, make the results of these experiments, and experiments with other myc containing viruses difficult to compare and interpret. C-myc activation does not appear to be limited to avian B-cell lymphomas since activated c-myc has been observed in other diseases induced by nonacute retroviruses. In one case, an adenocarcinoma induced by the ring-neck pheasant virus (RPV) was found to contain an activated c-myc gene (Simon et a1., 1984). RPV is classified as an avian leukosis virus (Fujita et a1., 1974; Temin et a1., 1976). This virus is unusual in that it is appears to be a recombinant virus between endogenous gag sequences of the pheasant, and ALV. Its disease potential is also unique in that it can induce a variety of short and 29 long latency neoplasms (Carter et a1., 1983). The molecular basis of oncogenesis by this virus is poorly understood; in some ways it appears to be analogous to the murine SFFV virus discussed earlier. The presence of an activated c-myc gene in this adenocarcinoma may be secondary to the initial transformation event, since c-myc was found activated in only one tumor (Simon et a1., 1984). In addition, several other lesions, including fibrosarcomas were observed in this one diseased chicken. An activated c-myc gene was not detected in any of the other lesions further suggesting that c-myc activation in the isolated tumor sample may not be significant. C-myc activation has also been implicated in T-cell lymphomas induced by REV. Over 90% of the REV induced B-cell lymphoma samples contained activated c-myc alleles (Noori-Daloii et a1., 1981; Swift et a1., 1985). The structural arrangement of the proviruses was consistent with a promoter insertion mechanism of activation (Swift et a1., 1987). It is interesting that the activation mechanism in T-cell lymphomas was slightly different from that observed in the B-cell lymphomas induced by both ALV and REV (Isfort et a1., 1987). As expected the level of c-myc expression was elevated in the T-cell lymphomas, but only 6-18 fold over normal c-myc as opposed to the 30- 100 fold increase observed in B-cell lymphomas. The difference in the relative levels of c-myc expression in the two tumor types may be due to the predominance of enhancer insertion as the mechanism of c-myc activation in the T-cell lymphomas. All the REV proviruses were found to be inserted 5' of the second exon of c-myc, and half of the 30 proviruses were situated in the opposite transcriptional orientation with respect to c-myc. Only a subset of the proviruses inserted in the same transcriptional orientation were found to actually utilize the 3' LTR to promote downstream transcription. In the other cases, transcription initiation mapped to a common cryptic promoter site present in the intron of c-myc. These observations indicate that activation of c-myc can occur via enhancer insertion irrespective of the transcriptional orientation. Enhancer insertion rather than promoter insertion appears to be the predominant mechanism by which c- myc is activated in REV-induced T-cell lymphomas. Enhancer insertion of c-myc is also the predominant mode of activation observed in lymphomas induced by murine retroviruses (Steffen, 1984; Corcoran et a1., 1984). Thus the enhancer element is not limited to avian retroviruses and appears to play an important role in myc activation. The fact that the same virus (REV) can alter the same gene (c-myc) in two different cell types (B-cells versus T-cells) by somewhat different mechanisms suggests that there may be other factors which dictate the precise mechanism of c-myc activation. Activation of c-myc in ALV induced lymphomas provided the first indication that abnormal expression of proto-oncogenes could be responsible for tumorigenesis. Since that time a variety of other neoplasms induced by non-acute retroviruses have been analyzed for the activation of cellular oncogenes. In addition to the c-myc gene, the c-erb B gene has been identified as an activated proto-oncogene in ALV induced erythroblastosis (Fung et a1., 1983). This leukemia and the 31 precise mechanism of c-erb B activation will be discussed in detail later. The mechanism of c-erb B activation is similar to the promoter insertion mechanism identified in B-cell lymphomas except that c-erb B transcription initiates from the 5' LTR of the integrated provirus rather than from the 3' LTR. Again, these subtle variations in activation mechanisms in different cell types suggests that there may be other factors which determine the precise mechanism by which a proto-oncogene can be activated. The c-Ha-ras gene is the only other avian proto-oncogene that has been found to be activated in an ALV induced neoplasm. The activated c-Ha-ras gene was observed in a nephroblastoma induced by MAV-l (myeloblastosis associated virus type -1; Westaway, 1986). MAV-l is similar to the ALVs used to induce B-cell lymphomas and erythroblastosis (Moscovici et a1., 1968). A promoter insertion mechanism appears to mediate c-Ha-ras activation by MAV-l, since LTR sequences are linked to the c-Ha-ras RNA. The proviral insertion, however, appears to be distal to the c-Ha-ras gene and is not detectable as a rearranged restriction enzyme fragment by Southern analysis. This tumor represents a unique class since an activated c- Ha-ras mRNA could only be identified in one of several nephroblastomas analyzed. Thus, c-ras activation may not be crucial to the formation of kidney tumors in chickens. A variety of tumors induced by nonacute murine retroviruses have been analyzed for activated oncogenes. Both c-myc and c-myb activation have been observed in lymphomas (Steffen, 1984; Corcoran et a1., 1985; 32 Shen-Ong et a1., 1984). The frequency of association of insertional activation is better in these cases than the correlation of c-Ha-ras and c-myc activation in avian nephroblastomas and adenocarcinomas, but is still poor. In general, an activated oncogene could be detected in less than 30% of the retrovirus induced tumors. This is in stark contrast to the lymphomas and leukemias induced in the chicken where there is a 80-100% correlation between tumorigenesis and activation of c-myc or c-erb B. While c-myc and c-erb B activation appear to play an important role in avian leukemogenesis, the weaker correlation in other systems suggests that other events may be necessary for the development of a full fledged tumor. Finally, cellular sequences with no homology to viral oncogenes have also been implicated in oncogenesis. These potential proto- oncogenes have been identified by virtue of their linkage to proviral DNA in a number of tumors. This approach, known as transposon tagging, takes advantage of the fact that tumors induced by non-acute retroviruses are clonal with respect to the integrated proviruses. Provirus integration sites which map to a common region in several independent tumor samples suggest that the adjacent or activated gene may be critical to tumor formation. In the avian system common integration sites have been found associated with RPV proviruses in nephroblastomas and fibrosarcomas (Simon et a1., 1984). The cellular sequences adjacent to the RPV proviruses, however, have not been characterized, nor has the precise mechanism of activation been determined. A similar strategy has been used to analyze a variety of 33 tumors induced by mouse retroviruses and several common loci have been identified. These include int-l, int-2, pim-l and mlvi-l (Nusse et a1., 1982; Peters et a1, 1983; Tsichlis et a1., 1983; Cuypers et a1., 1984). Perhaps the most notable is the int-l locus which has been shown to possess at least partial transforming ability ig_21;;a (Brown et a1., 1986). Like the c-myc and c-myb genes in murine lymphomas, the correlation between these new proto-oncogenes and the particular tumor induced is not absolute. More recent evidence suggests that some of these proto-oncogenes may work in concert with each other during tumor progression. Thus activation of more than one proto-oncogene may be necessary for the fully malignant phenotype to be manifested. The detection of activated proto-oncogenes may not be straightforward, and may be influenced by the stage of tumor development. This could explain, in part, the poor statistics observed between proto-oncogene activation and murine tumor formation. The idea that tumorigenesis is a multistep process is not a new one and is consistent with the current theories of cancer biology. The molecular characterization of neoplasms induced by non-acute retroviruses has extended our understanding of the role of cellular genes in oncogenesis, how they can be mutated, and how retroviruses can mediate this process in a very specific manner. In summary, retroviruses insert into the host genome as an intermediate step in the virus life cycle. In addition to acting as an insertional mutagen, they introduce strong transcriptional regulatory elements into the genome. Two types of viral elements, promoters or enhancers, can be 34 used to increase the transcription of a cellular gene. What determines the usage of one element over the other appears to be dictated by the cell type affected as well as the gene in which the crucial insertional lesion occurs. The net result is an overall increase in transcription and deregulation of the cellular gene. Although abnormal expression may be sufficient for development of the transformed phenotype, other structural changes either in the activated proto-oncogene itself or in other genes may contribute further to formation of the malignant tumor. We have seen different retroviruses induce the same neoplasm by activating the same genes. A single retrovirus can also be multipotent and induce different neoplasms by interacting with different cellular genes. It is these basic observations that have provided the foundation for our current understanding of oncogenesis. The molecular characterization of other tumors has extended these initial findings and led to the identification of yet another set of cellular genes important in retrovirus induced neoplasms. Analysis of naturally occurring tumors suggests that the same genes may be involved in nonvirally induced cancers. The question, however, remains as to what the normal functions of these genes are, and how deregulation contributes to oncogenic transformation. E. anggeaeaia by Acute Iraaaforming Viruses Oncogenesis by acute transforming viruses does not require the complex virus-cell interactions described for the non-acute retroviruses. Successful infection by the acute virus into the appropriate target cell is sufficient for tumorigenesis since activated 35 oncogenic sequences are intrinsic to their genomes. Continuous recruitment of target cells explains the rapidity and frequency of tumor formation characteristic of the acute transforming viruses. The highly tumorigenic nature of these viruses make them easy agents to isolate and monitor. It is not surprising that these retroviruses were some of the first to be characterized. How most of these viruses originated remains a mystery. Presumably they arose by recombination between a non-acute transforming virus and a cellular oncogene. This process is known as transduction. The limited number of independent acute transforming viruses isolated over the last 80 years suggest that transduction is a rare event. Analysis of the sequences and structure of known acute transforming viruses, suggest some of the events which may be involved (cf. Bishop, 1983; Varmus, 1984 for reviews of the transduction process). Several features of retroviruses make them amenable to the transduction of cellular sequences. First, retroviruses frequently undergo recombination. Viral gay sequences have been shown to be exchanged at an unusually high frequency during mixed infection (Vogt, 1971; Coffin, 1979). Second, the retrovirus genome is diploid. Heterozygote virions can occur which would allow two different genomes to be physically linked. Linkage between two different viral genomes should facilitate recombination. And finally, the DNA form of the virus integrates into the host genome. This is itself a recombination event and provides another way in which cellular and viral sequences can be physically joined. 36 In considering a mechanism of transduction, there are several features characteristic of viral oncogenes which should be accounted for. 1) Viral oncogenes are intrinsic to the viral genome and are therefore are always flanked by viral sequences. The positioning within the virus varies and may disrupt any of the structural genes. 2) Intron sequences that lie between the transduced exons of oncogenes are removed. This does not include sequences 5' of the first ' transduced exon since intron sequences presumably derived from cellular oncogenes are frequently observed at this point. 3) Viral oncogenes are not an exact copy of their cellular counterparts. In many cases only a portion of the cellular oncogene is transduced. The sequences not transduced may be untranslated sequences, like those described for c-myc, or coding sequences. The removal of coding sequences at either the amino- or carboxy-termini poses an additional problem since the initiation and/or termination sites are absent. In these cases, the oncogenic protein is expressed as a fusion protein with viral sequences at its amino- and/or carboxy-terminus. The correct reading frame of the oncogenic protein must be maintained in all cases, even when it is expressed as a hybrid protein. In addition to truncations, viral oncogenes contain other mutations internal to the coding sequences. These include non-conservative point mutations and internal in-frame deletions. 4) Examination of the recombination points between cellular sequences and the viral genome do not reveal any unique features suggestive of a recombination mechanism. In only a few isolated cases have short stretches of sequence homology been observed (Besmer, et a1., 1986). 37 A model for transduction was proposed by Swannstrom and coworkers (1983) based on the features of RSV. It is consistent with the structure of most acute transforming viruses and is described below. First, a non-acute retrovirus inserts itself adjacent to a cellular oncogene such that it is oriented in the same transcriptional direction. This step is similar to the insertional activation mechanism described for c-myc and c-erb B. The next step requires deletion of the 3'LTR. Adjacent cellular sequences of intron or exon origin may also be deleted. Removal of the 3' LTR allows for efficient transcription of a hybrid RNA molecule. The hybrid RNA is initiated in the 5' LTR, contains both viral and cellular sequences, and extends to the 3' end of the cellular oncogene. Introns are subsequently removed, and the chimeric RNA is packaged into virions. A second recombination is required to provide 3' viral sequences and complete the transduction process. The 3' viral sequences are supplied by the parental, non- acute retrovirus and require the formation of a heterodimer. Recombination is thought to be due to template switching of the DNA polymerase during reverse transcription, a process known as copy-choice (Coffin, 1979). In transduction of cellular oncogenes, the copy-choice mechanism does not appear to be facilitated by homologous sequences. The complexity of the transduction process coupled with the probability of each step occurring correctly may explain its rarity in nature. There is one variation on this proposed transduction mechanism which has been implied by some recent studies. It is that deletion of the 3' LTR may not be necessary to generate a functional transducing 38 virus. Initially, a deletion in the 3' LTR was invoked to generate a fusion transcript that is packageable. It was previously thought that the subgenomic gag RNAs could not be packaged because they lacked the appropriate packaging sequences (Pugatasch et a1., 1983). By analogy, any viral-fusion transcript generated by a subgenomic splice would also not be suitable for virus assembly. There are now at least two cases where subgenomic, spliced mRNA molecules are not only packaged but replicate as viruses (Ikawa et a1., 1986; Martin et a1., 1986). Thus deletion of the 3' LTR nay not be necessary for transduction if readthrough transcription and splicing can occur such that the subgenomic splice donor and a splice acceptor in the adjacent c-onc are used. This alternative method of transduction would not be very efficient. Transcriptional readthrough of the 3' LTR of integrated proviruses accounts for less than 10% of all viral transcription (Herman et a1., 1986). In addition, the spliced subgenomic viruses described above grow to low titers suggesting that the packaging efficiency is impaired. The low frequency at which readthrough transcripts are synthesized and their impaired packageability make them plausible, but unlikely candidates for intermediates in the transduction process. It has been difficult to rigorously test aspects of this model since there are few, if any, experimental systems available. Reconstitution experiments done in tissue culture have usually been facilitated by homologous sequences and are not directly comparable to the above mechanism. There are at present few animal host systems 39 which have been shown to transduce cellular oncogenes. The first is the ALV induced erythroblastosis system which transduces the oncogene c-erb B in 25 to 50% of the leukemia samples analyzed (Chapter 4 and Miles et a1., 1985). This system has been exploited for the purpose of understanding the mechanism of transduction, and the results are discussed elsewhere. Feline leukemia virus (FeLV) associated lymphomas also frequently contain transduced c-myc genes (Levy et a1., 1984; Mullins et a1., 1984, Neil et a1., 1984). These transductions are only observed in naturally occurring lymphomas and not in lymphomas resulting from injection of FeLV. Their analysis is consistent with the transduction scheme outlined above. In terms of c-myc activation, only very short truncated regions of exon 1 of c-myc is retained in the transduced viruses, supporting the idea that exon 1 plays a regulatory role in c-myc expression (Stewart et a1., 1986). The observation that the same cellular oncogene is transduced in several independently isolated acute transforming viruses suggests that these genes are indeed important in oncogenesis (cf. Bishop, 1983, for complete list). It is unclear how these overexpressed and potentially mutated cellular genes, be they insertionally activated or virally transduced, transform cells. Several things should be kept in mind when considering this question. First, the type of tumor produced and the specific cell types which are affected by expression of the oncogenic protein. Second, the actual oncogenic protein itself should be identified and its biochemical properties determined. Third, mutants altered in either their transforming ability or biochemical 40 properties should be analyzed since they may provide further insight into the function of the oncogenic protein. Finally, comparison of the mutated oncogene to its normal cellular counterpart should reveal features important in determining its oncogenic potential. The remainder of this discussion focuses on the erb B oncogene and its transforming ability. Although at least 20 other oncogenes have been identified, studies on erb B have greatly influenced our understanding of how normal cells become transformed. Erb B illustrates one of many ways in which this process may occur. F. Strugture and Functioa of Viral erb B v-erb B was first identified as the transforming component of the avian erythroblastosis virus, AEV (Bister and Duesberg, 1979; Lai et. a1., 1979; Roussell et. a1., 1979). At this time two strains of AEV had been described, strain R (AEV-R) and strain E84 (AEV-E84). Both strains were shown to induce a high incidence of erythroblastosis within a short period of time. Each virus transformed fibroblasts 1a gigrg and could induce fibrosarcomas if injected intramuscularly (Graf et a1., 1976, 1977; Rothe-Meyer et a1., 1933). Biochemical comparison of the viral proteins expressed in AEV-R and AEV-E84 suggested that these two strains were in fact identical (Hayman et a1., 1979). The remainder of this discussion will refer to these two virus strains as AEV-R. More recently, another strain of AEV, designated AEV-H, has been isolated (Hihara, et. a1., 1983). Like the other strains, AEV-H is capable of inducing erythroblastosis and causing fibrosarcomas 41 (Hihara et a1., 1983). AEV-H has also transduced c-erb B sequences. Its sequence content and biological properties, however, are distinct from AEV-R and AEV-E84. The AEV-R genome is 5.3 kb in length. Most of the 291 gene and a portion of the gag and gay genes of the helper virus have been deleted and substituted by 3 kb of AEV-specific sequence (Bister et a1., 1979; Roussel et. a1., 1979; Lai et. a1., 1979). Two mRNAs are transcribed from the AEV-R genome, a 5.3 kb genomic RNA and a 3.5 kb subgenomic RNA (Anderson et a1., 1980). These RNAs code for two distinct proteins (Hayman et a1., 1979; Lai et a1., 1980; Pawson et a1., 1980; Privalsky et a1., 1982). P75erbA is synthesized from the genomic RNA and is a gag-related protein of 75 kD. It contains the 5' half of the AEV-R specific sequences, designated as v-erb A. The subgenomic RNA contains the remaining 3' half of AEV-R specific sequences, designated as v-erb B, and codes for a glycoprotein of 68 kD. Molecular cloning of AEV-R proviral DNA verified the nature of the two AEV-specific genes (Vennstrom et a1., 1980). V-erb A and v-erb B appear to be distinct since they are derived from cellular sequences (c-erb A and c-erb B) located on different chromosomes and coding for different mRNAs (Roussel et a1., 1979; Saule et a1., 1981; Symonds et a1., 1984). The AEV-H genome contains only v-erb B sequences (Yamamoto et al., 1984a). Its genome is 7.8 kb in length and contains intact gag and pal genes. A portion of the gag gene has been replaced by approximately 2 kb of v-erb B specific sequences. Like the AEV-R genome, two mRNAs are synthesized, a full length genomic RNA and a 3.0 kb subgenomic RNA 42 containing the erb B specific sequences. This subgenomic RNA synthesizes an erb B protein of ca. 78kD. Both AEV-R and AEV-H have been molecularly cloned and their nucleotide sequence determined (Yamamoto et al., 1984b; Privalsky et a1., 1984). Although only a portion of the entire v-erb B sequence from AEV-R has been published (Henry et a1., 1985; Sealy et a1., 1983), the entire sequence of an AEV-R ts-mutant, ts-l34, allows the erb B sequences in AEV-H and AEV-R to be compared (Choi et a1., 1986). Both v-erb B alleles contain common intron sequences at their 5' junctions. AEV-H, however, has transduced approximately 50 nucleotides less than AEV-R. Directly adjacent to the c-erb B intron sequence is the 5' terminus of a c-erb B exon. A consensus splice acceptor marks the intron-exon boundary (Henry et a1., 1985). This splice acceptor is presumably the one utilized in synthesizing the subgenomic mRNAs in both AEV-H and AEV-R, and this same splice acceptor site is used to generate the insertionally activated c-erb B RNAs (described in Nilsen et a1., 1985). Comparison of the remainder of the v-erb B alleles indicate that they are closely related. They differ at 21 nucleotides, ll of which are conservative point mutations. AEV-R has also suffered an internal in-frame deletion of 21 amino acids near its carboxyl termini. Both AEV-R and AEV-H v-erb B genes appear to be truncated at their carboxyl terminus since their coding region is fused directly to sequences derived from the any gene. These features indicate that AEV- H and AEV-R resemble typical acute transforming viruses. That is, 1) they have transduced only a portion the c-erb B gene, 2) the 43 transduced c-erb B sequences are situated internal to viral sequences, 3) recombination has occurred within intron and exon sequences, and 4) a number of point mutations have accumulated and may contribute to the acute transforming ability. The v-erb B protein is expressed as a fusion protein which contains 6 amino-terminal amino acids of the gag gene. These sequences are fused directly to v-erb B as a result of subgenomic splicing and maintain the correct translational reading frame. In AEV-H, the subgenomic RNAs can be alternatively spliced in a manner similar to that described for the insertional activation of c-erb B (see Nilsen et a1., 1986), and therefore can produce two different proteins. Use of a viral splice acceptor and a cryptic splice donor 159 nt away results in the addition of 53 amino acids of any. AEV-H and AEV-R contain 546 and 567 v-erb B codons, respectively. Both terminate in stop codons immediately 3' to the erb B-viral junction. These codons are out of frame with respect to the any coding sequence and therefore are not true fusion proteins. Comparison with the insertionally activated c- erb B coding sequence indicates that AEV-H and AEV-R lack the last 34 and 74 amino acids of erb B, respectively. Primary translation products of ca. 61,700 MW and 69,000 MW are predicted for AEV-R and AEV-H, consistent with the 62 kD and 67 kD erb B related proteins detected in immmunoprecipitates of AEV-R and AEV-H infected cell extracts (Privalsky and Bishop, 1982; Hayman et. a1., 1983, Privalsky et. a1., 1983). 44 The deduced amino acid sequence of the v-erb B gene predicts a transmembrane glycoprotein (Privalsky et a1., 1983, 1984). A 23 amino acid transmembrane domain separates the amino-teminal extracellular domain (77 amino acids) from the cytoplasmic domain (430 or 473 amino acids). The orientation in the plasma membrane has been verified using antibodies specifically directed against the amino-terminus (Schatzman et a1., 1986). Synthesis of the erb B protein occurs on membrane bound polysomes and is transported from the rough endoplasmic reticulum to the cell surface (Hayman et a1., 1984; Beug et a1., 1984). The extracellular domain contains several sites for N-linked glycosylation. Experiments using glycosylation inhibitors indicate that the v-erb B polypeptide from AEV-R is synthesized as a 62.6 kD precursor and is modified to a 66 kD and 68 kD intermediate form before becoming fully glycosylated to the 74 kD erb B protein. Correct glycosylation of v- erb B does not appear to be important in either subcellular routing or transformation since selective expression of the glycosylation intermediates results in transformation (Schmidt et a1., 1985). Cell surface expression of v-erb B does appear to be important in v-erb B function since temperature sensitive (ts) mutants of AEV-R which are unable to incorporate erb B into the plasma membrane at the nonpermissive temperature also are not transforming in fibroblasts (Beug et a1., 1984). In general, only a small portion of the v-erb B protein actually reaches the cell surface. This is presumably due to the absence of a "consensus" signal sequence in v-erb B as a result of amino-terminal truncation (see below). 45 The first clue to the function of v-erb B was provided by demonstration of homology between v-erb B and the src gene family of tyrosine specific kinases (Privalsky et a1., 1983). Similar enzymatic activities among different oncogenes suggested that they may act by a common mechanism. No v-erb B associated kinase activity was initially detected in AEV-R transformed cells (Hayman et a1., 1983; Privalsky et a1., 1983; Hayman et a1., 1984). Development of more sensitive kinase assays and the use of enriched membrane fractions verified that v-erb B did possess an intrinsic kinase activity both in vivo and in vitga (Gilmore et a1., 1985; Decker, 1985; Hayman et a1., 1986). In frame mutagenesis which disrupts the structural integrity of the protein kinase domain suggests that kinase activity is essential for v-erb B transforming ability. v-erb B supplied the link between tyrosine kinases and cell proliferation when it was found to share homology with the human epidermal growth factor receptor (hEGF-R; Downward, et a1., 1984; and Ullrich, et a1., 1985). The human EGF-R is also a transmembrane protein possessing intrinsic tyrosine kinase activity (cf Carpenter, 1987; Schlessinger, 1986 for reviews). The tyrosine kinase activity in EGF-R however, is regulated by epidermal growth factor (EGF) and results in cell proliferation. Both v-erb B and hEGF-R contain related transmembrane and cytoplasmic sequences. V-erb B lacks the major portion of the extracellular domain responsible for binding EGF. The cytoplasmic protein kinase domain is the most highly conserved region between the two molecules displaying 95% amino acid homology. This 46 striking homology between v-erb B and the hEGF-R suggests that the c- erb B gene is equivalent to the avian EGF-R. This is a widely accepted view, despite the fact that very little is known about the c-erb B gene product or the avian EGF-R. Ligand-stimulated tyrosine kinase activity of hEGF-R is most likely an important event in signalling mitogenesis. Similarly, the transforming ability of v-erb B appears to be related to its tyrosine kinase activity. Therefore, the removal of the amino- terminus from c-erb B is thought to be a necessary step in making it oncogenic. Such an alteration would make the kinase ligand-independent and constitutively active. Other aspects of the v-erb B protein with regard to its regulation and function have been extrapolated from studies with hEGF-R. The hEGF-R contains three tyrosine residues which are major sites for autophosphorylation. These sites have been mapped to tyrosine residues 1173 (P1), 1148 (P2) and 1068 (P3), all within the carboxy-terminal region of hEGF-R (Downward et al., 1984b). Autophosphorylation at these sites has been shown to enhance the kinase activity of EGF-R (Weber et a1., 1984, and Bertics et a1., 1985). Tyrosine 1173 is the major site of phosphorylation in viva and is thought to play an important role in regulation. Analogous tyrosine residues have been located in v-erb B, although phosphorylation at these residues has not been documented. The v-erb B protein does, however, appear to be autophosphorylated (Kris et a1., 1985; Gilmore et a1., 1985; Hayman et a1., 1986; Decker, 1985). The major tyrosine residue (P1) is missing from both viral erb B products and is thought to be relevant in 47 determining oncogenicity (Downward et al., 1984b). An analogous Pl site was identified in the insertionally activated c-erb B gene (Nilsen et a1., 1985). Therefore removal of P1 does not appear to be necessary for erythroblast transformation (this topic is discussed in detail in the accompanying chapters). hEGF-R is also known to be phosphorylated at serine and threonine residues (Carlin et a1., 1982; Hunter et a1., 1981). Threonine phosphorylation is thought to regulate tyrosine kinase activity. Treatment with 12-O-tetradecanoylphobol-l3-acetate (TPA) stimulates protein kinase C resulting in an overall increase in serine and threonine phosphorylation. EGF-R kinase activity is also reduced by TPA treatment, and DNA synthesis is inhibited (Decker, 1984; McCaffrey et a1., 1984). Threonine residue 654 is a predominant site of phosphorylation associated with TPA treatment and may negatively regulate tyrosine kinase activity (Hunter et a1., 1984; Lin et a1., 1986). An analagous threonine residue is present in v-erb B at the same position -- near the cytoplasmic side of the transmembrane domain. The proximity of this residue to the plasma membrane makes it a likely target for regulating receptor activity and mitogenesis. Threonine phosphorylation provides a mechanism of down regulating EGF-R activity and may be important in regulating other cell surface receptors (i.e., the platelet derived growth factor receptor, PDGF-R). The importance of serine phosphorylation in receptor regulation has not been addressed but may be important since it has been shown to play an important role 48 in transmodulation of the adrenergic receptors (cf. Sibley et a1., 1985, for review). Although the biochemistry of the v-erb B gene product has not been extensively characterized, a great deal is known about the properties of AEV-R transformed cells. The primary target cell for erb B activity appears to be the erythroblast, although given the appropriate modifications other cell types can also be transformed (see Chapter 5). The majority of data regarding erythroblast transformation has been obtained from studies using AEV-R, which has transduced v-erb A as well as v-erb B. Although v-erb A is not required for erythroblast or fibroblast transformation, it has been shown to potentiate the transforming ability of v-erb B as well as other oncogenes in the tyrosine kinase family (Frykberg et a1., 1983; Sealy et al., 1983b; Damm et a1., 1987). This difference is manifested as an inhibition in differentiation and/or increased proliferation between AEV-R and AEV-H (or AEV-R mutants defective in v-erb A) transformed cells (Gandrillon, et a1., 1987). The nucleotide sequence of v-erb A shows striking homology to various steroid receptors including the human glucocorticoid receptor (Weinberger et a1., 1985), chicken estrogen (Krust et a1., 1986), and chicken progesterone receptors Conneely et a1., 1986). Recent cloning of the c-erb A proto-oncogene indicates additional homology with the tri-iodothyronine receptor (Sap et a1., 1986; Weinberger et a1, 1986). The precise role of v-erb A in transformation, however, remains unclear. The transduction of c-erb A sequences in addition to c-erb B sequences into the AEV-R genome 49 further illustrates the cooperation between potentially oncogenic sequences and their importance in selecting for a more malignant phenotype. The target cell for AEV transformation has been characterized using selective immunolysis of bone marrow cells. Antisera specific for various stages of erythroid cell differentiation reveals that AEV-R exclusively transforms cells at the BFU-E stage (Samarut, 1982). Further characterization of the transformed cells indicate that they display features similar to normal CFU-E and therefore must differentiate before becoming transformed. Transformed erythroblasts synthesize small amounts of hemoglobin, express erythroid specific H5, and contain erythroblast specific cell surface antigens (Beug et a1., 1979). Erythroblasts from leukemic animals form CFU-E colonies in semisolid media. Normal bone marrow cells can also induce CFU-E colonies if infected with AEV-R 1n vigzo (Graf, 1973).~ Colony formation does not require the addition of erythropoietin or other erythroid specific growth factors which may be present in anemic serum (Beug et al., 1982a). This is in contrast to normal CFU-E cells which absolutely require erythropoietin (epo) for colony formation. AEV-R infected erythroblasts are unique from erythroblasts transformed by other viruses in that they can be maintained in culture for prolonged periods. A number of continuous chicken erythroblast cell lines have been isolated from AEV-R transformed cells (Beug et al., 1982b). Expression of v-erb A is presumably responsible for the increased proliferative capacity of these cells. Erythroblasts transformed by v- 50 erb B alone (i.e., AEV-H) require special culturing conditions and a portion of these cells spontaneously differentiate (Beug et a1., 1985; Frykberg et a1., 1983). Interestingly, embryonic erythroid cells transformed by AEV-R also spontaneously differentiate in culture indicating that v-erb A expression may not be effective during early embryogenesis (Moscovici et a1., 1983). A number of AEV mutants have been isolated and characterized (Graf et a1., 1978; Beug et a1., 1982b). Most notable is the temperature sensitive mutant, ts-34, which contains a point mutaion in the protein kinase domain (Choi et a1., 1986). Erythroblasts transformed by this virus behave similar to normal CFU-E when shifted to the non-permissive temperature (Beug et a1., 1980). That is they require erythropoietin to differentiate and display antigenic markers characteristic of terminally differentiated erythrocytes. These cells have been especially useful in studying avian erythropoiesis. The fact that the ts mutation maps to the protein kinase domain further suggests the importance of tyrosine kinase activity in transformation. Several AEV mutants defective in erythroblast transformation have also been isolated (Royer-Pokora et a1., 1979; Beug et a1., 1980; Yamamoto et al., 1984a). These viruses retain their capacity to transform fibroblasts. The mutations of two host range mutants, td-359 and td-130, have been mapped to the carboxy-terminal portion of v- erb B. The v-erb B proteins synthesized by these two viruses are severely truncated at their c-terminus. The truncations, howvever, do not extend into the protein kinase domain and therefore should not 51 directly affect kinase activity. The ability of the v-erb B protein to transform one cell type and not another suggests that different configurations and/or interactions at the c-terminus may determine functionally significant differences in kinase.activity in specific cell types. Recent identification of other host range mutants further support this notion (see Chapter 5). AEV-R produces all the phenotypic alterations associated with oncogenic transformation of fibroblasts. These traits include focus forming ability of cell monolayers, elevated levels of hexose transport, increased production of plasminogen activator proteases, and the formation of colonies in soft agar. Similar to the search for kinase activity, special culturing conditions appear to be required to detect these properties. This is in contrast to some of the other tyrosine kinase-related oncogenes which readily transform fibroblasts (Ng et a1., 1986; Tracey et a1., 1985; Graf, 1973). Although this point is not dealt with in the literature it should be kept in mind when considering the function of erb B in the cell. Although the difference in fibroblast transforming ability may be a subtle one, it is in agreement with the weak kinase activity associated with it. This kinase activity may be less than normal due to inefficient transport of the protein to the cell surface. In either case, the kinase activity in erythroid cells must be sufficient for transformation. It is interesting that other src-related oncogenes can also transform erythroblasts but not any more efficiently than v-erb B (Kahn et a1., 1984; Palmieri et a1., 1984). Thus the v-erb B molecule may transduce 52 the most efficient proliferation signal in erythroid cells but not in others. This is further supported by the observation that AEV-R can infect and replicate in other hematopoietic cells, but only transforms erythroblasts. The mitogenic signal transduced by erb B, therefore may be specific for certain cell types and is not functional in other cell types. Different cell types may use different signalling pathways since subtle changes in the erb B protein alter its specificity. In view of its homology to a growth factor receptor, namely hEGF-R, there remain only a few possiblilities for what occurs in c-erb B mediated transformation. 1) c-erb B is not the EGF-R, but some other erythroid specific growth factor receptor; perhaps the erythropoietin receptor since AEV-transformed cells are epo-independent; 2) Erythroblastosis results from the inappropriate expression of a truncated EGF-R molecule, such that erb B mimics an erythroid (or cell) specific growth factor receptor; 3) Cell specific factors may regulate the kinase activity of v-erb B and therefore its transforming potential. The oncogenic potential of erb B in this case, would be defined by these factors. The latter two possibilities are not mutually exclusive and appear to be the most likely since hEGF-R is not expressed in murine or human hematopoietic cells. CHAPTER 1: NONACUTE RETROVIRUS INDUCTION OF AVIAN ERYTHROBLASTOSIS IN DIFFERENT STRAINS OF CHICKENS Results: A. Inaiaence af anythrablaagaaia 1n giffierant aniaken lines Susceptibility of erythroblastosis was tested by injecting RAV-l or RAV-2 (Rous associated virus type 1 and 2), prototype ALVs, into different chicken lines. We routinely injected 100 to 1000 infectious units of virus into the peritoneum of one-day old chicks. Our initial studies focused on line 151 since it had been shown to be highly susceptible to RAV-l induced erythroblastosis (Bacon et a1., 1981; Fung, et a1., 1983). Hatchability and egg production of this line, however, is consistently low and has limited the number of eggs available for inoculation at one time. Therefore, several hatchings were required to obtain sufficient numbers for sample collection. Data from similar hatches (hatches occuring within a two to three week time period) are grouped together. In addition to the low number of hatched 151 chicks available, a large number of chicks died of nonspecific lesions within two weeks of hatching. Death due to nonspecific lesions did not appear to be due to virus injection since both uninfected and infected 151 birds died of nonspecific lesions, but not other birds derived from other chicken lines (Table 1) and may be a feature characteristic of this particular inbred chicken line. Eighty to one- 53 .mHnmofiHmmm so: u «\z .wm>uomoo newuom honoumH ammocoH cam umouuonm mum mononucoumm cw muooesz .smwe owuuoEOou mm oommmumxo m“ cowuom usoumH womum>< m .coHumHsoosw umom memos ma um poemsflEuou ouo3 mucoafiuooxm .msoflmoa ofiuwooamuaoc on one sumac mafiuooaows m“ coconwocH .mwmoxsoa pacema>a I an umwmoummHnounu>uo I hum N .ooo uo hmnuoso um mauw> «0 mafia: msofiuooucw moa >Houmswxoumao sufl3 haammsouauoomuucw nonconsw one: mxofino a ¢\Z o O 0 mm «Hma N HmH >mm AoHH-Hev Os 0 mm 0 mm «Hma ~n>cm Assnmmv on o oo o om «Hma x Hms AcousmV so 0 ooa a Hm «Hma x Hma Aooummv ms ma m~ 0 mm mHmH Amm-mev so 0 ooH as mm Hma Aooanmmv me o as as me ems Aoosuemv co 0 km am as Hma H->¢m Assummv mm o so 0 as me x mHmH Aemnmav as o ooa mm s Hma >m¢ ¢\z o o o o «Hma x Hma «\z o 0 am ma ems oaoz mch c« mid NNum owuwooomucoz nonesz cflmuum Hazasvosa muowuom honoumq 0mmu0>¢ va mGOfimwd wouwfifioocH mxowfiu mmdwa coxofiso cmuoca acououufln ow mwmoummanounuaum mo coconwosH .H «Home 55 hundred percent of the 151 birds which survived nonspecific diseases and were injected with RAV-l, developed erythroblastosis. This level of susceptibility was maintained over a three year period of sample collection. The low viability of line 151 chicks prompted us to survey other chicken lines. We chose two lines, line 1515 and 1514, since they were healthier and maintained a genetic background similar to line 151. These lines are sublines of 151 (this line was identical to 151 until 1941) and differ in their subgroup specificity. Line 1514 is resistant to subgroup A virus (ie., RAV-l) infection, while line 1515 chicks are resistant to infection by subgroup B viruses (ie., RAV-2). As expected both lines showed little if any death due to nonspecific lesions, but were only moderately susceptible to erythroblastosis induction (Table 1). Twenty-six percent of 15I4 and twenty-three percent of 15I5 chicks developed erythroblastosis. Line 1515 was the only line tested in which lymphomas developed. In an effort to maintain the healthy characteristics of the 15I sublines but select for increased susceptibility to erythroblastosis, we tested the progeny of 151 X 1514. These chicks are healthy and maintain the high susceptibilty of RAV-l induced erythroblastosis characteristic of the parental 151 line. Ninety to one-hundred percent of RAV-l infected 151 X 15I4 chicks developed erythroblastosis when tested over a two year period (Table 1). 56 The latency of erythroblastosis induction is also shown in Table l. Latency was determined as the number of days post inoculation required for severe leukemia to develop. In many cases this was identical to the death date. The latencies for all the erythroblastosis varied between 34 and 100 days. The latencies in the more susceptible chicken lines, 151, and 151 X 15I4, averaged approximately 65 days or 8 weeks. Lines 1515 and 1514 had slightly longer latencies, 72 and 80 days, respectively. Experiments were terminated at 15 weeks since most of the birds had died of erythroblastosis by this time. The above description focuses on erythroblastosis induced by ALV. In order to compare this leukemia to that induced by AEV-R, we have injected AEV-R into two different chicken lines. Two week old chicks from either line 151 or a resistant line, line 1515 X 71, were inoculated with AEV-R. The erythroblastosis induced by AEV-R was pathologically indistinguishable from that of ALV. The kinetics of erythroblast accumulation and anemia induction were also similar. The latency of erythroblastosis development, however, was shorter than that observed for ALV. On average, erythroblastosis developed 19 or 22 days after AEV-R injection. Both lines were susceptible to erythroblastosis induction by AEV-R since all of the 151 and 67% of the 1515 X 71 birds developed erythroleukemia. We have used the highly susceptible 151 X 1514 chicken line to determine whether another class of non-acute retroviruses is capable of inducing erythroblastosis. We chose chicken synctial virus (CSV), a 57 member of the reticuloendotheliosis virus family, since it induces B- cell lymphomas indistinguishable from those induced by ALV (Witter et a1., 1979). Of the 25 birds inoculated, none developed erythroblastosis or any other neoplasm within the 15 week test period (Table 1). Thus avian erythroblastosis induction by non-acute retroviruses may be limited to the avian leukosis viruses. B. Gnoaa lesions Erythroblastosis is characterized by the uncontrolled proliferation of erythroblasts. The disease originates in the bone marrow, the only site of hematopoiesis in the chicken (Romanoff, 1960), but metastasizes to other tissues at the terminal phase of the disease (see below). As a result, gross morphological changes are observed in the bone marrow, liver, spleen, and occasionally in the kidney. The bone marrow turns red and loses its fat cells. Breakdown of the stromal network is apparent and the consistency of the marrow is increased. The liver becomes enlarged and reddened (Figure 1A Ery, compared with control, N). Enlargement of the spleen (Figure 1B) and kidney are also usually apparent. These organs can be enlarged two to three times that of their normal size. This produces the greatest burden to the animal and is presumably responsible for death. The alterations in gross tissue morphology are characteristic of erythroblastosis and are routinely used as criteria for diagnosis. 58 Figure 1. Gross lesions of ALV induced erythroblastosis, Liver (A) and spleen (B) from a typical erythroleukemic (Ery) and a normal (N) uninfected control were excised and photographed. Both samples were taken from line 151 chickens at 69 days post-hatching. 59 Tc 1'. an ' 60 C. Hiatalogy and Hematology Microscopic examination of leukemic tissues indicates that the changes in gross morphology are due to an infiltration of erythroblasts. Erythroblasts are immature erythroid cells and can be distinguished from other blast cells by their basophilic cytoplasm, large round nucleus, prominent nucleoli, and characteristic perinuclear halo. Typical examples are shown in Figure 2A, Eb. Mature erythrocytes, in contrast, are smaller, ovoid in shape, contain a tightly packed nucleus, and show no cytoplasmic basophilia. Histological examination of the liver and spleen from leukemic chickens indicate that the intravascular spaces of the liver and spleen, namely the hepatic sinusoids and splenic red pulp region, are heavily infiltrated with basophilic erythroblasts (Figure 2C and 2D). Similar infiltrations are observed in the bone marrow and bloodstream (Figure 2A, 2B). Erythroblasts comprise only a minor population of cells in normal bone marrow (<1%) and are rarely observed in the bloodstream of uninfected animals (<0.01%; Nelson et a1., 1980). However, they are the predominant cell type observed in leukemic bone marrow (Figure 2B) and constitute as much as 90% of all bone marrow cells. The number of erythroblasts circulating in the bloodstram can increase as much as 10,000 fold in severely leukemic chickens. D. v men 0 o a tos s Erythroblastosis development can be monitored by analyzing the blood of infected animals. Differential cell counts of blood smears 61 Figure 2. Histological examination of ALV induced erythroblastosis, Peripheral blood (A) and bone marrow (B) smears showing erythroblasts (Eb). Infiltration of liver sinuses (C) and the red pulp region of the spleen (D) with basophilic erythroblasts. Panels A and B, lOOX; panels C and D, 40X. 62 63 were used to follow the development of the leukemia. Blood samples were taken at regular time intervals after virus inoculation. The number of erythroblasts were scored per 100 white blood cells (WBC). Little if any changes in the white blood cell-population could be detected throughout leukemia development. Any gross changes in the percent of packed white blood cells (buffy coat) correlated with an increase in the number of erythroblasts. Examples of typical erythroblastosis development induced by ALV are shown for birds 23 and 63 (circles; Figure 3A, 3B). Erythroblasts are not seen in the circulating bloodstream for the first 67 or 70 days (Figure 3A, 33, respectively). These numbers are similar to erythroblast counts from uninfected animals. Erythroblastosis develops rapidly such that the number of erythroblasts increase dramatically over a two to five day period. During this period, referred to as the leukemic phase, as many as 1000 erythroblasts per 100 WBC can be observed in the bloodstream. Leukemic animals with erythroblast counts this high usually die within 24 hours. In only one case was there evidence of leukemia regression, and this particular animal relapsed into a fatal leukemic phase within three weeks of the initial appearance of erythroblasts in the bloodstream. Anemia development was also followed by measuring hematocrits or the percent of packed red blood cells (RBC) per unit volume of blood. Uninfected chickens have a hematocrit of 32% which decreases with age to approximately 30%. The development of anemia closely parallels erythroblastosis development (boxes, Figure 3A). No change in 64 Figure 3. Development of erythroblastosis, The number of erythrocytes (boxes) and erythroblasts (circles) in the circulating blood were measured at regular time intervals. The erythrocyte count is expressed as the percentage of packed red blood cells per unit volume (or hematocrit). A hematocrit of 32% is taken as a standard value for uninfected chickens. Erythroblast counts were determined from peripheral blood smears and scored as the number of erythroblasts per 100 white blood cells. Erythroblasts appeared in the blood of bird #23 (A) with no change in the number of erythrocytes. Bird #63 (B) also developed erythroblastosis with similar kinetics and was accompanied by severe anemia (decrease in hematocrit). I of Erythrocytes (Ec) (as 96 of Hematocrtts) # of Erythrocytes (Ec) (as 96 of Hematocrlts) 35 1500 30 - '-1000 25 - - 500 d ’ . P 20 " O 15 25 35 45 55 65 75 Days Post lnoculatlon 15 25 35 45 55 65 75 Days Post lnoculetlon # ot Erythroblasts (Eb) # of Erythroblasts (Eb) 4!!- Bird #23 EC -0- Bird #23 Eb @- Bird #63 Ec 0- Bird #63 Eb 66 hematocrit is observed until erythroblasts appear in the bloodstream. At this time, the percent of packed RBCs rapidly decreases and can reach levels as low as 10%. Anemia is routinely observed in erythroblastosis and accompanies approximately 80% of the leukemias induced by ALV. No significant change in the hematocrit level is observed in the remaining 20% of the ALV induced erythroleukemias (for example see boxes, Figure 3B). Discussion: In an effort to characterize the molecular basis of oncogenesis by non-acute retroviruses, we have identified two inbred chicken lines, line 151 and line 151 X 15I4, that are highly susceptible to erythroblastosis induction after injection of RAV-l. The incidences described here are higher than those previously reported and may be due to inoculation at an earlier time. Erythroblastosis samples from line 151 chickens have been extensively characterized (Fung et a1., 1983; Raines et a1., 1985; Beug et a1., 1986; Miles et a1., 1985). Indeed AEV-H, was originally isolated from an erythroblastosis sample induced in line 15I (Hihara et a1., 1983). We report here the identification of another line, line 151 X 1514, which is healthier and maintains the high susceptibility phenotype. We have used this line for all of our later studies and have found no variations at either the biological or molecular level. The high susceptibility to erythroblastosis in line 151 chickens appears to be dominant since only one of the parental strains carries 67 this trait. This observation is supported by backcrosses done in other laboratories (Robinson et al., 1985b). It is somewhat puzzling that two sublines of 151, namely 15I4 and 15I5 display only marginal susceptibility to erythroblastosis. Progeny-of 1515 X 71 crosses are entirely resistant to erythroblastosis induction by ALV and induce predominantly lymphomas (Bacon et a1., 1981). These observations suggest that susceptibility to erythroblastosis is most likely determined by more than one gene. The only genes which have been implicated in erythroblastosis susceptibility are the B5 and the B15 haplotype of the chicken major histocompatability complex (MHC; Bacon et a1., 1981, 1983). B5 and B15 are the two most prevalant haplotypes observed in line 151 and 1514. Again their influence appears to be indirect since erythroblastosis correlates with recessive B5 alleles or heterozygote B5/B15 alleles. They do not appear to be essential for erythroblastosis induction since two instances have been identified where the animals were homozygous for B13 (Robinson, et a1., 1985b). The presence of the B5 or B15 alleles, however, may contribute indirectly to erythroblastosis induction. Other host genes besides the B-haplotype of the MHC must be involved in erythroblastosis susceptibility, although the nature of these genes has yet to be determined. Lymphoid leukosis (LL) is the predominant neoplasm associated with ALV infection, yet LL was detected in only 4 birds. The latency of erythroblastosis (5 to 14 weeks) appears to be much shorter than that of lymphoid leukosis (4 to 10 months). In most cases, the high 68 incidence of erythroblastosis prevented detection of lymphomas. Assessment of the incidence of lymphomas in the surviving birds is also difficult since they were terminated prior to the reported latency of LL. Indeed, the few cases that were detected had latencies near the termination date (13 weeks). Therefore, the susceptibility of these chicken lines to ALV induced lymphomas remains undetermined. We have previously demonstrated a strong correlation between ALV induced erythroblastosis and activation of the c-erb B gene (Fung et a1., 1983, and Raines et a1., 1985). The acute transforming virus AEV- R also induces erythroblastosis and has transduced a portion of the c- erb B gene. The histological and hematological characterization of erythroblastosis induced by ALV and AEV-R suggests that they are similar. That both the cellular and viral erb B genes can induce the same neoplasm suggests they may play similar roles in oncogenesis. The erb B protein has been shown to represent a truncated epidermal growth factor receptor (EGF-R) molecule which retains the intracellular domain (Downward et a1., 1984). All activated c-erb B genes insert near a common c-erb B exon. This is also the first exon transduced by AEV-R. This exon marks the beginning of homology between EGF-R and erb B (Raines et a1., 1985: Nilsen et a1., 1985). Based on these observations we have suggested that interruption of the c-erb B locus at this particular exon is critical to activation of its oncogenic potential. This interruption removes the EGF-binding portion of the molecule and enables the transformed erythroblast to bypass its normal 69 regulatory signals. The result is uncontrolled proliferation of erythroblasts and ultimately the development of erythroblastosis. In addition to v-erb B, AEV-R contains another oncogene, v-erb A. Mutants of AEV-R suggest that functional v-erb B, but not v-erb A, are required for erythroblastosis induction (Frykberg, et a1., 1983 and Sealy et a1., 1983). No evidence for the involvement of c-erb A, the cellular homologue of v-erb A, has been detected in ALV induced erythroblastosis (unpublished result). AEV-H, another retrovirus which induces acute erythroblastosis, contains only a v-erb B gene. V-erb A, therefore, does not appear to be required for erythroblastosis induction. Subtle differences in the phenotypes of erythroblasts transformed in vitro by AEV-R and AEV-H, however, have been identified. Both AEV-H and ALV transformed erythroblasts spontaneously differentiate when cultured in vitro. AEV-R transformed erythroblasts, in contrast, maintain their proliferative ability (Beug et a1., 1985, Beug et a1., 1986). This data suggests that although erb A is not required for erythroblast transformation, it may play a role in arresting erythroid differentiation. These affects are not apparent in the leukemic animal and therefore may not be important in viva. Anemia does not appear to be directly involved in erythroblastosis since it does not always accompany its development. A similar phenomenon has been described for AEV-R (Graf et a1., 1976). Therefore, anemia may represent a non-specific response to either ALV infection or erythroblastosis induction. Indeed some ALVs are known to induce anemia with no other apparent lesions (Smith et a1., 1982). 70 Although there are many similarities between ALV and AEV induced erythroblastosis, two major differences remain - latency and lack of strain specificity. The latency for AEV induced erythroblastosis is short and ranges from 13 to 41 days. Erythroblastosis induced by ALV develops between 34 and 100 days, but on the average requires 8 weeks. In addition to its short latency, AEV-R induces a high frequency of erythroblastosis irrespective of chicken strain. ALV induces high incidence erythroblastosis in only two known strains (151, and 151 X 1514). The difference in strain specificity between AEV-R and ALV is best illustrated by the comparison of erythroblastosis induction in line 1515 X 71. ALV induces predominantly lympoid leukosis and virtually no erythroblastosis in this chicken line (Bacon et a1., 1981, 1983), while AEV-R is 67% susceptible to erythroblastosis induction. This may be a minimal estimate since older, more immunocompetent chicks were injected with AEV-R. The difference in latency and incidence of leukemia induction are what distinguish acute transforming viruses from the non-acute viruses and reflect the different mechanisms by which these two viruses transform cells. AEV-R carries its own oncogene and can readily transform target cells upon infection. Leukemia development results from continuous recruitment of newly transformed cells. Transformation by ALV, on the other hand, is a statistical event, requiring successful infection of the target cell as well as correct proviral insertion within the c-erb B locus. The increased latency and restrictive susceptibility associated with ALV induced erythroblastosis can be explained by the greatly reduced probability of these two events occurring. 71 The inability of REV to induce erythroblastosis in line 151 X 1514 chickens is surprising. Although REV resembles a mammalian retroviruses and is unrelated to ALV, it does induce B-cell lymphomas indistinguishable from those induced by ALV (Witter et a1., 1979). Therefore ALV and REV should infect common target cells. It is not clear whether REV can infect erythroid progenitor cells, the presumed target cell of this leukemia, since no virus of REV origin has been identified which is capable of transforming erythroblasts. B-cell lymphomas induced by REV and ALV contain activated c-myc genes. Both proviruses use similar mechanisms to augment c-myc transcription (Swift et a1., 1985). The structure of the REV genome and the mechanism of c- erb B activation suggest that REV may be unable to direct the synthesis of a functional erb B protein and therefore would not induce erythroblastosis. In erythroblastosis samples, ALV inserts itself into the middle of the c-erb B coding sequence thereby removing the c-erb B initiation codon. A translational start site present in the ALV provirus replaces the normal one. REV does not contain an analogous initiation codon. The activated c-erb B mRNAs resulting from REV insertion would lack a suitable initiation codon and therefore may not be translated. An alternate, more inefficient initiation codon within c-erb B itself may also be used to initiate translation, but in this case, the levels of erb B protein expressed may not be sufficient to transform erythroblasts. The inability of REV to infect the appropriate target cell and correctly activate c-erb B expression are two possible explanations for why injection of REV does not induce erythroblastosis in chickens of known susceptibility. REV based 72 retroviral vectors containing activated c-erb B genes should be useful in testing these two possibilities. ALV and AEV induced erythroblastosis represents the first example where the same identical neoplasm is induced by an acute and non-acute retrovirus through the action of the same oncogene. Although the modes of activation may differ, the end result is the same - elevated expression of an altered oncogenic gene product and development of the same neoplasm. Although retroviruses carrying other oncogenes can induce erythroblastosis, erb B is only associated with leukemias of erythroid origin (Palmieri et a1., 1985 ; Kahn et a1., 1984). The specificity of erb B for this cell type suggests that it may act to disrupt an important component of the erythropoietic differentiation machinery. CHAPTER 2: STRUCTURE AND EXPRESSION OF THE CHICKEN C-ERB B GENE. Results and Discussion: A. ure f e ch ke - b ene The chicken c-erb B gene was initially identified by its homology to v-erb B, the transforming gene of the avian erythroblastosis virus (AEV). Isolation and characterization of genomic clones from chicken DNA located this homology to 12 exons contained within a 24 kb region (Vennstrom et a1., 1982, and Sergeant et a1., 1982). A survey of inbred chicken DNAs, however, suggested heterogeneity in the c-erb B gene (Raines and Lewis, unpublished, and Fung et a1., 1983). The most obvious difference was characterized by the presence of three new Eco RI fragments (2.3, 5.1, and 6.2 kb in size) in some birds and not in others. In order to define this heterogeneity more precisely we constructed genomic libraries and isolated a variety of c-erb B containing clones. A summary of the structure of the c-erb B locus deduced from mapping genomic clones is shown in Figure 4. Analysis of these clones indicated that there were two alleles of c-erb B in the chicken, designated the alpha-and beta-alleles. The alpha-allele is identical to that previously described (Vennstrom et a1., 1982). The beta-allele, however, contains a 2.5 kb deletion in the intron region downstream of VBl, the first exon homologous to v-erb B. Additional Eco RI restriction enzyme site polymorphisms occur near the deletion 73 74 Figure 4. Molecular cloning of the alpha and beta-alleles of c-erb BI Molecular clones of the alpha and beta alleles of c-erb B were isolated from several different lambda phage libraries of line 151 and line 63 DNA. The insert regions of the clones are shown by arrows. The Eco R1 restriction map of the alpha allele is shown (vertical lines above open box). Additional Eco R1 sites present in the beta-allele clones (B clones) are marked by dotted lines (above open box). Other restriction enzyme sites (below box) are similar for both the alpha and beta- alleles and define the region of deleted sequences in the beta-allele (heavily dotted box). Coding sequence of v-erb B and the human EGF-R, and various domains are shown for comparison. Regions related to v- erb B (filled boxes) are based on heteroduplex analysis of the alpha- allele (Vennstrom et a1., 1982, and Sergeant et a1., 1982). Location of VBl, the first exon homologous to v-erb B, was defined by fine restriction mapping of the 4.5 and 2.3 kb Eco R1 fragments of the alpha and beta-alleles, respectively. Regions displaying homology to human EGF-R sequences are indicated: EGF-binding domain, open box; carboxy- terminal region of hEGF-R (C), moderately stippled box; 3' untranslated region of the insertionally activated c-erb B cDNAs, hatched box; erb B exons upstream of VBl as defined by clone 167 (see text), lightly stippled box; approximate location of polyadenylation sites, pAl and pA2. Horizontal bars denote probes used in Figure 6. Restriction enzyme sites shown are Bam H1 (B), Sac I (S), and Eco R1 (vertical lines above open box). <3 A g V l r A ‘ b E 7 A a; L 1 7 n 2: w l ’ a S— A K. v m m.w m mam m m m m m _ I I — _ an. e Pod—QC FE— Ne _ as.” 3 er: _ «q _ 2.: _ 3 no 9o \\ \ a... o 32.... ususniom .23.. 76 and further downstream. The deletion and restriction enzyme site polymorphisms account for the additional Eco RI fragments observed in certain chickens and not in others. The beta-allele appears to be functional since chickens homozygous for this allele have been identified (Raines and Lewis, unpublished, and Miles et a1., 1985). Insertional activation of the beta-allele of cerb B has also been observed in ALV induced erythroblastosis (Raines et a1., 1985). The c-erb B locus is now known to extend beyond the homology defined by v-erb B. The first indication of this was provided by the ‘demonstration of homology between the verb B gene and the human epidermal growth factor-receptor (EGF-R). The region of v-erb B homology spans a small portion of the extracellular domain of the EGF-R and extends through the transmembrane domain into the cytoplasmic domain. V-erb B lacks the last 74 amino acids of the EGF-R (Privalsky et a1., 1984). The striking homology between the protein kinase domain of EGF-R and v-erb B (98%) strongly suggests that c-erb B is identical to the chicken EGF-R. In an attempt to establish this identity we have characterized both the structure and expression of the normal c-erb B locus. Analysis of genomic clones extending beyond the 3' end of c-erb B is consistent with the sequence analysis of the insertional activated c- erb B (IA c-erb B) cDNAs (Nilsen et a1., 1985; and Goodwin et a1., 1986). Clone 63 (Figure 4) contains a large portion of untranslated sequence and two alternate polyadenylation sites, pAl and pA2. The use of both of these polyadenylation signals has been verified using 81 77 analysis of IA c-erb B containing and normal RNAs (data not shown). As predicted by the IA c-erb B sequences, the genomic locus also contains sequences encoding the additional 74 amino acids not present in the c- terminus of v-erb B. These carboxyl terminal sequences share 60% homology to the human EGF-R. Most notable is the the conservation of three tyrosine residues in c-erb B corresponding to the major autophosphorylation sites of the EGF-R (Downward et al., 1984b; Ullrich et a1., 1984, Nilsen et a1., 1985). The conservation of c-terminal regulatory sequences in the c-erb B gene provides further evidence in support of c-erb B being the chicken EGF-R. The primary difference between EGF-R and c-erb B is the presence of a large extracellular EGF-binding domain at the amino-terminus of EGF-R (Downward et al., 1984b; Ullrich et a1., 1984). It has been proposed that the normal c-erb B gene also encodes a similar amino-terminus, but that removal of the ligand binding domain is necessary for oncogenesis. If this hypothesis were true, sequences 5' of c-erb B should be homologous to EGF-R. Since VBl defines the point of divergence between c-erb B and EGF-R, we have focused our studies on sequences 5' of this exon. As an initial attempt to identify homology, we probed genomic clones spanning the region 5' of VBl (clones 10B and 107) with an EGF-R cDNA probe specific for the ligand-binding domain (LBD probe). Only a 4.2 kb Eco RI fragment hybridized at low stringency. Subsequent experiments, however, revealed additional hybridization 5' of this Eco RI fragment but not in the 3' 2.6 and 0.8 kb Eco RI fragments (Raines and Callaghan, unpublished) suggesting 78 a very large intron region between VBl and the next 5' exon (EB-1). The homology in this region was not comparable to that in the v-erb B region suggesting that perhaps this region is more divergent. This is somewhat surprising in view of the fact that chicken cells contain a receptor which binds murine EGF and therefore should contain a similar ligand binding domain (Kris et a1., 1985). Alternatively, this result could indicate that c-erb B is not the EGF receptor but some other related growth factor receptor. The weak and spurious homology between the two probes could be explained by the presence of a common cysteine- rich region in hEGF-R. This clustering of cysteine-residues is a motif common to other receptor molecules (Carpenter, 1987). The Drosophila EGF-R (DER) has recently been cloned and sequenced (Schejter et a1., 1986; Livneh et a1., 1985;). Although the kinase domain displays 55% homology, the extracellular domain is more divergent (33%). The similarity in the extracellular domain was comparable to that observed between DER and the human NEU gene, another human receptor molecule distinct from hEGF-R (Coussens et a1., 1985; Yamamoto et a1., 1986). Interestingly DER does not bind murine EGF and was cloned using v-erb B as a molecular probe. The physiological function of this Drosophila receptor molecule has yet to be identified as well as its ligand. If one assumes that a receptor is named based on the ligand it binds, then DER has been inappropriately assigned a name based on sequence homology. The same remains to be seen for the avian c-erb B gene which appears to be the most homologous to hEGF-R. 79 B. s o the c c e -e b cus In order to define the exons and extracellular coding region of c-erb B more precisely, we have analyzed several different tissue samples for elevated c-erb B expression. Of the limited number of normal tissues screened, low levels of c-erb B expression were consistently observed (data not shown). These results were consistent with levels of c-erb B expression reported by others (Gonda et a1., 1982). Liver RNA from leukemic birds that were prematurely sacrificed exhibited the highest levels of normal c-erb B expression (Figure 5, PL lanes). A ten to twenty-fold increase in expression is observed when compared to normal uninfected liver samples (Figure 5, lane N liv). Transformed erythroblasts at this stage of leukemia development are confined almost exclusively to the bone marrow and have not yet metastasized to the liver (see Chapter 6 for a detailed description of these samples). This is verified by detection of very low levels of the 3.6 and 7.0 kb IA c-erb B RNAs in these samples (Figure 5, lane 205; cf. Nilsen et a1., 1985 and Goodwin et a1., 1986, for a detailed description of these RNAs). The nature of the apparent elevation in c- erb B expression is unclear. Analysis of anemic liver RNA (Figure 5, lane AN) also reveals a moderate elevation in c-erb B expression suggesting that perhaps induction of c-erb B is related to hematopoietic stress induced by either successive bleeding or erythroblast transformation. In this case c-erb B expression may be elevated by production of its ligand and subsequent down regulation of the erb B receptor. An analagous down modulation of EGF-R by EGF 80 Figure 5. Northern blot analysis of c-erb B related RNAs in preleukemic and uninfected liver tiaaue- Poly (A)+ RNA was isolated from liver tissues of either ALV infected chickens prior to the development of erythroleukemia (PL samples, see Chapter 6 for description), normal uninfected chicken (N), or anemic chickens (AN). 5 ug of poly (A)+ RNA was fractionated on 1% formaldehyde agarose gels and electrotransferred to GeneScreen (New England Nuclear Corp., Boston, Mass.). A 1.7 kb Apa I-Sac I fragment from the insertional activated c-erb B cDNA was nick-translated and used as an erb B specific hybridization probe (for description see Figure 11, probe T). Hybridization and washing were as previously described (Raines et a1., 1985). The §§ze of the transcripts were determined relative to the mobility of P-labelled, Hind III digested lambda DNA markers. The location of the 7.0 and 3.6 kb erb B related transcripts typical of insertionally activated c-erb B leukemia samples are indicated by arrows. 81 82 treatment has been shown to increase the level of EGF-R mRNA (Clark et a1., 1985). Three c-erb B related RNAs, 5.8, 8.6, and 12.0 kb in size, were detected in all of the samples analyzed. This is in contrast to the c- erb B RNAs reported by others (Vennstrom et a1., 1982; Gonda et a1., 1984; Nilsen et a1., 1986). In order to identify potential differences in coding sequences we hybridized normal RNA to a probe specific for erb B RNAs terminating at the second polyadenylation site (probe C). This probe hybridized to only the 8.6 and 12.0 kb c-erb B RNAs (Figure 6C) and not to the 5.8 kb RNAs (Figure 6A) suggesting that the 5.8 kb RNA terminates at the first polyadenylation signal and the 8.6 and 12.0 kb RNAs terminate at pA2. The difference in size between the 5.8 and 8.2 kb RNAs is consistent with the use of similar promoters and different polyadenylation signals. We assume the difference between the 8.6 and 12.0 kb is due to a difference in 5' c-erb B sequences since they both contain similar 3' untranslated sequences. In order to document transcription of sequences 5' to VBl, we hybridized Northerns to a 0.6 kb Eco RI-Pst I genomic fragment (RP probe). This was the smallest genomic fragment identified which hybridized to the hEGF-R derived LBD probe, and therefore was expected to produce the best hybridization signals. All three c-erb B related transcripts hybridized to the RP probe suggesting that this fragment did indeed.tontain exon sequences (Figure 6B). Two new size classes of RNAs, 2.3 and 2.5 kb, were detected with the RP probe. These transcripts appear to hybridize exclusively to sequences upstream of 83 Figure 6. Northern blot analysis of normal c-erb B RNAs, Northern analysis of poly (A)+ RNA (5ug) from preleukemic sample 215 was performed using three different hybridization probes: a) a 1.7 kb Apa I-Sac I fragment identical to the erb B specific probe in Figure 5; b) RP probe, a 0.6 kb Eco Rl-Pst I fragment derived from a portion of a 4.2 kb Eco R1 fragment of genomic DNA (see Figure 4, vertical bar, and text); and c) a 1.0 kb Eco R1 fragment of genomic DNA situated 3' of the first polyadenylation signal of c-erb B (Figure 5, vertical bar). 84 PL215 85 V31 since they were not detected with erb B specific probes. The levels of expression of these novel transcripts is similar to the other erb B related RNAs in both preleukemic and normal tissues suggesting that they may be coordinately regulated. The presence of multiple c-erb B related transcripts suggests that the c-erb B transcription unit is a complex one. The differential hybridization described above suggests that more than one promoter is utilized and alternate polyadenylation is involved, as well as possible alternate splicing. This is slightly different from the hEGF-R which produces only two transcripts in normal cells, 5.6 and 10.5 kb (Ullrich, et a1., 1984; Ishii, et a1., 1984; Merlino, et a1., 1985). These RNAs have been shown to differ in their 3' untranslated sequence. No transcripts analagous to the 12.0 kb c-erb B RNA have been detected in human tissues. Most of the hEGF-R studies have been done in A431 cells, an epidermoid carcinoma cell line, which contains an amplified EGF-R gene. An abherrant 2.9 kb EGF-R RNA is observed in A431 cells. This RNA hybridizes exclusively to the ligand-binding domain of EGF-R and encodes a secreted protein (Lin et a1., 1984; Merlino et a1., 1985). The detection of an analagous transcript in normal as well as leukemic chicken tissues suggests that c-erb B may be alternately processed such that the smaller 2.3 and 2.5 kb transcripts encode a secreted ligand-binding domain. Further hybridization experiments, cDNA cloning and sequence analysis should determine whether this gene is related to c-erb B, or encodes another closely related gene. 86 C. n e ext e u ar oma he h ke -erb B W RNA from one of the preleukemic liver samples (sample 215) was used to synthesize a cDNA library in lambda gt-10. We selectively enriched for erb B sequences upstream of VBl by using a synthetic primer complementary to sequences within the transmembrane domain of IA-cerb B (Figure 7A). Approximately 100,000 clones were screened with the RP probe and yielded three positive clones. Restriction mapping and Southern hybridization indicated that one clone (clone 166) contained an 80 bp insert, while the other two clones (clones 165 and 167) contained 1.6 and 2.0 kb Eco RI inserts, respectively. The restriction enzyme maps of these two clones are shown in Figure 7B. Based on the restriction enzyme map and hybridization data, clone 167 was the only clone which contained erb B sequences. The remainder of our studies have focused on the characterization of this clone since it overlapped with erb B sequences and was the largest clone identified. The insert DNA was used to probe southern blots of both genomic DNA as well as DNA from the genomic lambda clones. The pattern of hybridization on Eco RI restricted DNA from clones 10B, 107, and 121 is shown in Figure 8A. This pattern is consistent with the presence of erb B sequences since both the 2.3 and 5.1 kb Eco RI fragments of the beta-allele of c-erb B are detected. In addition all Eco RI fragments situated 5' of VBl also hybridize. These include the 0.8, 2.6, and 4.2 kb fragments as well as other 5' sequences. This is in contrast to our initial analysis using the hEGF-R LBD probe which only detected the 87 Figure 7. e ective cDNA c on n o c-erb se uences located ' of VBL, A). Nucleotide sequence of a 25 residue synthetic oligonucleotide primer used to prepare a lambda gt-lO cDNA library from poly (A)+ RNA (2ug) from PL215 liver tissue. The complementary c-erb B sequence was taken from Nilsen et a1., (1985), and situated 236 nt downstream from the beginning of erb B coding sequences. B). Restriction enzyme map of two c-erb B cDNA clones (167 and 165) obtained from the PL 215 cDNA lambda library. Clones are aligned based on common restriction enzyme sites. The erb B related region is represented by a solid box. The restriction enzymes shown include: A, Apal; B, Bam H1; Sp, Sph I; and St, Stu I. 237 2:61 5 ' CCTGTGCCTGGTTGTGGTTGGTCTA 3 ' cDNA sequence 3 ' GGACACGGACCAACACCAACCAGAT 5 ' synthetic primer B. clone l l r 167 Pv Sp 8 0.1 kb clone . r 165 Sp 8 89 Figure 8. Southern blot analysia using cDNA clone 162, The 2.0 kb insert from clone 167 was gel purified, nick-translated, and used to probe Southern blots containing genomic lambda DNA clones (A), or genomic chicken DNA (B). Lambda phage DNA was prepared from clones 10B, 107, and 121 (see Figure 4). Cloned inserts were liberated by digestion with Eco R1 (10B) or Eco R1 and Sal I. Genomic DNA was extracted from an uninfected chicken tissue and was digested with either Eco R1 (R1), Bam H1 (B), or Sac I (S). The digested DNAs were electrophoresed, transferred to nitrocellulose and probed with the insert from clone 167. Molecular weights were based on relative mobility compared to Hind III/Eco R1 digested lambda DNA markers. The 2.3 kb and 5.1 kb bands hybridizing to clone 10B correspond to the beta-allele of c-erb B (see Figure 4, and text). The genomic chicken DNA is homozygous for the alpha-allele, and therefore recognizes only the 4.5 kb and 12.0 kb bands of c-erb B. 90 H1BS kb 12.6 C I; mow PNF how 52 6 44 2 12 63 54 22 .6. ..t 0.8 0.8 91 4.2 kb Eco RI fragment in these clones. A similar hybridization pattern was obtained when the 167 insert of clone 167 was hybridized to Eco RI digested genomic DNA (Figure 8B). Therefore the sequences present in clone 167 correspond to c-erb B sequences and span the region 5' to VBl. These sequences appear to be derived from a single locus since no additional hybridization was detected in genomic DNA aside from that which can be accounted for by c-erb B (figure 8B). We have recently subcloned the insert from this clone into the Bluescript phagemid vector for subsequent sequence analysis. Preliminary sequencing data indicate that clone 167 begins precisely at the site of the synthetic oligonucleotide primer (and therefore, contains 260 nt of erb B) and extends 5' into c-erb B and the extracellular sequences. Further sequence analysis should indicate whether the c-erb B gene is homologous with the ligand-binding domain of the hEGF-R. Based on the size of the 167 clone, we estimate that approximately 1.0 kb of 5’ c-erb B sequence is missing, a portion of which is probably coding. Recent immunoprecipitation data suggests that the primary translation product of c-erb B is 150 RD, 30 kD larger than the hEGF-R (N. Maihle, unpublished). If this estimate is correct, approximately 800 bp of the c-erb B coding region is missing in clone 167. C-erb B coding sequence does appear to be present since a portion of this clone can be expressed as a fusion protein in bacteria. We are currently making antisera in order to further characterize the nature of the extracellular domain of the c-erb B gene product. 92 The identification of c-erb B sequences 5' to VBl support the idea that amino-terminal truncation is necessary for activation of the oncogenic potential of c-erb B. Activation of c-erb B, then, results in both qualitative and quantitative changes in its expression. The fact that it is a membrane associated tyrosine-specific kinase makes it a likely candidate for signalling proliferative responses in normal and transformed cells. The remaining chapters deal primarily with the mechanism of c-erb B activation and variations on that theme. In most cases the c-erb B gene is assumed to be the chicken analogue of the hEGF-R. Although this assumption may not be correct, the concept remains the same; truncation and ligand-independence of the normal receptor can lead to constitutive enzymatic activity and oncogenesis. CHAPTER 3: TRANSCRIPTION FROM THE 3' LONG TERMINAL REPEAT OF INTEGRATED ALV PROVIRUSES Results: A. Identification of two novel erb B related RNAs in ALV induced erythroblastosis We have previously analyzed the RNA from two ALV induced erythroblastosis samples containing insertionally activated c-erb B genes. These samples contain two size classes of c-erb B mRNA, a 3.6 and 7.0 kb, which differ primarily in their 3' untranslated regions (Nilsen et a1., 1985; Goodwin, et a1., 1986). In an effort to extend these initial findings we have analyzed c-erb B expression in six other ALV induced erythroblastosis samples known to contain insertionally activated c-erb B (IA c-erb B) alleles. Hybridization of Northern blots with an erb B specific probe was consistent with our initial findings. A 3.6 and 7.0 kb erb B mRNA was detected in all of the leukemic RNA samples analyzed (Figure 9A). Upon closer examination, other minor erb B related transcripts could be seen (Figure 9A, arrow). This RNA is a minor erb B species in samples 139 and 019 and comprises approximately 2 to 5% of the total erb B related RNAs. Sample 103 is unique in that two novel erb B species are present which constitute as much as 25% of all of the erb B mRNAs. 93 94 Figure 9. Detect on of nove -erb re ated RNAs n eukemic sam e containing insertionally activated c-erb B genesI Poly (A)+ RNA was isolated from three ALV induced erythroblastosis samples (019,139,103) known to contain insertionally activated c-erb B alleles (see Raines et a1., 1985). Poly (A)+ RNA (Sug) was fractionated on 1% formaldehyde agarose gels and electrophoretically transferred to GeneScreen (New England Nuclear, Inc., Boston, Mass.). The blots were probed with sequences specific for erb B, 5' intron sequences, or 3' untranslated sequences (panel A). The erb B specific probe has been previously described (Figure 5), and contains 1.7 kb of erb B coding sequences. The intron specific probe and 3' untranslated probes (3'UT) are diagrammed in panel B. The intron probe is a 1.4 kb Pst I-Apa I fragment located upstream of the first exon homologous to v-erb B (designated VBl). It spans the region where the ALV provirus is known to insert (dotted boxes). The approximate site of integration for ALV proviruses in leukemic samples 019, 139, and 103 are indicated by arrows, and are taken from Raines et al. (1985). The 3' UT probe is a 1.0 kb Eco R1 fragment containing untranslated sequences (hatched box) downstream of the first polyadenylation site of the c-erb B gene (pAl). pAl and pA2 are identical to the sites defined by Goodwin et a1., (1986). Restriction enzyme sites shown include: A, Apa I; P, Pst I; RI, Eco RI. Solid boxes represent coding exons. 95 oaoa one... .5 .n cob... an 3 \thl silt. 2. <4— ._ r. cap a; nop- .5 .n :93... m aco cob... m :3 a.» . t. . ’ °Oh . 0.5 A V to . .L a: BI-O BSt 6L0 66l- 96 Initially we thought that these minor erb B related RNAs represented expression of the normal uninterrupted cerb B gene. This, however, did not appear to be the case since the sizes of the minor erb B RNAs differed from the Sizes of the normal c-erb B transcripts identified in uninfected tissues (Vennstrom et a1., 1982; Gonda et a1., 1982; Goodwin et a1., 1986). The sizes of the additional erb B transcripts were different in various leukemic samples and ranged from 3.8 kb to 5.0 kb. Our previous DNA analysis revealed that the approximate size of the novel transcripts correlated with its proviral integration site within the c-erb B gene. Both samples 139 and 019 contain ALV proviruses inserted within 400 bp of the first exon of c- erb B (designated VBl since it is the first exon showing homology to the v—erb B oncogene). These samples contain a similar 3.9 kb novel erb B RNA. Sample 103, on the otherhand, contains an ALV provirus inserted approximately 1.3 kb 5' of VBl and synthesizes a 5.0 kb novel RNA species. Thus samples containing proviruses inserted close to VBl consistently contained smaller erb B transcripts than those known to contain proviruses inserted further upstream of VBl. The above observations suggested that the difference in size between these novel erb B transcripts and the 3.6 kb erb B RNA could be due to the presence of intron sequences. To test this possibility, we hybridized parallel northern blots with a probe specific for the intron region 5' to VBl (Figure 9A and 98). Indeed the novel 3.9 kb erb B RNA of samples 139 and 019 comigrate with an RNA containing c-erb B intron sequences. Similarly, the 5.0 and 8.4 kb erb B RNAs unique to sample 97 103 also hybridized with the intron specific probe. The intron specific probe detected an additional 7.2 kb RNA species in samples 139 and 019. Detection of this species with an erb B probe may have been obscured by the predominance of the 7.0 kb erb B related mRNA. The size difference between the two intron containing RNAs of all three samples was similar to that seen between the 7.0 and 3.6 kb RNAs. Since the latter two RNAs were known to differ in their 3' untranslated regions, we hybridized a blot containing sample 103 to a probe which would distinguish between the untranslated region present in the two erb B transcripts (Figure 98). Only the novel 8.4 kb and 7.0 kb erb B transcripts were detected suggesting that the smaller 3.6 kb and 5.0 kb erb transcripts utilize the first polyadenylation signal of c-erb B (pAl) while the other larger transcripts terminate at the second polyadenylation site (pA2; Figure 9A). B. Transcription of intron sequences in insertionally activated c-grb B sample§ Sl analysis was used to determine the precise amount of intron sequence present in the novel erb B transcripts. A 1.9 kb Eco Rl-Stu I fragment spanning the intron region was labelled at the 5' Stu I site and used to probe leukemic RNA samples. The Stu I site is located 55 nt 3' to the splice acceptor site of VBl (Figure 10B), therefore the labelled probe should hybridize to all erb B related mRNAs. A 55 nt 81 resistant fragment is detected in all leukemic samples as well as in uninfected tissues (Figure 10A), and indicates removal of intron 98 Figure 10. S1 nuclease analysis of erb B related RNAs in erythroleukemic and normal tissuesI (A) A 5' end-labelled Eco Rl-Stu I fragment was hybridized to 1 ug or 5 ug poly (A)+ RNA from several erythroleukemic samples (139 to 103), or normal uninfected (N) tissues, respectively, and digested with 81 nuclease. Sl nuclease resistant fragments were separated on a 5% denaturing polyacrylamide gel, and detected by autoradiography. Undigested probe is also shown (probe). (B) Diagram of the 1.8 kb Eco Rl-Stu I fragment used as probe in (A). This fragment contains 55 nt of VBl, including the splice acceptor (SA), and 1.7 kb of upstream intron sequences (dotted box). The star denotes location of radioactive label. The location of inserted ALV proviruses in erythroleukemic samples 103, 019, and 139 are designated designated as arrows and are taken from Raines et a1., (1985). Restriction enzyme sites are RI, Eco RI; St, Stu I. 1800 m prob. 100 sequences due to splicing. Additional nuclease resistant fragments are present in each leukemic sample and are larger than the 55 nt fragment. This observation verified our initial results suggesting that the novel erb B related RNAs contain intron sequences. The size of the nuclease resistant fragment should provide an estimate of the amount of intron sequence transcribed. Sample 139 and 019 protect a 330 and 360 bp fragment while sample 103 protects a 1.3 kb fragment. Interestingly, this amount of intron sequence corresponds to the approximate site of integration relative to VBl and can account for the size differences between the 3.6 and 7.0 kb transcripts. The relative intensities of the intron containing fragment and the 55 nt exon fragment, however, do not correlate with the ratios observed in the Northern analysis. We suspect this difference is due to inefficient formation of the 55 nt hybrid relative to the longer hybrids and is not representative of the true abundance of the corresponding RNAs. Discussion: Insertional activation of c-erb B results in the expression of two size classes of c-erb B RNAs, 3.6 and 7.0 kb. Synthesis of these RNAs is initiated in the 5' LTR, proceeds through the entire viral sequence and into the cerb B gene, and terminates at one of two possible polyadenylation sites. Splicing removes all of the erb B introns and most of the viral sequences. The resulting 3.6 and 7.0 kb erb B mRNAs contain 5' viral sequences linked directly to erb B coding sequences. 101 The transcripts differ only in the amount of 3' untranslated sequence present. We report here the detection of novel erb B related transcripts in several other ALV induced erythroblastosis samples which contain insertionally activated c-erb B alleles. These samples contain the previously characterized 3.6 and 7.0 kb mRNAs as well as other novel transcripts. These transcripts are not the predominant erb B mRNAs and are distinct from the previously described transcripts in that they contain intron sequences. The amount of intron transcribed directly correlates with distance between the inserted ALV provirus and the first exon of cerb B (VBl). Two novel transcripts are consistently observed which, like the other erb B mRNAs, differ in their 3' untranslated regions. Based on these observations we speculate that these novel erb B RNAs are the result of transcription initiated in the 3' LTR of the inserted provirus. This is in contrast to the 3.6 and 7.0 kb erb B transcripts which are transcribed from the 5' LTR. 3' LTR promoted transcripts would readthrough the downstream intron sequence, into the cerb B gene, and terminate at one of the two c-erb B polyadenylation sites (pAl or pA2). Splicing of these transcripts would remove all the internal erb B intron sequences but not those situated between the inserted provirus and the first exon (VBl). As a result the 3' LTR promoted transcripts would contain intron sequences and although two size classes of RNAs would be synthesized in a single leukemic sample, the amount of intron sequence present would determine the relative size variability among different leukemic samples. 102 The level of expression of the novel erb B transcripts in samples 019 and 139 appears to be approximately 2 to 5% of the total erb B mRNAs. The predominant erb B mRNAs present in these samples result from transcriptional readthrough of the 3' LTR. Other studies indicate that these transcripts account for 5 to 20% of all virus related transcription (see chapter 4). Extrapolation from these estimates indicate that the transcriptional activity of the 3' LTR is less than 1% of the total viral transcription. This estimate is in agreement with the level of 3' LTR promoted transcripts from randomly integrated ALV proviruses (Herman et a1., 1986). We cannot, however, rule out the possibility that the 3' LTR promoted erb B RNAs are less stable than those initiated in the 5' LTR. The inclusion of intron sequences or the absence of additional viral sequences could contribute to RNA instability. This does not seem likely since other 3' LTR promoted transcripts, namely those of activated c-myc, lack additional viral sequences and are known to be stable. In addition, c4erb B intron sequences are included in all of the erb B containing retroviruses with - no apparent affect on their levels of expression (Yamamoto et al. 1984b; Henry et a1., 1985; and unpublished). In contrast to the 019 and 139 samples, the novel transcripts of sample 103 were expressed at relatively high levels compared to the 3.6 and 7.0 kb transcripts. This sample is atypical and differs from 019 and 139 in that the ALV provirus associated with the c-erb B locus is deleted (Raines et a1., 1985). Southern analysis indicates that the 103 provirus is missing approximately 1.2 kb of viral sequences, but 103 both LTRs appear to be intact. Northern analysis is consistent with this observation and reveals an aberrant genomic viral RNA of 6.5 kb (unpublished). Although the precise location of this deletion has not been mapped, it does not appear to encompass sequences 5' of the viral splice donor site (up to nucleotide 326) since the 3.6 and 7.0 kb erb B mRNAs indicate that the transcripts are properly spliced. This internal deletion in the provirus may be responsible for the relative increase in the 3' LTR promoter activity. Internal proviral deletions and a subsequent increase in 3' LTR promotion are not unique to this erythroleukemia sample. Both ALV and REV proviruses inserted in the c-myc locus of B-cell lymphomas are deleted. In most cases the deletions do not affect the LTRs and the 3' LTR is used to initiate c-myc transcription. Unlike sample 103, no viral transcripts initiated from the 5' LTR have been detected in B- cell lymphomas (Hayward, et a1., 1981; Robinson, et a1., 1985; Swift et a1., 1985; Ridgeway et a1., 1985). These studies suggested that internal deletions within the provirus could suppress transcription from the 5' LTR. In vitro studies indicate that retroviruses may contain a positive regulatory element which affects the promoter activity of the 5' LTR. Transfection experiments using plasmid which contain variable amounts of 5' viral sequences linked to an indicator gene suggest that the 5' end of the gag gene (up to nucleotide 532) is required for efficient viral transcription (Norton et a1., 1985). The ggg gene has also been shown to contain an enhancer sequence 900 nt downstream from the 104 promoter site (Arrigo et a1., 1987). Interestingly, the deletion of this sequence inhibits transcription of the 5' LTR but does not totally suppress it. Deletion of these sequences in sample 109 could account for the relative levels of expression of the.erb B related transcripts. The apparent enhancement of the novel erb B mRNAs would not be due to increased transcription from the 3' LTR, but rather from the reduction of transcription from the 5' LTR. The exclusion of 5' LTR promoted transcripts in proviruses associated with activated c-myc genes suggest that other factors must also be important in determining which LTR is predominantly active. Other studies also indicate that certain promoters arranged in tandem are preferentially used. They suggest that epigenetic factors actually select for transcription from one promoter and in doing so supress the activity of the other (Emerman et a1., 1984). Changes in chromatin structure may mediate this epigenetic suppression by exposing the preferred promoter and closing the other. In the case of c-myc activation, extremely high levels of c-myc expression may be necessary for oncogenesis. The inefficiency of transcriptional readthrough from viral RNAs initiated in the 5' LTR may not produce sufficient amounts of c-myc RNA to transform B-cells. C-erb B activation, on the otherhand, may not require high levels of expression. Instead the 5' viral sequences may be necessary for expression of a functional protein since these sequences contain the presumed translational start site for erb B (Nilsen et a1., 1985). Therefore the use of one LTR over the 105 other may be dictated by the particular oncogene that is activated and the tumor samples which result select for these epigenetic factors. Insertion of the ALV provirus into the c-erb B locus disrupts synthesis of the normal c-erb B gene product, i.e., the chicken epidermal growth factor receptor (EGF-R) such that its amino terminal sequences are removed. The normal translational start site is presumably replaced by one found in the gag gene. Only truncated c- erb B mRNAs initiating from the 5' LTR would contain the gag start codon. Another potential initiation codon is located 6 amino acids from the beginning of the erb B coding sequences. 13 vitrg transcription-translation analysis of the insertionally activated c- erb B cDNAs indicate that this AUG can be utilized to initiate translation however, it does not appear to be the preferred start site for translation (N. Maihle and H. J.Kung, unpublished results). This observation raises the question of whether the 3' LTR promoted c-erb B RNAs are important in oncogenesis. We believe that they are not important based on the following data. REV, a virus whose splice donor site is situated 5' of the gag translational start site, is unable to induce erythroblastosis in our highly susceptible 151 X 15I4 chicken line (see chapter 1). REV efficiently infects and activates the c-myc locus resulting in B- or T-cell lymphomas (Swift et a1., 1985; Isfort et a1., 1987). By analogy, REV may insert into the c-erb B locus and activate c-erb B transcription. Translation of the activated c-erb B mRNAs, however, may be inefficient due to the absence of an appropriate initiation codon. A sufficient amount of erb B protein is not 106 synthesized and therefore erythroblast transformation does not occur. Although we have not tested this hypothesis directly, in vigrg construction of c-erb B containing REVS may be useful in determining whether infectivity or translational efficiency is responsible for the inability of REV to induce erythroblastosis. ALV proviruses inserted into the c-erb B locus resemble other randomly integrated proviruses. Approximately 15% of all viral transcripts readthrough into the downstream sequences and less than 2% are initiated from the 3' LTR. This study represents the first example where the transcriptional activity of an intact ALV provirus can be be determined for a specific integration site, namely the c-erb B locus. We have obtained a molecular clone of one such provirus and are in the process of determining its transcriptional activity in vitro. Mutational analysis of this and other ALV proviruses such as those in sample 103 should provide further insight into the differential transcriptional activity of the ALV LTRs. CHAPTER 4: ON THE MECHANISM OF C-ERB B TRANSDUCTION: NEWLY RELEASED TRANSDUCING VIRUSES RETAIN POLY A TRACTS OF ERB B TRANSCRIPTS AND ENCODE C-TERMINALLY INTACT ERB B PROTEINS Results: A. Strugture 9f the Transduceg Proviruses We have previously shown that the majority of erythroblastosis samples induced by ALV contain proviral insertions within the c-erb B locus (Raines et a1., 1985). The proviral insertion sites all map at or near the 5' end of the VBl exon, an exon where homology with v-erb B begins. About 25% of the erythroblastosis samples, however, revealed the presence of transduced erb B viruses. The present report is concerned specifically with the structural characterization of these transduced proviruses. Transduced erb B proviruses can be readily identified by their lack of introns and have a restriction map more compatible with c-erb B cDNA or v-erb B than with the c-erb B genomic locus. Here, we present the Bam HI digestion data to illustrate the approaches used to screen tumor samples for the presence of transduced proviruses. Bam HI cleaves c-erb B cDNA three times (Figure 11C); an internal 0.72 kb Bam H1 fragment (probe BB) was used as a probe. As shown in figure 1A, samples carrying the transduced erb B gene (lanes with tumor numbers) reveal an additional 0.72 kbp fragment, whereas in an uninfected (lane 107 108 Figure 11. The presence of transduced erb B proviruses in ALV induced erythroblastosis, A and B). DNA isolated from ALV induced erythroblastosis samples were digested with Bam HI and analyzed by Southern blotting (Raines et a1., 1985) with probe BB (A) and probe BB (B). N: normal uninfected line 151 chicken DNA; IA: representative leukemic samples which carry a c-erb B allele which is insertionally activated by an ALV provirus. Numericals indicate erythroblastosis samples. C). The structure of the 7.0 kb insertional activated c-erb B cDNA (Goodwin et a1., 1986). Probe AB and probe BB correspond tho the Apa I-Bam HI and Bam HI-Bam HI fragments of the cDNA. Probe T is derived from a 1.7 kb Apa I-Sac I fragment and contains the majority of the erb B coding sequence (black box). Regions and sites diagrammed include: A, Apa I; B, Bam HI; 8, Sac I; 3' UT, 3' untranslated region (hatched box); pAl, first polyadenylation signal in c-erb B; pA2, last polyadenylation site in c-erb B; LTR-gag, ALV-derived sequences (open box). VI le6 0998 ZSLG LVOO - vets ' til. probe as pA2 ZZZZfliiiiiigf’)5553555555552 probe AB probe BB probe T 110 N) sample or a sample containing an insertional activated c-erb B gene (lane IA) the exons covered by BB probe remain in an unspliced form and are distributed over a 6.0 kbp Bam HI fragment. Sample 9132 has a Bam HI fragment slightly larger than 0.72 kb- More detailed analysis revealed structural rearrangement within the transduced erb B sequences (data not shown). The 0.72 kb Bam H1 fragment represents a common diagnostic fragment for all the transduced erb B proviruses. To differentiate between individual transduced proviruses, we used probe AB, which was derived from the very 5' end (a 0.35 kb Apa I-Bam H1 fragment) of c- erb B (Figure 11C) and should detect the variable 5' viral-erb B junctions in different transducing viruses. Indeed, junction fragments of different sizes (0.8 to 4.5 kb) are detected in transduced samples and in most of them, multiple bands are seen, indicating the presence of more than one transduced erb B provirus. B. ss of ansduced e b Proviruses We then analyzed the expression of these transduced proviruses. A complex pattern of erb B related transcripts is detected in many of the samples (Figure 12A). This is in stark contrast to the simple pattern observed in leukemic samples resulting from insertional activation of c-erb B locus (cf. lane IA), where, as previously shown (Nilsen et a1., 1985), only two transcripts, 7.0 and 3.6 kb in size, are seen. The complex expression pattern of transduced proviruses, however, is not unexpected, since for a given provirus, at least two transcripts (a 111 Figure 12. Expression of erb B transducing viral RNA in ALV induced epythroblastosis samples andaaecondarv leukemiaaa RNA samples isolated from the original leukemia (panel A and E1 samples in panel B) or secondary leukemias (E2 and E3 samples in panel B), were subjected to Northern analysis (Radinsky et a1., 1987) using probe T (Figure 11C). E2 samples are acute erythroblastosis samples derived from injection of El leukemic extracts. E3 samples are acute erythroblastosis samples derived from the E2 leukemic extracts. AEV, leukemic liver sample from a bird infected with AEV-R. Other notatations are similar to those described in Figure 11. ._. onoa 6998 0998 ZLLB 8998 ZZlB VSLB lit 5...; FL 3.. .L . .m .< 113 genomic and a spliced RNA) can be generated (see below) and since in many samples, multiple transduced proviruses are present. Another striking difference between the transduced and insertional activated samples is the level of expression. In general, the former is about 10-20 fold higher than the latter and reaches a level comparable to AEV infected samples (lane AEV). This is most likely due to the fact that transduced erb B transcripts result from direct transcription of the 5' LTR, whereas additional readthrough of the 3' LTR is required to generate the IA c-erb B transcripts. The latter process is estimated to occur in only 15% of the viral transcripts (Herman et a1., 1986). To verify that the observed novel transcripts did originate from the transduced proviruses, viral extracts from some of these samples were made and inoculated into embryos. Of the four samples tested, all but one (9141) induced erythroblastosis. RNA from the resulting erythroblastosis was compared to that of the original inoculum. As shown in Figure 12B, erb B related transcripts of similar size are observed in the original samples (9134El, 8660E1, 8669E1) as well as the newly generated leukemic samples (E2 and E3). This conclusively documents the viral nature of these transcripts. B. e 5' and 3' unctions of the transduced c-erb B se uences To probe the possible mechanism underlying the frequent transduction of the c-erb B gene, we sought first to identify the 5' and 3' boundaries of the erb B genes in these viruses. 81 protection analysis of the leukemic RNAs was performed using genomic probes; probe 114 PSt for the 5' boundary and probe RR for the 3' boundary. Probe PSt is a 1.1 kb Pst I-Stu I fragment which contains 55 nucleotides of the VBl exon including the splice acceptor (SA) and approximately 1.1 kb of the intron sequences 5' of VBl exon. RNA from all the transduced leukemic samples protected a 55 nt fragment, which represents a spliced erb B RNA (Figure 13A). Importantly, all the transduced samples also contain protected fragments larger than 55 nt. These fragments are likely to be derived from the transduced viral genomic RNA and provide a measurement of the extent of intron sequences incorporated into the individual viral genomes. These data are diagrammatically summarized in Figure 13D. The 9134E1 provirus was subsequently sequenced (see below) and shown to contain 326 nt of intron sequence, in complete agreement with the 81 analysis data presented here. A similar strategy was taken to define the amount of 3' sequences of the c-erb B locus incorporated into the transduced viral genomes. A 2.6 kb Eco Rl fragment (probe RR) which encompasses the last 260 nt of the erb B coding sequences and 2.3 kb of the 3' untranslated region was used to porbe leukemic RNA. A summary of the protected fragments and where they map relative to the erb B coding sequence is shown in Figure 13D. All except 9112 and 9132 protected fragments larger than 260 nt indicating that the carboxy-terminus of the erb B protein expressed by these viruses is intact, a feature different from the existing AEV-H and AEV-R isolates. The 9132 sample did not protect any fragments; further mapping analysis located the breakpoint 5' of the Eco R1 site and the junction point of AEV-R and AEV-H (data not shown). 115 Figure 13. The 5' and 3' junctiops of erb B transducing vipusaa, A and B). 81 nuclease protection analysis of RNA samples isolated from ALV erythroleukemias. Sample notations are the same as those described in Figure 11; probe, is undigested probe. C). Diagram of the 5' and 3' exon-intron map of the chicken c-erb B locus (not drawn to scale) showing probes used to for 81 analysis. PSt probe and RR probe are derived from a 1.1 kb Pst I-Stu I fragment and a 2.6 kb Eco RI-Eco RI fragment, respectively, and are situated in the region indicated. Regions and sites depicted include: exon sequences, solid boxes; VBl, the first exon with homology to v-erb B; 3'UT, 3' untranslated region (hatched box); pAl and pA2, major polyadenylation sites; *, location of radioactive label; R, Eco RI; P, Pst I; St, Stu I. D). A summary of the extent of 5' and 3' erb B sequences incorporated into the transducing viral genomes. This is a composite summary based on 31 protection analysis (A and B), restriction enzyme mapping, cloning, and sequencing. Numericals below the diagram represent data derived solely from $1 analysis. The numericals above the diagram represent data either deduced or confirmed by cloning and sequencing of the proviruses. Only the most intense bands which were reproduced in several independent experiments are depicted in panel D. The 2.6 and 1.1 kb bands present in panel B could result from polyadenylation and therefore may not represent 3' junctign points; only those verified by sequencing are included in D. 9134E1 denotes a breakpoint due to deletion rather than recombination. The dotted line adjacent to 9141 indicates the the 3' boundary of 9141 proviral derived DNA clones. The AEV-R and AEV-H boundaries are based on published sequence (Henry et a1., 1985; Yamamoto et al., 1984b). rQ—Q <— un.a , 33. , . 38 III p.131. 88 .- 83 I d a. were _ . 2.2.. w m M2 H1 RR probe pA1 RR probe 1 e 2600 m 11” I18 e «up. «Fr. .1100 nt 55 m PST probe “4 III - 5‘ Sr :11 PSt probe RIP -/ l—i" I 117 One limitation of the 81 analysis described above is that it measures contiguous sequences present at the 5' and 3' ends of erb B and does not distinguish whether the breakpoint is due to recombination with viral sequences, deletion (see below) or polyadenylation. Thus, they represent the minimal amount of erb B flanking sequences incorporated into the viral genone. Also, due to the presence of multiple proviruses in the same leukemic samples, we cannot draw individual maps of the erb B containing viruses. C. Molecular cloning and sequence analysis of the transduced proviruses The limitation of the analysis described above prompted us to isolate individual proviral clones for detailed characterizations. From genomic libraries of the leukemic samples, we have isolated clones representing three transduced proviruses, 9141, 9134E1, 913483. Detailed restriction mapping and sequencing were performed for these proviruses and in two cases (9134E1 and 913483), all the erb B derived sequences including the sites of recombination have been determined. In this paper, we will focus on the recombination sites of these proviruses, in the hope to better understand the transduction process. In chapter 5 the transforming potentials of 9134El and 913483 will be described. 1. t c ure e 914 rov rus The 9141 provirus possibly represents the largest transducing genome thus far identified (Figure 14). It is at least 12 kb long and contains 6.0 kb of ALV sequence at its 5' end, interrupted by a small 118 Figure 14. Structure of c-grb B transduced proviral clones 9141I 9134El and 913483 Molecular clones carrying transduced c-erb B proviral sequences were isolated from genomic DNA of erythroleukemic samples (9134El and 9141) or transformed fibroblast DNA (913483). The 913483 transformed fibroblast cell line is described in detail in Chapter 5. Viral and erb B regions were lggated by restriction enzyme digestion and Southern blot analysis with P-labelled probes corresponding to either viral sequences (open box), erb B containing sequences (solid box), or 3' untranslated sequences of c-erb B (hatched box). Only the Bam H1 restriction map is shown here. The 9134E1 and 913483 intron sequences (dotted box) were determined by DNA sequence analysis, and the precise junction between viral sequences and erb B sequences are shown (arrow, above provirus). The amount of c-erb B untranslated sequence (hatched box) and its 3' recombination point (arrow, below provirus) was similarly determined. The intron region of 9141 was estimated based on 81 analysis (Figure 12A and 12D). The 9141 provirus clones did not extend beyond the 3' untranslated sequences. The approximate point of divergence was estimated based on Southern analysis of 9141 DNA and Northern and 81 analysis of 9141 RNA. The sizes of erb B related transcripts in Figure 11 are similar to the insertionally activated cDNAs suggesting that the viral transcripts terminate in cellular sequences near the estimated point of divergence. Larger transcripts may result from alternate splicing or polyadenylation at yet another site downstream from the point of divergence. The viral and erb B splice donor (SD), and splice acceptor (SA) are shown as well as the cryptic splice donor (‘SD', see Nilsen et a1., 1985), and the two c-erb B polyadenylation signals (pAl and pA2, Goodwin et a1., 1986) are also shown. — m GGTCATGGGATCCCA GTGOCATTAMTTA 3227 m deletlon 9134E1 3 o—erba , AAAAIAAAGTI’TATACATAAGGMMATA1 almAGGAGG gov—g TGAGTGATCACAG m CACAGATATTAAT ”1er A / 139 ll dOlOlIOfl SD 913483 ILTRI gag pol ‘ ——— - erb 8 ~ WTGTCMGGAAATGCCMAutMAflZACAGGAGGC‘ITATA .nv — 120 deletion of about 1.0 kb in the gay region. The 5' junction point between ALV and erb B lies in the intron region about 150 bp 5' of the VBl exon. The entire erb B coding sequences downstream from VBl are retained in a completely spliced form. Interestingly, virtually the entire 3' untranslated region of the 7.0 kb IA cerb B transcript (Figure 11C) is transduced, suggesting that the transcript co-terminal with the 7.0 kb transcript is the precursor engaged in recombination. Our clone terminates at a site 0.3 kb 5' from the downstream poly A (pA2) signal. The upstream poly A signal, pAl, is retained. Since both LTRs are required for integration, we presumed that there should be a 3' LTR at the 3' end of this provirus. Exhaustive screening as well as Southern analysis of the 9141 leukemic DNA failed to provide such an evidence. It is thus likely, that 9141 provirus represents a vestige of a transduced virus which has lost the 3' LTR following integration. The defective nature of the 9141 provirus together with the unusually large size explain why extracts of 9141 failed to induce acute erythroleukemia. The defective provirus, however, is responsible for the development of the original 9141 leukemia. Southern analysis reveals that the 9141 provirus is the only transduced genome in the 9141 leukemic sample. This provirus contains multiple splice sites; at least eight transcripts can be generated via differential processing. The predicted sizes match closely with those identified in Northern analysis. The two major transcripts, the 7.0 and 3.6 kb message, are virtuallly identical in size and content to the oncogenic transcripts 121 found associated with IA c-erb B. It is, therefore, not surprising that 9141 provirus is leukemogenic, despite, the fact that no infectious virus can be produced. The monoclonal nature of 9141 provirus (unpublished data) further supports this notion. 2. ngpcture of the 9143B; provirus The 9134El provirus (Figure 14) is the predominant component of the original 9134 erythroblastosis and of leukemia samples resulting from injection of 9134 extracts. It has one of the smallest leukemic viral genomes thus far identified with a total size of 4.5 kb. This size is consistent with the RNA analysis (Figure 12A). The 5' ALV sequence constitutes only 541 bp; the breakpoint being situated 161 bp downstream of the gag AUG, a region that has previously implicated in encapsidation of ALV RNA (Pugatesh et a1., 1983). The ALV sequence is juxtaposed with 326 bp of erb B intron with no significant homology at the junction. The erb B intron sequences retained in 9134 (Figure 14 and 15A) is larger than those of AEV-R and AEV-H, and also differs at several nucleotide positons. We have also sequenced the relevant portion of intron from a c-erb B genomic clone derived from line 151 chicken DNA and found that they are identical to those associated with 9134El. Thus, the intron sequence of 9134E1 is a faithful copy of the c-erb B locus from line 151 chickens. The erb B coding portion is identical to that of the IA c-erb B cDNA indicating that no point mutations have occurred. The most intriguing part of this provirus perhaps lies at its 3' end, where 929 bp of the 3' UT (untranslated) region of the c-erb B locus adjoins the last 44 bp of the gag coding 122 Figure 15. Nuclaotide Sequgnce of the 5' and 3' viral-erb B junctippa ip 9134El and 913483, A). Sequences surrounding the 5' recombination point of the 9134E1 provirus including viral sequences and all of the erb B intron sequences. Vertical lines indicate the junction between viral and intron sequences in AEV-R, AEV-H, 913483 and 9134El. Only divergent sequences are indicated; * denotes deletion; SA, splice acceptor site. B). The 3' recombination points of 9134El and 913483 viruses. The boundary of the 3227 nt deletion present in the 9134El provirus and the two erb B polyadenylation signals pAl and pA2 (underlined) are indicated. Arrows illustrate the possible scheme whereby the DNA synthesized by reverse transcriptase of wild type RAV-l RNA switches template to the polyadenylated erb B RNA via oligo(A) homology. — m “m —-’ 913431 WMIIIMWmoma AEV-l 9134!]. WWW 913493 9134!]. W AIV-I pp 913481 WWW RSV-I c e Alli-I — a ' ' to... 9134!}. WWW AIV-l ‘1' IIVdI A 3227 m deietlen I pA2 — or! I — 00000000 ACAUMOO ::: 729 m ::: AGAAMAUAAAOUUUAUACAUMOAMAAU WAAMAMAMAA... 9194!: ---OCUAUCOCOAOOAAUCUMAAAAUUACAgOAOOCUUAUMOCAOCC — AV-t m —. pA1 UUUACAGACUCUAAQQAAAflGUCAAOOAAAUOCCAAAAAAAAAAMAAMMAAAAMMMAAA... 9 I 3 4 8 I 124 region of ALV (Figure 15B). The recombination point is within a string of 23 A's (interrupted by one T). This oligo (A) is apparently derived from the poly A sequence of an erb B transcript utilizing the pA2 signal. We have also sequenced the app gene of the RAV-l provirus that was originally used to generated the erythroblastosis. Most interestingly, the point of recombination lies within a stretch of 6 A's which are found in the RAV-l genome. These 6 A's can thus potentially serve as a homologous recombination site during reverse transcription. The 9134El provirus has suffered a large deletion of 3227 bp including the pAl signal. As a consequence, the virus is more compact and contains one internal polyadenylation signal (pA2). The 9134E1 provirus is fully infectious and leukemogenic (figure 12B and chapter 5), despite the presence of pA2 internal to the virus. 3. Sgpggture of the 913483 provirus 913483 probably represents an independent virus isolate present in the 9134 leukemia extracts. It was undetectable in the original leukemia sample by DNA or RNA analysis and became apparent only upon selection for fibroblast-transformation or sarcomagenesis. The structure and the transforming properties of the 913483 and 9134E1 provirus are distinct. 913483 has much larger 5' ALV-related sequences; it includes intact gag and pal genes, and a segment of gay, which is joined to 98 bp of erb B intron sequence. The 5' junction point contains an overlapping pentanucleotide common to both app sequences of ALV and erb B intron sequences. These pentanucleotides are likely to be involved in homologous recombination. The erb B 125 coding portion contains an in-frame deletion of 417 bp, which renders the virus sarcomagenic, but not leukemogenic (see chapter 5). Most strikingly, the erb B sequence also terminates within a stretch of poly A residues and the 3' recombination point occurs in ALV ah! sequences identical to that shown for 9134El (Figure 14 and 153). One important difference, however, is that the poly A tracts in this case is longer (31 residues) and is derived from an erb B transcript utilizing pAl rather than pA2 signal. This suggests that 9134El and 913483 evolved through independent recombination events and that the oligo A homology represent a site of frequent recombination. Discussion: A. The Proposed Mechanisms for Oncogene Transgucpion Viral oncogene transduction is a well-documented phenomenon but the mechanism is still not fully understood. Several models have been proposed (Goldfarb et a1., 1981; Swannstrom et a1., 1983; Herman and Coffin, 1987; and Roebroek et a1., 1987). A multi-step model that has received general acceptance (Swannstrom et a1., 1983) contains the following features: 1) Proviral integration within or upstream of a protooncogene in the same transcriptional orientation; 2) deletion of the 3' proviral DNA sequences including the 3' LTR thereby fusing the proto-oncogene to the viral transcriptional unit; 3) transcription from the 5' LTR to generate a fusion transcript encompassing both proviral and proto-oncogene sequences; 4) packaging of the chimeric transcripts and wild-type viral genomes into heterodimeric virus 126 particles; and 5) template switching between the heterodimer by reverse transcriptase during viral DNA synthesis, such that the 3' viral sequence is restored to the chimeric molecule. The overall consequence of this process is the capturing of the proto-oncogene into the central portion of the viral genome, and retention of the terminal cis-acting viral elements, essential for replication. This model predicts that the 5' joining of viral and cellular sequences occurs at the DNA level (Step 2) and the 3' joining at the RNA level (Step 5). The majority of transduced viral genomes analyzed contain 5' recombination points which are situated in the intron region of a proto-oncogene (thus retaining a splice acceptor site) and a 3' recombination point lying within an exon region (for review see Besmer, 1983) or, in one case, a poly A tract (Huang et a1., 1986), supporting this model. There are, however, several interesting exceptions. For instance, in some recently identified myc and src variants (Ikawa et a1., 1986; Martin et a1., 1986), the 5' recombinatiOn point coincides with sequences generated by splicing. This raises the possibility that RNA splicing across the viral and cellular sequences may serve as an alternative pathway for gene fusion (i.e., Step 2). Herman and Coffin (1987) recently demonstrated that readthrough transcripts carrying the entire ALV provirus and the adjacent downstream cellular sequences can be synthesized and packaged efficiently. This raises another interesting possibility since the readthrough transcript can serve as a precursor for recombination at both the 5' and 3' end, presumably during reverse transcription. This model is attractive in its simplicity since no proviral deletion step needs to be invoked. It 127 also predicts a higher frequency of oncogene transduction in cases where readthrough transcription into the proto-oncogene is a necessary step, such as in c-erb B activation. In the following paragraphs, we shall discuss the structural data of erb B transducing viruses obtained in this report in the context of these models. B. The Mechanisms of c-erb h Ipahsduction l. e tio a ctiv tio r t te - b T sdu tion All the transduction models described above postulate that proviral insertion near a cellular oncogene is the first step involved in oncogene transduction. Yet for most of the transduced viral oncogenes, it is difficult to experimentally recreate this rare evolutionary step. Thus, ALV induced erythroblastosis, where both insertional activation and transduction are frequently observed, presents a unique opportunity to examine such an issue. Our findings that all the proviruses involved in the insertional activation of erb B are in the same orientation as the erb B gene, and cluster in the intron region which coincides with the 5' junctions of all the transduced erb B viruses analyzed in this report lends strong support to this thesis. 2. o t n o t e 5' June 0 ' letio Prov r e e e a t e NA ve As discussed above, the second step of oncogene transduction involves the fusion of the viral and erb B sequence into a single 128 transcriptional unit. This is where the proposed models differ most significantly. The model of Herman and Coffin (1987) which suggests that the readthrough viral-erb B transcript is a transduction precursor is appealing and may account for the unusually high frequency of erb B transducrion. The large size of the primary viral-erb B readthrough transcript (35 kb), however, makes it unlikely that this hybrid RNA transcript can be efficiently packaged. The second model which suggests 5' fusion via RNA splicing is also attractive for erb B transduction, since c-erb B is activated through splicing and the viral-erb B fusion RNAs are present in all leukemic samplews. We, however, have no evidence that this is the case. Our data, especially based on direct cloning and sequencing analysis, clearly show that most, if not all, transducing erb B viruses retain intron sequences and intact 5' splice acceptor sites. We interpret this to mean that efficient packaging of the ALV-erb B transcript requires a viral "encapsidation signal", which at least in part resides in a region between the splice donor (SD) site and the 5' junction of 9134El. Our results are most consistent with the generation of a fusion transcript by deletion of proviral and erb B intron sequences at the DNA level. Examination of the 5' junction structures, however, does not indicate a unified mechanism for such a process. In the case of 913483, homologous recombination involving a pentanucleotides, has occurred. By contrast, no obvious homology is detected at the junction of 9134El. 129 3. o t o t e ' Ju i ' em late w tch W h A t everse anscri tas Once deletion of the 3' proviral sequence has occurred, the ALV- erb B fusion transcript can be generated by initiation at the 5' viral LTR promoter and termination at one of the erb B polyadenylation sites, either pAl or pA2. By virtue of maintaining the encapsidation sequence, this fusion message should be effectively packaged into virions together with the wild-type ALV RNA, which are amply abundant in the leukemic cells since most of them harbor more than one provirus. Upon subsequent infection, template switching by reverse transcriptase permits the incorporation of 3' viral sequence into the fusion transcript. It is most interesting that in two different isolates, 9134El and 913483, the template switch occurs within the poly A tract of erb B RNA and at a common site within the ALV genome. The six A residues present in the ALV 22! gene apparently facilitate the switching process. While we cannot completely rule out the possibility that 9134El and 913483 arose through a common ancestral virus, the distinct 5' junctions and the different poly A tracts used in 3' recombination strongly suggest that they involved independent transduction processes. The presence of a poly A tract within a retroviral genome as demonstrated here, together with a similar finding in the Fujinami virus (Huang et a1., 1986), provide strong evidence for a 3' recombination that takes place at the RNA level. Both 9134El and 913483 contain internal polyadenylation signals, a feature that in theory should impede the transcription of viral genomic RNA. 130 Literature reports on this point, however, are not converging. Some internal poly A signals appear to be detrimental to virus production (Shimotohno et a1., 1981; Sylla et a1., 1986; Joyner et a1., 1983; Bandyopadhyay et a1., 1984), whereas others have no effect. We found that at least in the 9134E1 case, RNA transcripts terminating prematurely at the internal poly A site could be identified in infected cells yet, the virus still was released at moderately high levels (unpublished data). It is also significant that, unlike AEV-H and AEV-R, all but two of these newly generated transduced erb B proviruses retain the c- terminal coding sequences including the putative major tyrosine autophosphorylation site. This suggests that the loss of the c- terminus of c-erb B is not a necessary step involved in transduction and more likely is a consequence imposed by selection for fibroblast- transformation properties. This subject is addressed more fully in the next chapter. In summary, our analysis of ten newly generated erb B transducing viruses reveal that the 5' junction site occurs in the intron region of erb B, thereby retaining a functional splice acceptor site. This suggests that the insertional activated erb B transcripts, despite their abundance in leukemic cells, are probably not the immediate precursors for transduction. The 3' junction site resides predominantly in the 3' untranslated region of c-erb B and, in two cases, within the poly A tracts of the c-erb B RNA molecules. This 131 reinforces the view that the second recombination occurs at the RNA level and suggests that it may be facilitated by homologous sequences. CHAPTER 5: IDENTIFICATION OF NEW C-ERB TRANSDUCING VIRUSES: DELETIONS IN THE C-TERMINAL DOMAIN ACTIVATE SARCOMAGENIC POTENTIAL Results: A. s a e oten ia o leu e i am es ontaini ew c- rb a V 88 We have identified several new c-erb B transducing viruses from different ALV induced erythroblastosis samples. We have shown that most of these retroviruses carry the entire c-erb B coding region including an intact carboxy-terminal domain similar to the insertional activated c—erb B gene product (IA c-erb B). This is in contrast to the v-erb B containing retroviruses, AEV-R and AEV-H, which lack c- terminal erb B sequences and terminate in the 22! gene of the ALV genome. We have used the leukemia samples carrying the new c-erb B transducing viruses to test the oncogenic potential of c-terminally intact c-erb B. Plasma and liver homogenates from three different leukemia samples were injected into chick embryos. All induced short latency erythroblastosis but at low incidences (Table 2). Because of the apparent loss of virus titer from the original tumor extracts, we used homogenates from the secondary leukemias (E2) for all subsequent experiments. One E2 extract, 9134E2a, contained an unusually high titer of focus forming units, when assayed on chick embryo fibroblasts (CEF). This was surprising since most of the newly released erb B transducing viruses were unable to transform fibroblasts (Tracy et al., 132 Table 2. Induction of Erythroblastosis From Leukemia Samples Containing Transduced c-erb B Viruses Originating Chicken Source 8669 Plasma 8660 Plasma 9134 Liver Number With Erythroblastosis/ Number Inoculated 4/9 1/10 5/8 Latency Period+ 14-19 7—15 0.1 m1 of liver homogenates or plasma was injected intravenously into 16—day embryos. +Latent period is days required for erythroblastosis to develop. 134 1985 and Beug et. a1., 1986). In order to characterize the fibroblast transforming component of 9134E2a more closely, we attempted to amplify and purify this component through further propagation and selection by transformation as illustrated in Figure 16. Transformation was selected for ih vitro by colony formation in soft agar, and in vivo by the ability to induce sarcomagenesis. Northern analysis was used to monitor the c-erb B transducing viruses during purification. As previously described (see chapter 4), the original 9134El sample contains two erb B-related transcripts (4.3 kb and 4.5 kb) which correspond to the genomic and subgenomic viral transcripts (Figure 17). Similarly sized erb B transcripts are only observed in the secondary erythroblastosis (E2a and E2b) and not in transformed fibroblasts or sarcomas (83a, 83b, 84a, 84b, or F3). Instead smaller erb B RNAs are present suggesting that a new smaller erb B virus is present. The erb B transcripts in the fibroblast selected samples (F3, 84a, and 84b) differ from those directly selected in viva (83a and 83b) suggesting that two distinct viruses are responsible for the high focus forming ability of the inoculum. B. Datection of deletions in the erb B sequences of the transduced viphsea The smaller erb B transcripts in the sarcoma and fibroblast samples suggested that they may have undergone structural rearrangements. In order to address this possibility we determined the integrity of the erb B sequences using 81 analysis. We focused 135 Figure 16. Purification scheme for three different c-erb B transducing viruses in erythroleukemic chicken 9134I Erythroblastosis (E) samples, fibrosarcomas (S), and infected chicken embryo fibroblasts (F) were used to purify three different c-erb B transducing viruses. The route of injection or infection is indicated, and the number of tumor bearing animals generated/number injected is noted in brackets. Only samples which were used for passage were characterized further and are as illustrated. _ BE BE «me... 83. «ms. 53.— «32> nmvnpa b 9:620 .30. coo own was; .3303... _ as... 23. 93“. 2;... «<3. 9:... «<3. 21— 05:20 sea. to. 25> vanpa b 3.. 73.. , Sfl TE 3.. 8.. an“. 8..— ..o:o:... 5.5:... — _ 3.32.... 35. i... - q Tam , 8m _ n n. 3:: 8.8.... .3.3— fig. _ sum cum _ a... are... _ 3.81... 5.. 5.33 143:5. 19.4.3.2“. E £223.32? 33 05.3.9 .80- to. 137 Figure 17. Northern analysis of erb B related RNAs in sarcomas induced hy 9134 viral extracts Poly (A)+ RNA was isolated from either the original 9134 erythroleukemic sample (E1), or secondary erythroleukemias (E2a and E2b), sarcomas (83a, 83b, 84a, 84b) or transformed fibroblasts (F3). Poly (A)+ RNA (2 ug) was fractionated on 1% formaldehyde agarose gels and electrotransferred to GeneScreen (New England Nuclear, Boston, Mass.). Hybridization was done using an erb B specific probe (a 1.7 kb Apa I-Sac I fragment derived from c-erb B coding sequences). The position of the 4.3 kb subgenomic 9134E1 virus is indicated. 138 9134 139 primarily on sequences outside of the protein kinase domain since mutations in this region have been shown to abolish fibroblast transformation (Ng et a1., 1986). Probe A, a 1.8 kb Nco I-Bgl II fragment, was used to locate deletions within the 3' end of c-erb B (Figure 18B). Two Nco I sites are located 57 nt apart and are situated 277 and 334 nt upstream of the c-erb B termination codon. No 81 nuclease resistant fragments smaller than 277 nt were observed in El, E2b, 83a, and 83b samples indicating that these viruses had retained their c-terminal coding sequences. The transforming viruses that were selected in vitro (samples F3b, 84a and 84b) did protect smaller fragments of 208 nt and 263 nt. A deletion at this point would remove approximately the last 20 amino acids of erb B. A 720 bp Bam HI fragment (probe B) was used to detect mutations occurring between the protein kinase domain and the c-terminal region (Figure 18B). As expected for viruses containing intact erb B sequences, only the full length probe was protected in most samples. In this analysis we have included AEV-R RNA, a virus known to be deleted in this region, as a control for hybridization due to reannealing. Two novel 81 resistant fragments are observed in the 83a and 83b samples suggesting that two deleted c-erb B transducing viruses are present. Based on their size (91 nt and 172 nt) we would predict that approximately 240 or 268 amino acids would be removed from their c-termini. The results using probe A suggested that one of the erb B viruses present in the 83 samples contained intact c-terminal sequences; 140 Figure 18. 81 nuclease ahalysia of transduced erb B sequencea in RNA from newly isolated sarcomas and transformed fibroblasts, A). RNA was isolated from erythroleukemic samples (E), sarcomas (S), or infected fibroblasts (F). RNA from AEV-R infeCted (leukemic) chicks was used as control. 1 ug poly (A)+ or 30 ug total RNA were hybridized to either probe A, a 3' end-labelled Nco I-Bgl II fragment, probe B, a 3' end- labelled Bam H1 fragment, or probe C, a 5' end-labelled Bam H1 fragment, treated with 81 nuclease, and separated on a 6% denaturing polyacrylamide gel. Sizes ogzprotected fragments were determined relative to the mobility of P-labelled Hinf I and Hae III digested PhiX174 markers. Undigested probe (probe) is also shown. B). Diagram of insertionally activated c-erb B cDNA indicating probes used to detect deletions in erb B sequences. * denotes the location of the radioactive label; dotted box, protein kinase domain; black box, transmembrane domain; hatched box 3' untranslated region; thick hatched box, 5' viral derived sequences; pAl and pA2, first and last polyadenylation sites of c-erb B; P1, P2, P3, tyrosine residues analagous to the autophosphorylation sites present in EGF-R. Restriction enzyme sites include B, Bam HI; N, Nco I; Bg, Bgl II. 172 ° .- .- pmbOB 7 / pAi II S. a i me at 142 therefore it seemed likely that one of the deletions identified with probe B was internal. 81 analysis with the 720 Bam HI fragment labelled at its 5' end (Figure 18, probe C) was used to map the 3' boundary of this internal deletion. A 110 bp fragment was protected indicating that one of the two deleted c-erb B transducing viruses in 83a and 83b contained an internal deletion. C. tablishment of cell ine ex ressi in 1e ansd e ' B VI’US The detection of multiple Sl resistant fragments in the tumor RNAs indicates that they are heterogeneous with respect to the c-erb B transducing viruses they contain. It is interesting that similar viruses of erb B transducing viruses were consistently observed only after their initial selection and that viruses selected in vitrp (F3 and 84) were distinct from those selected in vivo (83). We purified the predominant transforming component from each of these two selection procedures by cloning in soft agar. F3b is one of three independently isolated colonies which were identical. A single virus, containing the 20 amino acid c—terminal truncation in c-erb B, was expressed in this cell line and was the predominant c-erb B transducing virus in both 84a and 84b samples. We have designated the c-erb B transducing virus in this cell line 913484. The F3b cell line which contains the S4 virus has been used for all of our subsequent studies. Two other soft agar clones were also isolated. The virus in these clones was distinct from 913484 but was unstable with passaging and therefore has not been characterized further. 143 The sarcomagenic components present in 83a and 83b were similarly isolated by first infecting fibroblasts with sarcoma homogenates and selecting for colonies in soft agar. Clones containing a single erb B virus were determined using 81 analysis with probe B (Figure 18B). This probe detected both deleted c-erb B transducing viruses in the 83a and 83b inoculum (Figure 18, probe B, lane 83a). Several clones retained both of these viruses even after soft agar selection. A common erb B virus was present in all of the clones suggesting that this virus was responsible for fibroblast transformation. We have designated this virus as 913483 and have used cell line F4A6, a cell line containing only this virus, as a source of the 913483 virus. Further Sl analysis using probe C indicated that this virus was the one which contained an internal deletion within erb B (data not shown). We estimate that this deletion encompasses approximately 438 nt. In order to compare the mutant viruses to one that contained all of the erb B coding sequence, we established a cell line (ElM) expressing the molecularly cloned 9134El virus (the molecular cloning of the 9134E1 virus is described in the chapter 4). We have verified the intactness of the erb B coding region by sequencing and have found no nucleotide differences between it and the insertionally activated c- erb B (IA c-erb B) sequence. A chemically transformed cell line, Qt-6, was cotransfected with a plasmid carrying the entire 9134El provirus and SV2-neo. G418 resistant cells were selected, pooled, and infected with RAY-l. Immunoprecipitations of 35S-labelled ElM cells with an 144 erb B specific antisera indicates that 9134El synthesizes two major erb B related proteins, 82 kb and 84kD (Figure 19, lane ElM), which are indistinguishable from those expressed in cell lines containing IA c- erb B cDNA clones (Maihle, Raines, and Kung, unpublished results). D. o reci itati o e b e ated roteins We have also immunoprecipitated the erb B related proteins from the 913483 and 913484 containing cell lines (Figure 19). All of these experiments used antisera directed against the protein kinase domain of erb B and therefore should not affect the immunoreactivity of the c- terminal mutants. The slightly smaller erb B related proteins detected in the 913484 cell line was in agreement with our 81 analysis; approximately 20 amino acids are missing at the c-terminus of c-erb B. We assume that the truncated protein terminates in the adjacent viral sequences, similar to the erb B products of AEV-R and AEV-H. The 62 kD and 64 kD erb B related proteins in 913483 suggest that the internal deletion within c-erb B is in frame, since a frameshift into the adjacent erb B sequences predict an even smaller protein product. E. Molecular cloning and nucleotide sequence analysis of epb B trahagpging viruseaI In order to verify the presence of an internal in-frame deletion we molecularly cloned the 913483 provirus from infected CEF DNA and sequenced the entire erb B derived region. The structure of the 913483 provirus is shown in Figure 20 and is consistent with that predicted by Northern and 81 analysis. The virus is approximately 10.3 kb long and 145 Figure 19. Immunoprecipitations of erb B related roteins in cel lines infected byhdifferent c-erb B containing viruses, Uninfected fibroblasts (CEF), and fibroblasts in ected with AEV-R, AEV-H, 913483, 913484, or 9134El were labelled with 5S-methionine. Extracts were immunoprecipitated with anti-erb B serum. The positions of erb B related proteins are indicated. 146 147 Figure 20. Structure and aequence of the 913483 provipus, The 913483 provirus was molecularly cloned from infected chicken embryo fibroblast DNA. The structure of the provirus is shown. Viral and c-erb B related regions were determined by restriction enzyme mapping and Southern analysis. The precise erb B content was determined by DNA sequence analysis. The 5' and 3' viral-erb B junction sequences are described in Chapter 6. The sequences defining the 139 amino acid in- frame deletion within erb B are shown below the diagram. Other sequences include: viral sequences (open box), erb B intron sequences (dotted box), erb B coding sequences (solid box), 3' untranslated region of c-erb B (hatched box). The position of potential splice acceptors (SA), splice donors (8D), and polyadenylation signals (pAl) are also shown. B, Bam HI; 8, Sac I. case—on an amp Eu .3 new .2: .3 so. .3 9a <0<<0ho<00h< ._.._.G._.._.o._.<._.ooo .2. = _.m< :.>m< «wen—6 amen—b Pmenwm 156 irrespective of whether it is terminal or internal, is sufficient to alter the oncogenic potential of c-erb B. The R2 region does not appear to be required for erythroblast transformation since AEV-H and AEV-R, two extensively characterized erythroblastosis inducing viruses, lack the last 37 and 74 amino acids of erb B, respectively (Yamamoto et al., 1984b; Choi et a1., 1986). Similarly the c-erb B gene product of 913484 lacks the ultimate 20 c- terminal amino acids. The Rl region, on the otherhand, is required for erythroblast transformation since erythroblastosis defective mutants of AEV-R and AEV-H (td359 and tdl30) contain deletions in this region (Damm, et a1., 1987; Yamamoto et al., 1983b). Truncations do not appear to be necessary for erythroblast transformation since 9134El encodes a c-terminally intact protein and induces erythroblastosis. This observation has been recently verified using a replication competent retrovirus expressing IA c-erb B sequences (Pelley, Moscovici, and Kung, unpublished). An intact R2 region has been implicated in determining host susceptibility to erythroblastosis induction (Gammett, 1986). C- terminally intact transducing viruses similar to 9134E1 were found to only induce erythroblastosis in 151 related chickens and not K28 chickens. C-erb B transducing viruses missing the R2 region, such as AEV-R, do not display this specificity. We have not observed a difference in host susceptibility in SPAFAS versus line 151 X 15I4 chickens. Erythroblastosis has been induced in both lines using 9134 homogenates as well as others which are known to contain c-terminally 157 intact transducing viruses. Therefore host susceptibility to c- terminally intact c-erb B transducing viruses does not appear to be restricted to 151 related chickens. The disease specificity of 9134El supports the idea that intact R2 sequences are inhibitory to fibroblast transformation. Indeed their removal is sufficient to activate sarcomagenesis (illustrated by 913484 or AEV-H). The 913484 virus maps the inhibitory sequence to the last 20 amino acids of erb B. This region contains a tyrosine residue analagous to the major autophophorylation site (P1) of the human epidermal growth factor receptor (hEGF-R). Phosphorylation on this tyrosine appears to be important in regulating EGF-dependent kinase activity of EGF-R (Downward et al., 1984b). By analogy, removal of this tyrosine residue from c-erb B, may regulate fibroblast transformation. The tyrosine autophosphorylation sites for IA c-erb B have not yet been determined. However, they do appear to be important, since replacement of the Pl analog with 3 other amino acid residues render it weakly sarcomagenic (Pelley and Kung, unpublished). The P1 autophosphorylation site may not be the only tyrosine residue responsible for regulating sarcomagenesis since the Pl site is retained in 913483. The 913483 virus contains an internal deletion of 139 amino acids which encompasses an alternate tyrosine residue (P3) which is also autophosphorylated in EGF-R (Downward et a1., 1984b). Therefore, removal of any one of these autophosphorylation sites may be sufficient to alter the transforming ability of c-erb B. Site-directed mutagenesis of this residue should determine its significance in 158 sarcomagenicity. Alternatively, an internal deletion may disrupt the structure of erb B protein in such a way that the Pl site is no longer functional. The above possibilities are not mutually exclusive. The observation of other c-erb B transducing viruses which do not transform fibroblasts, yet contain internal deletions indicate that only select deletions are capable of activating the transforming potential of c- erb B. Analysis of the c-erb B kinase activity and the respective sites of autophosphorylation in the newly isolated c-erb B transducing viruses should provide insight into the potential role of P1 in sarcomagenesis. The internal deletion in 913483 not only activated the sarcomagenic potential of c-erb B but also altered its disease specificity. 913483 infected birds succumbed to hemangiosarcomas shortly after injection. Other c-erb B transducing viruses containing similar internal deletions affecting the R1 domain induce angiosarcomas (Gammett et a1., 1986), a similar disease of endothelial cell origin. Of these viruses only 913483 has been shown to transform fibroblasts and induce sarcomas. This variation may account for the different manifestations of endothelial cell transformation observed in vivo. The erb B protein is a truncated version of the human epidermal growth factor receptor. Although it lacks the amino-terminal EGF- binding region, it retains the transmembrane domain (Downard et a1., 1984; Ullrich et a1., 1984). Oncogenesis is presumably due to the expression of a truncated receptor kinase molecule whose activity has become ligand-independent. Our studies suggest that the erb B molecule 159 is active in at least three cell types - erythroblasts, fibroblasts, and endothelial cells. The ability to transform these cell types is regulated by distinct domains of the c-terminus suggesting that different signal transduction pathways may be involved. The analysis of the erb B proteins displaying altered disease specificity should aid in defining the role of c-erb B in signal transduction. CHAPTER 6: EVIDENCE FOR DIFFERENTIATION OF ALV TRANSFORMED ERYTHROBLASTS IN VIVO Results: A. de tif cation of c e th oc es releuke i ase of V nduced er ob to The primary cell type observed in ALV induced erythroblastosis is the erythroblast (Eb). It is an immature erythroid cell which can easily be identified from other blast cells by its large nucleus, basophilic cytoplasm and perinuclear halo (Figure 22A). In addition to the erythroblast, other erythroid cell types can be observed in the peripheral blood of leukemic chickens. Most notable is the polychromatic erythrocyte (PC) which is characterized by its condensed (polychrome) nucleus, slightly basophilic cytoplaSm, and somewhat ovoid shape (Figure 23B). It represents an intermediate in erythroid differentiation and occurs between the immature erythroblast and the terminally differentiated erythrocyte. The PC observed in erythroblastosis contains a larger cytoplasm than the polychromatic erythrocytes seen in normal uninfected peripheral blood. In monitoring for erythroblastosis development, we observed an unusually large number of these cells in the bloodstream prior to the appearance of erythroblasts. A typical example is shown in Figure 22B. Differential counts of PC and Eb erythroid cells indicate that the PCs appear two to three weeks prior to the development of leukemia 160 161 Figure 22. B ood smears from releukemi and leukemic h ckens Blood smears of the same bird taken at 50 (A) and 68 days (B) post inoculation. Polychromatic erythrocytes (PC) and erythroblasts (Eb) are indicated, 100x. 163 development of leukemia (Figure 23). The number of PCs reaches peak levels one to two weeks before the appearance of the first erythroblasts. As the erythroblasts start to appear in the bloodstream, the number of PCs diminishes becoming a minor component of the total erythroid precursors (less than 10%) at the terminal stage of leukemia. No changes are observed in the erythrocyte population during the accumulation of PCs in the bloodstream since hematocrit values remain the same during this time. This is in contrast to the appearance of erythroblasts which is usually associated with a concomitant decrease in hematocrit levels. The period over which erythroblasts infiltrate the bloodstream and metastasize to other hematopoietic organs, we define as the leukemic phase. The two to three week period where the polychromatic erythrocyte is the predominant erythroid precursor observed in the bloodstream, we refer to as the preleukemic phase. A preleukemic phase was consistently observed in all birds developing ALV induced erythroblastosis. The number of PCs and the time at which they appeared in the bloodstream varied between individual cases. B. o 0 ho and istolo o releu emic chick s In order to characterize this phenomena more closely, we sacrificed ALV infected birds at the preleukemic stage. Gross examination and histology indicated that the liver, spleen, and kidney were normal and showed no evidence of leukemia. This is consistent with the differential counts which detected few if any erythroblasts during this time period. The bone marrow, on the otherhand, was 164 Figure 23. Appearance of circulating polychromatic erythrocytes prior to erythroleukemia development define a preleukemic phase, The number of polychromatic erythrocytes (circles) or erythroblasts (boxes) per 100 white blood cells were determined at regular time intervals after ALV inoculation. The transient increase in polychromatic erythrocytes in the circulation of bird 63 illustrates the two phases of erythroleukemia. Day 60 to 69. the period over which the polychromatic erythrocytes increase defines the preleukemic phase and day 70 to 74, the period in which erythroblasts accumulate, define the leukemic phase. # Erythroblasts 1 500 1250 -' 1000 n 750 " 500 - 250 - 1 0.1 20 Bird #63 30 40 Days Poet Inoculation 250 r- 200 -150 -1OO y. '50 I PO 80 # Polychromatlc Erythrocytes (PC) 4!- Erythroblasts + PC 166 morphologically similar to that of erythroleukemic birds as evidenced by the deep red color, increased consistency, and loss of fat. Examination of bone marrow smears revealed a mixture of erythroid precursors consisting of primarily erythroblasts and polychromatic erythrocytes (Figure 24B). The number of erythroblasts in preleukemic bone marrow is much less than in the leukemic bone marrow (Figure 240). Similar to the blood, the PCs are the predominant erythroid precursor. This is in contrast to normal bone marrow which consists primarily of more mature reticulocytes and erythrocytes (Figure 24A). The gross appearance of the bone marrow is presumably due to the destruction of the stromal network as a result of the abnormal proliferation of PCs. The sudden increase in PCs in the bloodstream may be a consequence of the deterioration of this stromal network. C. Ihaerrional activation of the c-erb B gene ih preleukemic samples Analysis of the preleukemic birds indicated that dramatic changes were occurring in the erythroid population prior to the onset of erythroblastosis. In order to determine whether these changes were related to erythroblastosis we analyzed DNA samples from preleukemic and leukemic tissues. We have previously shown that the erythroblastosis induced by ALV is due to the activation and mutation of the host oncogene c-erb B by proviral DNA insertion into a 4.5 kb Eco RI fragement situated at the 5' end of c-erb B (Fung et a1., 1983; Raines et a1., 1985). As a consequence, the restriction enzyme pattern of the activated c-erb B gene in the transformed erythroblasts is different from that of the normal c-erb B gene. The presence of an 167 Figure 24. Comparison of bone marrow smears from birds in preleukemia and leukemic phases. Bone marrow smears of preleukemic (B), leukemic (C), and uninfected chickens (A) showing the predominance of polychrome erythrocytes, erythroblasts, or erythrocytes. A and B, 60X; C, 100X. 169 altered c-erb B fragment readily distinguishes transformed erythroblast DNA from normal DNA and thus provides a molecular marker for erythroblast transformation. Furthermore, the altered c-erb B fragments vary in size between different leukemia samples, due to the fact that the sites of proviral insertion differ. A clonal population of transformed erythroblasts originating from a single proviral integration event is identified by its characteristic altered c-erb B fragment. Thus identification of this altered c-erb B fragment should allow us to define the relationship between preleukemia and leukemia during erythroblastosis development. DNA from bone marrow, liver, and enriched PCs or Ebs or erythrocytes from the blood were digested with Eco RI and probed with the 4.5 kb Eco RI fragment (R4.5). ALV has Eco RI sites at each of its termini, therefore insertion into the 4.5 kb Eco RI fragment will generate two altered c-erb B fragments rather than one. This type of analysis has been extensively used to map proviral integrations sites and is described in detail elsewhere (Raines et a1., 1985). Leukemic DNA from liver, bone marrow, and enriched erythroblasts contain the same altered c-erb B fragments (Figure 25, L330 samples). This is consistent with the gross morphology and histology of erythroblastosis (see Chapter 1) and indicates that a clonal population of erythroblasts has infiltrated these organs. Also in agreement with the histology is the absence of altered c-erb B fragments in preleukemic liver samples indicating that erythroblasts have not yet metastasized to this organ. Altered c-erb B fragments were observed in the bone marrow and enriched PCs of preleukemic birds. This suggest that the PCs present in the bone 170 Figure 25. Structural alterationa in the c-erb B locus of preiehkemia and leukemic DNA samples. DNA was extracted from the bone marrow (bm), liver (liv), or enriched polychromatic erythrocytes (PC), erythroblasts (Eb) or erythrocytes fractions (ec), of peripheral blood from leukemic (L330) or preleukemic (PL207, PL205) chickens. DNAs were digested with Eco R1 and subjected to Southern blot analysis using a genomic 4.5 kb Eco R1 fragment as probe. The endogenous 4.5 kb fragment is indicated. Two novel erb B related Eco R1 fragments are indicative of an insertion by a single ALV provirus (see Raines et a1., 1985, and text). Two pairs of additional fragments (a and b) are indicated in bm sample PL207. 171 3'.In . ,-8Ia ... .. .2. En no on En oma; mow 4Q do on En >= NON 4Q av. 172 marrow and bloodstream are indeed related to erythroblastosis. The PCs most likely represent a more differentiated form of the transformed erythroblast since it is a more mature erythroid cell than the erythroblast. Therefore, transformed erythroblasts may differentiate into PCs as an initial step toward erythroblastosis development. Little if any terminal differentiation appears to occur since no altered c-erb B fragments were detected in the erythrocyte population (lane ec). This, however, may be misleading since the transformed PCs and Ebs represent only a minor fraction of the total red cell population. Interestingly, sample PL207 contained two clonal populations of transformed erythroblasts as evidenced by four altered c-erb B fragments in the bone marrow (figure 25, PL207). Only one clone, however, appeared to be released into the bloodstream as PCs. This may represent a case where the structure of the bone marrow has been destroyed at one site of erythroblast transformation but not at the other leading to selective release into the bloodstream. This may explain the apparent abundance of the nonreleased clone (a) over the released clone (b) in the bone marrow. To verify that the altered c-erb B fragments associated with the preleukemic samples resulted in c-erb B activation, we compared the erb B related RNAs from leukemic and preleukemic tissues (Figure 26B). As previously described (Nilsen et a1., 1985), both the liver and bone marrow of leukemic birds expressed high levels of the 3.6 and 7.0 kb insertionally activated c-erb B RNAs (IA c-erb B). Only normal c-erb B transcripts (5.8, 9.0, and 12.0 kb) were detected in preleukemic liver 173 Figure 26. Northern blot ana s o ukemic and releukemi ssue samples, Poly (A)+ RNA was extracted from liver (liv) and bone marrow (bm) tissues of preleukemic (PL 207) and leukemic (L019) chickens. 5 ug poly (A)+ RNA was subjected to Northern analysis. Hybridization was done with an erb B specific probe encompassing 1.7 kb of the erb B coding sequence (probe T, defined in Figure 11). The 7.0 and 3.6 kb erb B related RNAs typical of insertionally activated c-erb B erythroleukemia samples are indicated. Note that at this exposure little, if any, of the normal c-erb B transcripts can be detected. kb 7.0 3.6 "liv- 174 L019T bm rs» e ”- ’41.” V PL207 liy bm 175 samples after long exposures (data not shown, see Chapter 2). No IA c- erb B RNAs were observed in preleukemic liver RNA as would be expected from the absence of transformed erythroblasts in this organ. The c- erb B related RNAs in the preleukemic bone marrow were similar to those in the leukemic animal; both contain the 3.6 and 7.0 kb IA c-erb B transcripts and are expressed at comparable levels. Thus the predominant cell type in the bone marrow of preleukemic chickens, the polychromatic erythrocyte, contains an activated c-erb B gene similar to the transformed erythroblast. The timely appearance of this cell type and its obvious relation to transformed erythroblasts suggests that it is an intermediate step in the manifestation of erythroblastosis. Discussion: Erythropoiesis occurs almost exclusively in the bone marrow of hatched chicks (Romanoff, 1960) and it is there where the target cells for erytrhoblast transformation reside. Studies with acute transforming viruses indicate that erythroid progenitor cells at the BFU-E stage are the target cells for erythroblast transformation (Samarut, 1982). We assume the erythroblasts which accumulate in the bone marrow and bloodstream of leukemic birds, however, resemble CFU-E cells indicating that transformation of erythroid cells does not arrest erythroid differentiation. In addition to displaying differentiation markers similar to CFU-E cells (Beug et al., 1985a), ALV tranformed erythroblasts retain their capacity to differentiate ih vitro. They differ from normal CFU-E in that they display an increased 176 proliferative capacity and do not require erythropoietin for differentiation or self-renewal. As a result, only a portion of the cells become committed to differentiate and mature into erythrocytes. In view of these properties, it is not surprising that polychromatic erythrocytes are observed in viva. The presence of the activated c- erb B gene in PC cells strongly suggests that they originated from transformed erythroblasts and are the result of partial differentiation in viva. Analysis of preleukemic blood samples and leukemic blood samples from the same chicken should indicate whether the two cell types are directly related. The large number of PCs and the destruction of the bone marrow in preleukemic chickens suggests that these cells display an increased proliferative capacity in addition to the capacity to differentiate. Like the erythroblasts, PCs from the peripheral blood of preleukemic animals differentiate in culture (Beug and Raines, unpublished). The predominance of the polychromatic erythrocytes and not erythroblasts at this stage suggest that the balance between the ability to self-renew and differentiate typical of leukemic erythroblasts is disrupted. In the preleukemic stage this balance appears to be similar to normal erythroid homeostasis in that proliferation is coupled directly to differentiation. At the leukemic stage the proliferative signal is favored over the differentiation signal, as if the two had perhaps become uncoupled. This uncoupling may be related to the erythropoietin independence characteristic of transformed erythroblasts. 177 The distinction between the cell types present at the leukemic and preleukemic stages suggests that some other event may be necessary for erythroblast transformation. This event may be unrelated to erythroblastosis and may be a nonspecific response due to disruption of normal hemostatic regulation. Perhaps a specific growth factor required for erythroid differentiation is depleted by the leukemic phase. Alternatively, a second event may occur in the transformed erythroblast itself, resulting in the selection of a highly proliferative subclone of erythroblasts. This latter possibility is consistent with the multistep models of tumor progression and metastasis (Buick et a1., 1984). Indeed a similar selection appears to occur in ALV induced lymphoid leukosis where transformed follicles are detected as early as 4 to 8 weeks post inoculation, but only one or two develop into a lymphoma 4 to 6 weeks later (Cooper et a1., 1969; Neiman et a1., 1980). Selection of a subpopulation of transformed erythroblasts may also explain why preleukemia has not been reported for erythroblasosis induced by acute transforming viruses. ALV induced erythroblastosis is due to the clonal expansion of a transformed erythroblast population and requires a long latency period. Erythroblastosis induced by acute transforming viruses is polyclonal and occurs over a short latency period. The continuous recruitment and selection of highly proliferative transformed erythroblasts by acute transforming viruses may therefore, obscure the detection of a well defined preleukemia in birds infected with acute transforming viruses. 178 C-erb B activation appears to be a key step in erythroblast transformation since it has been associated with all ALV induced erythroblastosis samples analyzed to date (Raines et a1., 1985, and Chapter 4). The presence of an insertionally activated c-erb B gene in the preleukemic polychrome erythrocytes suggests that it is one of the first steps in erythroblast transformation. Most significantly, the IA c-erb B protein bears a striking resemblance to a truncated form of the epidermal growth factor-receptor (EGF-R). The erb B protein lacks the extracellular EGF-binding domain of EGF—R, and therefore is unable to bind EGF. It retains, however, the protein kinase domain of EGF-R and a c-terminal regulatory region. As a result of amino-terminal truncation, the erb B protein is constitutively active and can therefore trigger a proliferative response in the transformed erythroid cells. The large number of PCs and erythroblasts present in leukemic and preleukemic birds is indicative of an increase in the proliferative response of erythroid cells. Interestingly, several other oncogenes of the tyrosine kinase family are also capable of inducing erythroblastosis. This suggests that an active protein kinase activity is involved in producing this proliferative signal. The constitutively active protein kinase molecules are thought to mimic the erythropoietin receptor since the transformed erythroblasts are epo-independent and a portion of the leukemic cells terminally differentiate. Further characterization of the polychromatic erythrocytes in preleukemic chickens should aid in identifying these signals and how they are coupled in normal erythropoiesis. SUMMARY Avian leukosis virus (ALV) is a naturally occurring virus which induces a variety of neoplasms in chickens. B-cell lymphoma is the predominant neoplasm induced, although nephroblastomas, hemangiosarcomas, and erythroblastosis have also been observed. Characterization of B-cell lymphomas suggested that proto-oncogenes, upon activation, could be oncogenic. In this case the proto-oncogene c-myc was activated by proviral insertion. In an effort to explain the multipotency of ALV we have identified two chicken strains, line 151 and 151 X 1514, displaying an unusually high susceptibility to ALV induced erythroblastosis. Molecular characterization of these leukemic samples (over 50 total) indicate an absolute correlation between erythroblastosis induction and the activation of the proto-oncogene c- erb B. The c-erb B gene was first identified by homology to v-erb B, the transforming gene of the avian erythroblastosis virus. More recently it has been shown to be related to the gene encoding the human epidermal growth factor receptor (hEGF-R). ALV induced erythroblastosis results from two different types of c-erb B activation - insertional activation and transduction. Insertional activation of c-erb B is the predominant mechanism of c-erb B activation and accounts for 75% of the samples analyzed. These samples contain an ALV provirus inserted within one of two c-erb B alleles, alpha and beta. The proviral integration sites are clustered 179 180 in a region very close to the first exon displaying homology to v-erb B (designated VBl). Unlike the proviruses associated with activated c- myc genes, the c-erb B associated proviruses are intact and always situated in the same transcriptional orientation as c-erb B. Transcription of the insertional activated c-erb B gene initiates in the 5' LTR of the integrated provirus, although a small amount of 3' LTR promoted erb B RNAs can also be detected (less than 1%). Transcription terminates at one of two polyadenylation sites and can utilize alternate splicing. Two different splicing reactions predict the synthesis of different insertional activated erb B proteins. Both are identical in erb B content, but contain either six amino acids of gag or six amino acids of gag plus 53 amino acids of gay at their amino terminus. The erb B sequences of both proteins begin precisely at the 5' boundary of VBl and terminate in sequences displaying homology to the carboxy-terminal portion of hEGF-R. Transduction of c-erb B is the other mechanism of c-erb B activation and accounts for approximately 25% of the erythroblastosis samples analyzed. This is an unusually high frequency for oncogene transduction and has enabled us to study the mechanism of transduction more closely. Our results are in agreement with the model of transduction proposed by Swannstrom et a1. (1983), and suggests that insertional activation of c-erb B is probably a key step in c-erb B transduction as well as deletion of the 3' LTR. The 5' recombination site consistently mapped to the intron region 5' to VBl, while the 3' recombination occurred in the last exon of c-erb B. Disruption of c- 181 terminal sequences were observed in only two cases. Nucleotide sequence analysis of the precise junction site at the 5' and 3' end of two transduced retroviruses indicate that homologous sequences may be involved. Most notable is the frequent recombination between the poly (A) tract of c-erb B mRNA and a AAAAAA sequence in the gay gene of RAV- 1. This provides direct evidence for an RNA intermediate in transduction and strengthens the notion that the second recombination utilizes a "copy-choice" mechanism. The insertional activated and transduced erb B genes encode virtually identical proteins. The c-erb B coding sequences of both begin precisely at VBl and end at a common termination codon. This protein bears a striking homology to the hEGF-R. The hEGF-R contains three domains, an extracellular EGF-binding domain, a short transmemebrane domain, and a cytoplasmic domain. The activated c-erb B sequences are homologous to the carboxy-terminal half of hEGF-R and lack the EGF-binding domain. The cytoplasmic protein kinase domain is retained as well as the ultimate c-terminal regulatory sequences. This homology suggests that c-erb B is the avian counterpart of EGFcR. Our preliminary characterization suggests that the normal c-erb B product does contain a large extracellular domain, although it remains to be seen whether this domain is homologous to the EGF-binding domain. In any case, activation of c-erb B appears to result from a very specific amino-terminal truncation such that this extracellular (ie., ligand- binding domain) is removed. The effect of this truncation on c-erb B function is not known but is presumably due to constitutive protein 182 kinase activity. Further biochemical analysis of the activated and normal c-erb B gene products should address this question. The involvement of c-erb B in ALV induced erythroblastosis was correlative and did not demonstrate that activated c-erb B was responsible for erythroblastosis. We have used the transduced erb B viruses to determine more directly the oncogenic potential of c-erb B. Our results indicate that introduction of an activated c-erb B protein into the appropriate target cell is sufficient for erythroblast induction. In addition we have isolated two mutant c-erb B transducing viruses which display altered oncogenic potentials. Molecular characterization of these viruses indicate that the c-terminal sequences of c-erb B are important. These studies support the notion that c-terminal sequences of EGF-R are important in regulating receptor function. The c-erb B mutants suggest that this regulation may vary in different cell types. The differential regulation of the c-erb B protein has far reaching implications and suggests that a single growth factor receptor may use different signal transduction pathways in different cell types, or different receptors may transduce similar signals. Indeed the biology of erythroblastosis and the phenotype of transformed erythroblasts suggest that the activated c-erb B protein can mimic an erythroid specific growth factor receptor. Further characterization of both the normal and activated c-erb B products should extent our current knowledge of growth factor receptors and their role in leukemogenesis. APPENDIX A: C-ERB B ACTIVATION IN AVIAN LEUKOSIS VIRUS INDUCED ERYTHROBLASTOSIS: CLUSTERED INTEGRATION SITES AND THE ARRANGEMENT OF ALV PROVIRUS IN THE C-ERB B ALLELES M. A. Raines, W. G. Lewis, L. B. Crittenden, and H. J. Kung Proc. Natl. Acad. Sci. USA (1985) 82:2287-2291. Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2287—2291, April 1985 Biochemistry c-erbB activation in avian leukosis virus-induced erythroblastosis: Clustered integration sites and the arrangement of provirus in the c-erbB alleles (mime/cellular oncogene/epidermal growth factor receptor/promoter bet-tin) Manual-rm A. Runes”, WYNNE G. Lewrs“, LYMAN B. Cruncuoersii, AND HerG-JIEN Kuno‘ii ‘Department of Biochemistry. Michigan State University, East Lansim. Mi 48824; and $0.8. Department of Agriculture, Regional Poultry Research laboratory. East Lansing, MI 48823 Communicated by Charles J. Amtzen, December 6, I984 ABSTRACT Thereisconsiderable evidence thatlinksthe activation of cellular genes to oncogenesis. We previously re- ported that structural rearrangements in the cellular oncogene c-erbB correlate with the development of erythroblastosis in- duced by avian leukosis virus (ALV). c-erbB recently has been shown to be related to the gene encoding epidermal growth factor receptor. We now have charuterlzed the detailed mech- anisms of c-erbB activation by ALV proviruses. We report here that the ALV proviral integration sites are cluflered 5' to the region where homology to v-erbB starts, suggesting that interruption in this region of c-erbB is important for its activa- tion. The proviruses are oriented in the same transcriptional direction as c-erbB and usually are full-size. The latter finding - h in contrast to the frequent deletions observed within the c- ave-linked proviruses in B-cell lymphomas. We have also identified a second c-erbB allele, which differs from the previ- ously known allele primarily by a deletion in an intron region. This allele is also oncogenic upon mutation by an ALV provi- rus. Avian leukosis virus (ALV), a naturally occurring cancer vi- rus of chickens, can induce a variety of neOplasms, including B-cell lymphomas, erythroblastosis, nephroblastomas, fr- brosarcomas, etc. (1, 2). In the past few years, the mecha- nisms of ALV oncogenesis have been characterized in some detail (3-8). it was shown that ALV induces B-lymphomas by activation of the host oncogene c-myc (3). This activation of c-myc is accomplished by the insertion of an ALV provi- rus, which carries strong promoter/enhancer sequences, near the c-myc gene. We recently reported evidence suggest- ing that ALV induces erythroblastosis by a similar mecha- nism, with proviruses inserted near another host oncogene, c.erbB (9). c-erbB is the cellular homolog of one of the onco- genes carried by avian erythroblastosis virus (AEV), an acute oncogenic retrovirus known to induce rapid erythro- blastosis in chickens (l, 10). The data indicate that, upon activation, c-erbB can assume an oncogenic role similar to that of its viral counterpart. In our previous communication, we showed a strong correlation between ALV-induced structural alterations of the c-erbB gene and the develop- ment of erythroblastosis (9). No alteration of c-erbA (the cel- lular homolog of the other oncogene of AEV) was found in any of the samples analyzed. Furthermore, in all the leuke- mic samples analyzed to date, transcription of c-erbB but not c-erbA is highly elevated (unpublished data). The data sug- gest that activation of the c-erbB gene alone is sufficient to cause erythroblast transformation. Although these studies provided important insights into the involvement of the c- erbB locus in the development of erythroblastosis, little was 'I'bepublicationcostsofthisarticle weredefrayedinpartbypagecharge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. 51734 solely to indicate this fact. known about its activation mechanism, since the position, orientation, and structure of the adjoining ALV proviruses had not been examined. In addition, the c-erbB gene ap- peared to be more complex than previously reported. Using restriction endonuclease analysis, we had detected polymor- phism in the c-erbB locus, a feature atypical of most cellular oncogenes. At least seven different EcoRl-digestion patterns of c-erbB were identified (ref. 9 and unpublished data). Sev- eral of the erbB-related fragments could not be accounted for by the published map of c-erbB (10). The nature of these polymorphic elements—whether they represented different alleles, members of a gene family, or pseudogenes—had not been explored. We report here our detailed characterization of an addi- tional 37 erythroblastosis samples induced in line 151 chicks by ALV infection. Our data may be summarized as follows: (i) A 100% correlation of c-erbB structural alteration with the development of erythroblastosis was observed. The great majority of the proviral integration sites are clustered in a region at the 5' end of the first exon homologous to v- erbB, suggesting that disruption of the c-erbB locus in this region is important for its activation. (ii) Most of the provi- ruses appear to be full-length and oriented in the same tran- scriptional direction as c-erbB. One such provirus was mo- lecularly cloned and shown to be completely intact. This finding contrasts with the analogous studies with B-lympho- mas, where c-myc-linked proviruses usually carry large dele- tions. (iii) A second c-erbB allele was identified. This allele is also potentially oncogenic and can be mutated by an ALV provirus to cause erythroblastosis. MATERIALS AND METHODS Collection and Analysis of Erythroleukemic Samples. RAV- 1, a prototype ALV, was used to inoculate l-day-old line 151 chicks. The development of erythroblastosis and collection of leukemic samples were similar to those described previ- ously (9). DNA was extracted from quick-frozen bone marrow or liver samples as described by Maniatis et al. (11). DNA sam- ples (25 ug) were digested with restriction enzymes under conditions recommended by the supplier (Bethesda Re- search Laboratories). Digested DNAs were ethanol-precip- itated, dissolved in 10 mM Tris Cl, pH 8.0/1 mM EDTA, and then electrophoresed in 0.7% agarose gel, transferred to ni- trocellulose, and hybridized with the appropriate radioactive probes (9, ll). Abbreviations: ALV. avian leukosis virus: AEV, avian erythroblas- tosis virus;-LTR. long terminal repeat; EGF, epidermal growth fac- tor: kb, kilobase(s). iPresent address: Department of Molecular Biology and Microbiolo- gy. Case Western Reserve University, Cleveland. OH 44106. ’To whom reprint requests should be addressed. 228719? 2288 Biochemistry: Raines et al. Radioactive Probes and Molecular Hybridhation. All re- striction fragments were purified by agarose gel electropho- resis and electroelution prior to radiolabeling (11). Hybrid- ization probes were synthesized from the isolated DNA frag- ments by nick-translation, and hybridizations were carried out under conditions identical to those previously reported (9). Filters were washed in 30 mM NaCl/3 mM sodium cit- rate, pH 7/0.1% NaDodSO, at 65°C, dried, and exposed to x-ray film. Molecular Cloning and Restriction-Enzyme Mapping. Par- tial EcoRl digests of liver DNA were size-selected on su- crose density gradients and ligated to the arms of phage vec- tor EMBL-4 (12). The recombinant phages were packaged and screened by probes specific for v-erbB and the ALV long terminal repeat (LTR) as described (11, 13). Restriction- enzyme mapping of recombinants was performed by single and double digestions, followed by Southern blot (14) analy- sis with ALV- and v-erbB-specific probes. RESULTS a and B Alleles of c-erbB. Vennstrom and Bishop (10) pre- viously have isolated and characterized c-erbB clones, de- rived from a genomic library of an outbred Leghorn chicken. The EcoRi and exon maps at this allele, designated the :1 allele of c-erbB, are shown in Fig. 1.4. Our subsequent stud- ies revealed restriction-fragment polymorphisms of c-erbB in different inbred lines of chickens (9). The most obvious dif- ference is the presence of the 45- and 12.0-kilobase (kb) EcoRl fragments in some birds and the presence of the 2.}, 5.3-, and 6.4-kb EcoRI fragments in others. By cloning and fine-structure mapping, we now have identified a second c- erbB allele, B, that can adequately account for these poly- morphic variations (data to be published elsewhere). The EcoRI map of the B allele is summarized in Fig. 1A. The major difference between a and B lies in the intron region next to VBl, the first exon homologous to v-erbB. The B allele has a deletion of ~25 kb in this region, with the ap- pearance of a new EcoRI site near the boundary of this dele- tion. An additional EcoRI site specific for the B allele is lo- cated further downstream and splits the 12-kb fragment pres- ent in the 0: allele into 5.3- and 6.4-kb fragments. Aside from these two differences, the a and B alleles are very much alike. Neither of the polymorphic variations seems to affect the coding region of c-erbB, although conclusive evidence awaits the direct DNA sequence comparison of the two al- leles. In an extensive survey of the inbred chickens main- tained at the United States Department of Agriculture Re- gional Poultry Research Laboratory, we found that most lines (e.g., His, 153, 7, and 63) carry a alleles. B alleles were identified in line 151 and RLC (and in K28; H. Robinson, personal communication). Among the 15, birds surveyed, 65% are homozygous for a, 10% are homozygous for B, and the remaining 25% are heterozygous for a and B. Activation of the a Allele by ALV Proviral Insertion. We previously have shown that a c-erbB structural alteration correlates with the development of erythroblastosis (9). One fragment from an altered c-erbB locus was molecularly cloned, and it was shown by direct sequencing that the alter? ation is due to the insertion of an ALV LTR about 1.6 kb upstream from the VB] exon. To determine whether LTR insertion near the VB] exon is a general activation mecha- nism, we have analyzed 37 additional ALV-induced erythro— blastosis samples. The VBl exon is located inside the 4.5-kb EcoRI fragment of the an allele (and the 2.3-kb fragment of the B allele). Thus, to determine whether ALV provirus in- sertion occurs in this region, the 4.5-kb EcoRi fragment of the a allele was subcloned and used as a probe (the R45 probe, Fig. 2A). The strategy of this experiment is illustrated in Fig. 2A. Ifthe ALV proviral insertion occurs near VBl as 1 8 4 Proc. Natl. Acad. Sci. USA 82 (I985) A 13 H—H——-I-—-—O-4—I-—-t—+O— V81 08 05 a L44 1251'] as 1 no 1324291115 C Q 5| i may , 5.: L as J 1 1L4 : s. s s as; 9.22 ‘3' “3 -- 8. wail-‘- ., s Basra seam” '1 v vvvvv V'V'é" , 5 ' :88 V81 g-e C LTR M m m LTR Q53 5 an sag? R ‘\ 8.0 fl/ ” R ., ‘/ \‘. './' in ”(VB-1 l I II x ale a" a 0.8 2.3 8.3 FIG. 1. (A) EcoRI restriction map of the a and B alleles of c- erbB. The wallele map is according to that reported by Vennstrom and Bishop (10) and Sargeant et al. (15). The B-allele map was estab- lished by the isolation and restriction enzyme mapping of overlap- ping clones of this allele (data to be published elsewhere). Solid box- es show regions homologous to v-erbB. Size and approximate loca- tion of exons are based on previously reported heteroduplex anal- ysis (10, 15). The location of the first exon homologous to v-erbB, designated VBl, is defined more accurately by fine restriction-en- zyme mapping of the 45- and 2.3-kb EcoRI fragments. The vertical bars denote the EcoRI cleavage sites; ---, deleted sequences: t, EcoRI sites present only in the B allele. (B) Proviral integration sites in the c-erbB gene of erythroblastosis samples. Positioning of the integration sites in different samples (indicated by arrowhead with corresponding sample number) is based on the sizes of EcoRl re- striction fragments as described in the text. The integration sites in a or in B are placed according to their relative distances from the 5' EcoRI site of the 45- or 2.3-kb fragment, respectively. (C) Restric- tion enzyme map of clone x139. Clone 1139 was isolated from a genomic library derived from leukemia sample 139. Restriction frag- ments were ordered based on their single- and double-digestion pat- terns as well as on their hybridization to specific cellular and viral probes. EcoRl, R; BamHl, B: Hindlll, H; Sac l, S; Sal l, Sal. The bottom line represents cellular sequences of the B allele. Solid boxes denote exon sequences. Wavy lines indicate the arms of the A vec- tor. Dotted line indicates the point of insertion of the ALV provirus (top line). LTRs are shown as boxes; gag. group-specific antigens: pol, polymerase: env, envelope glycoproteins. depicted, we should see an interruption of the EcoRI 4.5-kb fragment by the provirus, resulting in two fragments (X and Y) detectable with probe R45. Since there is an EcoRI site present in the ALV LTR, fragment X should contain a por- tion of the LTR, and fragment Y should contain the comple- mentary part of the LTR. As a result, the sum of X and Y should be equal to 4.5 kb plus the size of an ALV LTR, which is 0.34 kb. Thus, one would anticipate seeing two a]. tered fragments, with their sum being ~4.8 kb. (This calcula- tion was based on the a allele, but the same argument holds for the B allele, except that the sum should be 2.6 kb.) The following data (Fig. 28 and Table 1) clearly demonstrate that this is indeed the case. Tire left panel shows EcoRldigested Biochemistry: Raines er al. A lkb v31 R R ‘ ‘ k—Y—a r._r HR4'§__.| 5" V8 31 r——-ALV-—i ——-"——-$==q3--—-1 nausruuuasuuunums-J Ls- ”M‘QNIQ"... '- :8... I ,_:-.‘. r‘... h.- D'- F. ,,_ to. ”0’- . .- '- a. I ’ D . ’ t L R45 C I an a u a as s: n a] 4.5-'1’- 4.5-cl- 45-... —_i i >~—>‘hj , . a ’ r 31 5F VB DNA samples from chicks of the an type. In the normal con- trol (lane N) probe R45 hybridizes to the 4.5-kb EcoRI frag- ment as expected. In other lanes with leukemic samples, two additional bands X (solid arrowheads) and Y (open arrow- heads) can be identified (the larger fragments are arbitrarily designated X). In every case, X and Y total approximately 4.8 lrb (Table 1). Analysis of samples from chicks heterozygous for the a Table 1. Size of viral-cell junction fragments of proviruses inserted in the «2: allele Fragment size, kb Fragment size, kb Sample EcoRl Sac 1 Sample EcoRl Sac l 89 4.3, 0.5 12.0, 4.2 28 3.2, 1.6 10.5, 5.3 103 4.1, 0.7 11.0, 4.1‘ 22 3.2, 1.6 ND 94 4.0, 0.7 12.0, 4.2 53 3.1, 1.6 11.5, 5.3 93 4.0, 0.7 11.0, 4.3’ 96 3.3, 1.6 11.0, 5.3 64 3.7, 1.0 12.0, 4.5 35 3.2, 1.7 11.0, 5.2 88 3.8, 1.0 11.5, 4.7 41 3.2, 1.7 ND 92 3.6, 1.1 11.5, 4.6 40 3.1, 1.7 105, 5.4 49 3.4, 1.4 11.5, 4.8 47 3.1, 1.7 10.5, 5.4 60 3.4, 1.4 9.6, 4.1' 49 3.0, 1.8 10.6, 5.3 41' 3.4, 1.4 11.8, 4.6 42 3.0, 1.8'i 10.5, 5.4 42' 3.3, 1.4 10.5, 5.2 30 3.0, 1.81 10.2, 5.4 39 3.4, 1.4 11.0, 5.0 86 3.0, 1.81 10.5, 5.5 38 3.3, 1.5 11.0. 5.2 82 3.0, 1.9’ ND 48 3.4, 1.5 10.5. 5.2 61 3.0. 1.9* 11.0, 5.5 67 3.3, 1.6 11.0, 5.3 98 2.8, 2.0l 10.8, 5.4 Shown are the 30 typical cases in which direct proviral insertions into the «1 allele were found. Cases involving the processed erbB gene (see Discussion) and the proviral insertions in the B allele are not included. EcoRI and Sac l junction fragments are determined as described for Figs. 2 and 3, respectively. Samples are arranged in order of their integration sites relative to VBl. ND. not determined. ‘Deleted provirus. *Integration site within VBl. 18 5 Proc. Natl. Acad. Sci. USA 82 (1985) 2289 FIG. 2. EcoRI-digestion analysis of the proviral integration sites. (A) Schematic diagram of proviral inser- tion upstream from VBl. Also shown are probes used in this study, and the regions they detect. Probe R45 rep- resents the 4.5-kb EcoRI fragment of the a allele. Probe SP is a 0.8-kb EcoRl—Psr I fragment derived from the 5' end of R45. Probe 31 is a 1.6- kb Pvu II fragment located in the 3' intron region of R45. A 0.7-kb BamHI—Sac I fragment specific for LTR V81 . x 3 I a a “can? & . the 5’ end of v-erbB was used as the #5- - F- VB probe. This probe recognizes the a.— .g‘ 4.5. and 12.0-kb EcoRl fragments of _ __ the a allele (9); for clarity. only hy- 23 .1. - -« bridization to the 4.5-kb fragment is "'5 2< included in C. (B-D) Southern blot L analyses of EcoRI-digested DNA . ». from normal uninfected (N) and R45 erythroblastosis samples (numbers 03 above lanes). Filters were hybridized with the probes indicated at the bot- D I ll es ts ml toms of the autoradiograms. Leuke- ‘ aria-specific bands that show rear- rangements within the 4.5-kb EcoRI fragment (B and C) or the 2.3-kb EcoRI fragment (D) are indicated by solid or open arrowheads (X and Y ’- >- fragments. respectively; see A). Sizes of the rearranged bands in B are sum- r, marized in Table l. B-D are compos- ' . M ites of five gels, among which the mi- ’ ' ' gration properties of the fragments R45 differ slightly. and B alleles are shown at right in Fig. ZB. The 4.5- and 2.3- kb fragments present in the normal control represent the a and B alleles, respectively. The panel shows samples in which alteration of the a allele is observed. Again, the sum of fragments X and Y is 9«4.8 kb. Sample 49 carries four rearranged fragments which pair into two sets of X and Y; presumably, this sample contains DNA from two clonal pop- ulations of leukemic cells, each harboring an ALV provirus near VBl but at a slightly different site. It is noteworthy that the intensity of the 45okb band is reduced relative to the 2.3- kb band in a few samples. Since these samples are from birds heterozygous for a and B, disruption of the a allele should correlate with the loss of the 4.5-kb band, assuming that all cells in the samples are transformed erythroblasts. Analysis of bone marrow samples that contain ~80% erythroblasts (i.e., samples 38 and 49) does show significant reduction in the intensity of the 4.5-kb band relative to the 2.3-kb band. The residual 4.5-kb band is presumably derived from the un- disrupted a allele present in the untransformed leukocytes in the bone marrow. The experiment described above indicates that there is a high frequency of proviral integrations near the VBl exon and in the EcoRI fragment, but it does not reveal whether the proviral integration sites are located upstream or down- stream from the V81 exon. To examine this, we hybridized the same DNA blot as in Fig. 2B to the following region- specific probes (see Fig. 2A): 5F (the 5' flanking sequence), 31 (3' intron), and VB (v-erbB). Examples of such hybridiza- tions are shown in Fig. 2C; probe 31 detects exclusively the longer (X) fragment, whereas probe 5F hybridizes more strongly to the shorter (Y) fragments. This indicates that the interruption due to proviral insertion is in the 5' half of the EcoRI 4.5-kb fragment. Hybridization with probe VB de- tects only fragment X in most cases, as shown for samples 67 and 88. This result suggests that VB] is linked to its down- stream intron sequence, implying that the ALV provirus must integrate on the 5' side of VBl. In sample 82, both frag- 2290 Biochemistry: Raines er al. ments X and Y are detected by probe VB, suggesting that, in this case, the ALV provirus is integrated within VB]. Based on the sizes of fragment X (or Y) and the information regard- ing their relative positions to VB], the individual proviral integration sites can be determined. They are summarized in Fig. 13. It is apparent that the ALV proviral integration sites are clustered in a region immediately upstream from VB]. In those cases (e.g., sample 82) where proviral integration with- in VB] is suspected, the sizes of fragments X and Y match very well with what is predicted if there is a disruption inside VB]. Based on these data, we conclude that in erythroblas- tosis samples, the ALV provirus preferentially integrates just 5' to or within the region where homology to v-erbB starts. Activation of the B Allele by ALV Proviral Insertion. Hav- ing found that the 0: allele is frequently mutated by proviral insertion near VB], we were interested in determining whether the B allele could be interrupted similarly. Using the strategy described above, we were able to show for three aB heterozygous samples that insertion near the B allele occurs. As shown in Fig. 20, the sum of X and Y in these cases equals 2.6 kb (as opposed to 4.8 kb for the a allele). In addi- tion, the intensity of the unaltered B 2.3-kb band is largely reduced compared to that of the unaltered a 4.5-kb band. The locations of the three integration sites relative to VB] map to the same region as those for the 0: allele (Fig. 18). Since the alterations in the B allele are the only ones detect- able in these leukemia samples, the data indicate that inser- tional activation of the B allele can also induce erythroblasto- srs. To conclusively document that ALV proviral insertion in- deed occurs in the B allele, we have isolated a c-erbB clone, M39, from a genomic library of partially EcoRI-digested DNA from leukemia sample 139. This clone carries a 16.4-kb insert. A battery of enzymes was used to construct a restric- tion map, which is summarized in Fig. 1C. The map is in complete agreement with the insertion of an intact ALV pro- virus in the 2.3-kb EcoRI fragment of the B allele, with the provirus oriented in the same transcriptional direction as the c-erbB gene. That the ALV provirus is intact was further substantiated by in vitro transfection of chicken embryo fi- broblasts with the M39 DNA, resulting in the release of in- fectious virus (unpublished data). The integration site of the provirus fits exactly that determined by the Southern analy- sis [Fig. ZD (lane 139) and Fig. 1B]. The Structure and the Orientation of the Provirus. The finding that an intact ALV provirus is present near the c- erbB gene deviates from the previous observations that the ALV proviruses linked to the c-myc gene in B-lymphomas frequently show large deletions, especially near and includ- ing the 5' LTR (4-6). It was postulated that active transcrip- tion of an upstream promoter (in this case, the 5' LTR) may significantly affect the strength of the downstream promoter (3' LTR)—a phenomenon described as promoter occlusion (16, 17). Therefore, removal of the 5' LTR appears to be necessary for efficient utilization of the 3' LTR for down- stream promotion of the oncogene. It was therefore of inter- est to find that A139 carries a full-length ALV provirus. To see whether this is generally true for other leukemic-cell DNA, Sac I digestion was conducted. As shown in Fig. 3, Sac I has a single cleavage site near the 5' terminus of ALV DNA. The Sac I map of c-erbB surrounding the proviral inte- gration sites is also shown. For the undisrupted a allele, probe R45 should detect two fragments, 8.0 and 3.5 kb long. Upon proviral integration, the 8.0-kb fragment is disrupted into two fragments, due to the presence of the additional Sac I site in ALV DNA. If the provirus is full-length (8 kb), the sum of the two new Sac I fragments should approximate 8.0 + 8.0, or 16.0 kb. This appears to be the case for the majority of the leukemic samples (Fig. 3 and Table 1). It is also note- 1 8 6 Proc. Natl. Acad. Sci. USA 82 (I985) 3 LTR 3,0 urn ~~\.. __/" O. s 0.0 T s as s 1 1 4 : fl—l- 34.5 N «9495239640335 8-0 Odo-Ia”; - a. a. Q . -- ‘ 0 FIG. 3. Sac I digestion analysis of proviral DNA structure. (Up- per) Sac I (S) restriction map of an intact ALV provirus integrated 5' to VB] and in the same transcriptional orientation as the a allele. Solid boxes represent exons. Sizes of the full-length provirus and the two Sac 1 fragments of the uninterrupted allele are given (in kb) above the provirus diagram and the restriction map, respectively. The region detectable by R45 is shown. (lower) Southern hybrid- ization of Sac I-digested normal (N) and erythroblastosis DNA with R45 probe. Erythroblastosis sample numbers are above lanes. The sizes of the rearranged bands in leukemia samples are listed in Table ]. worthy that EcoRl digestion analysis presented above indi- cates that both LTRs might be intact, since the EcoRI sites of the LTRs appear to be present in all cases. Although rigor- ous proof that the proviruses are intact has to come from transfection studies such as those described above for M39 clone, the preponderance of full-sized proviruses in erythro- blastosis samples indicates that the presence of an intact pro- virus may not be unique to the DNA of sample 139. This data suggests that promoter occlusion, if it occurs in this case, is not absolute and that its effect is not sufficient to block the activation of c-erbB by a 3' LTR. Alternatively, the provirus may utilize the 5' LTR as the promoter to activate the c-erbB gene. ‘ Sac I analysis also provides important information regard- ing the orientation of the proviruses. For example, sample 88 gives two Sac I, viral-cell junction fragments of about 11.5 and 4.7 kb. The relative intensity of the two bands suggests that the 11.5-kb band is the downstream fragment and the 4.7-kb band, the upstream one. Hybridization to probe 31 (Fig. 2) invariably detects the larger of the two tumor-specif- ic bands, confirming this assignment (data not shown). We know from the data in Table 1 the sizes of the EcoRI junction fragments and, hence, the location of the integrated provirus (Fig. 1B). These data together allow the viral Sac 1 site to be unambiguously placed near the 5 ' end of the inserted provi- rus; the provirus therefore is oriented in the same transcrip- tional direction as c-erbB. The calculated distances from the viral Sac I site to the LTRs agree very well with the intact ALV map, further confirming this alignment. All the provi- ruses surveyed by Sac I analysis in this study are oriented in the same direction as c-erbB. DISCUSSION The studies described here suggest that ALV activates c- erbB in erythroblastosis by a mechanism very similar to its activation of c-myc in B-iymphomas: the proviruses are ori- ented in the same transcriptional direction as the host onco- gene and are clustered either at or immediately 5 ' to a region (~15 kb) corresponding to the start of the viral oncogene. Although exceptions to this general activation scheme exist Biochemistry: Raines et al. (7), this mechanism, referred to as promoter insertion, repre- sents the predominant one used by the ALV provirus. Retic- uloendotheliosis virus, another avian retrovirus, also uses promoter insertion as the major mechanism of c-myc activa- tion in B-lymphomas caused by this virus (ref. 18 and unpub- lished results). In contrast, almost all the proviruses in mouse mammary tumor virus-induced mammary carcinomas are arranged either in the opposite orientation or down- stream from the putative oncogene. Furthermore, the inte- gration sites are spread over a large (20-kb) region (19). In this case, presumably, the LTR enhancer is involved in acti- vation. Clearly, in different systems, different activation mechanisms are favored. What dictates the mode of viral in- tegration as well as oncogene activation is unclear, but it may be related to the intrinsic pr0perties of the virus (e.g., the strengths of the enhancer and promoter), the oncogene in question (the local conformation and the structural require- ments for activation), or a combination of both. In the case of c-myc activation, most of the ALV and reticuloendothe- liosis virus DNA integrations result in truncation of the c— myc transcript and removal of the first noncoding exon (ref. 20 and unpublished results). It was postulated that the first noncoding exon may contain a negative-controlling element that inhibits either the transcription or translation of the gene (20-22). The situation with c-erbB is less clear, since the coding capacity of the gene has not been defined fully. How- ever, recent evidence strongiy suggests that the c-erbB prod- uct is closely related or identical to the epidermal growth factor (EGF) receptor (23—25). Since the region of homology involves the carboxyl-tenninai portion of the EGF receptor and v-erbB, one would expect the c-erbB coding sequences to extend significantly further upstream from VB], where the proviral integration sites are concentrated. All the acti- vated c-erbB products would, therefore, represent truncated versions of the normal protein. It is, then, interesting that the starting points of all the activated c-erbB genes studied here map very close to the point of insertion of v-erbB sequences in AEVR and AEV". This suggests that inten'uption in this region of c-erbB probably is important for activation. The requirement to interrupt the c-erbB locus and generate a truncated product perhaps also imposes a need for the pro- moter-insertion (as opposed to enhancer-insertion) type of proviral activation, since, in this region, no cellular promoter is present to_ be activated by the LTR enhancer. Whether c-erbB is identical to EGF receptor or not, the chicken c-erbB locus is a complex one; the region homolo- gous to v-erbB spans more than 20 kb and contains at least 12 exons. Added to this complexity is the presence of stnrctural polymorphisms in different lines of chickens. Using cloning and hybridization studies, we have identified a second allele, B, which differs from the previously known allele, a, primar- ily by a deletion in the intron region. The a and B alleles can account for the majority but not all of the polymorphic erbB elements observed in chickens and must constitute the major c-erbB locus, since we have not found any chickens that lack both alleles. Among the few aB heterozygotes studied, the B allele appears to be as susceptible to proviral insertion as the 0: allele (3/7 vs. 4/7; Fig. 3 A and B). The present communication is primarily concerned with the typical promoter-insertion mechanism of c-erbB activa- tion, which accounts for 90% (34/38) of the cases examined. We previously reported an atypical case where a single al- tered c-erbB fragment contains an LTR and v-erbB related exons but no intron sequences, as if the activated c-erbB message had been reverse-transcribed and reinserted into the host genome (9). We have again observed this phenome- non in the present study; the altered c-erbB fragment of the four remaining cases possesses these features (unpublished data). One such fragment was cloned, and structural analysis confirms the processed nature of the erbB gene. The linkage 187 Proc. Natl. Acad. Sci. USA 82 (1985) 2291 point between the provirus and the processed erbB gene again maps near VB]. The generation of such a processed gene can occur either intracellularly, as has been suggested for the formation of other pseudogenes (26), or via virus in- termediate (ref. 27 and H. Robinson, personal communica- tions). In either case, promoter-insertion probably was in- volved in the initial activation of the oncogene (4, 5). When we include these cases in the category of promoter-insertion activation, we find a striking statistic for c-erbB activation: In the 37 chicks with erythroblastosis (38 proviruses) ana- lyzed, there is a 100% correlation between c-erbB alteration and erythroblastosis development. Furthermore, virtually all proviruses found linked to c-erbB are clustered in a small chromosomal region and are uniformly aligned in a config- uration compatible with the promoter-insertion type of acti- vation. We thank J. Vitkuske and R. Wagner for excellent technical as- sistance; J. Dodgson, S. Conrad, and M. Fluck for critical reviews of the manuscript; and T. Vollmer for assistance in manuscript prep- aration. This work was supported in part by a grant from the Leuke- rnia Research Foundation and by Grant CA33158 from the National Cancer Institute. H.-J.K. gratefully acknowledges support from the Faculty Research Award of the American Cancer Society. 1. Weiss, R. A., Teich. N. M.. Varmus, H. E. a Coffin, J. M. (1982) Mo- lecular Biology of Tumor Virus (Cold Spring Harbor Laboratory. Cold Spring Harbor, NY). 2. Crittenden. L. B. a Kung. H. J. (1983) in Mechanism of Viral Leuke- mogenesis, eds. Goldman, J. M. & Jarret, O. (Churchill-Livingstone, Edinburgh. Scotland). Pp. 64-88. 3. Hayward, W. S., Neel. B. G. & Astrin, S. M. (1981) Nature (London) 290, 475-480. 4. Payne. G. S., Courtneidge. S. A.. Crittenden, L. B.. Fadly. A. M.. Bishop. J. M. a Varmus, H. E. (1981) Cell 23, 311-322. 5. Neel, B. G.. Hayward. W. 8.. Robinson. H. L., Fang. J. & Astrin, S. M. (1981) Cell 23, 323-334. 6. Fung. Y.-K.. Fadiy. A. M.. Crittenden, L. B. a Km. H. J. (1981) Proc. Natl. Acad. Sci. USA 78, 3418-3422. 7. Payne. G. 8., Bishop, J. M. & Varmus, H. E. (1982) Nature (London) 295, 209-213. 8. Fung. Y.-K.. Crittenden. L. B. a Kong. H. J. (1982).]. Virol. 44, 742— 746. 9. Fung, Y.-K.. Lewis. W. G.. Crittenden. L. B. & Kung. H. J. (1983) Cell 33. 357-368. 10. Vennstrom, B. a Bishop. J. M. (1982) Cell 28, 135-143. 11. Maniatis. T.. Fritsch. E. & Sambrooit. J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory. Cold Spring Har- bor. NY). 12. Murray. N. E. (1983) in Lambda ll, eds. Hendrix. R. W.. Roberts. J. W.. Stahi. F. W. a Weisberg. R. A. (Cold Spring Harbor Laboratory. Cold Spring Harbor. NY). Pp. 395-432. 13. Hohn. B. & Murray. K. (1977) Proc. Natl. Acad. Sci. USA 74. 3259- 3263. 14. Southern. E. M. (1975) J. Mol. Biol. I, 503-517. 15. Sargeant. A., Sanie. S.. Leprince. D.. Begue. A.. Rommens. G. a Ste- helin. D. (1982) EMBO J. 1, 237-242. 16. Adhya, S. & Gottesman. M. (1982) Cell 29. 939-944. 17. Cullen. B. R.. Lornedico, P. T. a Ju. G. (1984) Nature (London) 37, 241-245. 18. Noori-Daioii, M. R.. Swift. R. A.. Kung. H.-J.. Crittenden. L. B. a Witter. R. L. (1981) Nature (London) 194, 574-576. 19. Nusse. R.. VanOoyen. A.. Cox. D.. Fung. Y. &. Varmus, H. E. (1984) Nature (London) 307, 131-136. 20. Shih. C.-K.. Linial, M.. Goodnow. M. M. & Hayward. W. S. (1984) Proc. Natl. Acad. Sci. USA 81. 4697-4701. 21. Saito. H.. Heyday, A. C., Wiman. R.. Hayward. W. S. a Tonegawa. S. (1983) Proc. Natl. Acad. Sci. USA fl, 7476-7480. 22. Taub. R.. Moulding, C.. Battey. J.. Murphy, W.. Vasicek. T.. lanior. G. & Leder. P. (1984) Cell 36. 339-348. 23. Donward. J.. Yarden. Y.. Mayes. 15.. Scrao. G., Totty. N., Stockwell, P.. Ulnich. A.. Schlessinger, J. a Waterfieid, M. D. (1984) Nature (Lon- don) M, 521-527. 24. Merlino. G. T.. Xu. Y.-H.. Ishii, W.. Clark. A. J.. Semba. R.. Too- shima, K.. Yamamota. T. & Pastan. 1. (1984) Science 224, 417-419. 25. Lin. C. R.. Chen. W. S.. Kruiger. W.. Stoiarsky. L. S.. Weber. W.. Ev- ans. R. M.. Verma, l. M.. Gill. G. N. a Rosenfeld. M. G. (1984) Sci- ence 224, 843-848. Sharp, P. A. (1983) Nature (London) 31. 471-472. Bishop, J. M. (1983) Annu. Rev. Biochem. 52, 301-354. .38 APPENDIX B: C-ERB B ACTIVATION IN ALV-INDUCED ERYTHROBLASTOSIS: NOVEL RNA PROCESSING AND PROMOTER INSERTION RESULT IN EXPRESSION OF AN AMINO- TRUNCATED EGF RECEPTOR T. W. Nilsen, P. A. Maroney, R. G. Goodwin, F. M. Rottman, L. B. Crittenden, M. A. Raines, and H. J. Kung Cell (1985) 41:719-726. Cell. Vol. 41. 710-726. July 1”. Copyright 0 1”!) by MIT $92-$741851070719-08 $02.00/0 c-erbB Activation in ALV-Induced Erythroblastosis: Novel RNA Processing and Promoter insertion Result in Expression of an Amino-Truncated EGF Receptor Timothy W. NIieen,‘ Patricia A. Maroney,‘ Raymond G. Goodwin: Fritz II. Rottman,‘ Lyman B. Crittendenfi Ilaribeth A. Raines} and Hslng‘iien Kung‘t ' Department at Molecular Biology and Microbiology Case Western Reserve University, School at Medicine Cleveland, Ohio 44106 iUnited States Department of Agriculture Reqional Poultry Research Laboratory East Lansing. Michigan 48823 tDepartment of Biochemistry Michigan State University East Lansing, Michigan 48824 Summary ALV-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the in- sertion of an intact provirus. integrated proviruses are oriented in the same transcriptional direction as c-erbB, and expression of truncated c-erbB tran- scripts ls observed. Evidence, including sequence analysis oi cDNA clones, indicates that transcription of truncated c-erbB mRNA is initiated in the 5' LTR of the integrated provirus. This transcript is processed through a series of remarkable splicing reactions to yield viral peg and em sequences fused to erbB se- quences. These results establish a novel pathway of promoter insertion oncogenesis that stands In con- trasttothepathwaysueed lntheectivstionoic-myc In B lymphomas. introduction Avian leukods virus (ALV) is a replication competent retrovirus that laclre a transforming gene but causes a va- riety of neoplasms in chickens, including 8 lymphomas and erythroblastosis. usually after a long latent period (Weiss et al.. 1982; Crittenden and Kong, 1983). Analysis oi genomic DNA from neoplastic tissue has provided a molecular explanation of tumor induction by ALV. ln partic- ular, ALV induces B lymphomas by activating the cellular proto-oncogene c-myc (Hayward et al.. 1981; Payne et al.. 1982). This activation results from the insertion of an ALV provirus, which carries strong promoter/enhancer func- tions, near the cm gene. Several lines oi evidence indi- . cate that a similar mechanism may activate the cellular protooncogene c-erbB in ALV-induced erythroblastosis (Fung et al.. 1983; Raines et al.. 1985). The c-erbB locus was initially defined by homology to the transforming gene v-erbB identified in avian erythro- blastosis virus (AEV) Melee et al.. 1982; Venstrom and Bishop, 1982). Subsequent comparison of the predicted amino acid sequence of v-erbB with portions oi the human epidermal growth iactor (EGF) receptor amino acid se- quence revealed striking homology and raised the possi- bility that c-erbB was, in fact, the gene coding for the EGF receptor (Downward et al.. 1984b). The EGF receptor con- tains three. domains. an extracellular EGF binding do- main. a short transmembrane domain, and a cytoplasmic domain that possesses protein kinase activity (Hunter, 1984). The v-erbB gene is homologous to sequences en- coding a small portion oi the extracellular domain, the transmembrane domain, and the entire cytoplasmic do- main excluding a short sequence that codes for the 32 carboxy-terminal amino acids oi the human protein (Yamamoto et al..1983;Ullrich et al.. 1984; Lin et al.,1984; Xu et al.. 1984). Both the c-erbB gene and the EGF recep- tor gene map to the same chromosome in humans (Spurr et al.. 1984; Davies et al.. 1980; Shimizu et al.. 1980), and our own recent evidence (see below) indicates that, in chickens, the EGF receptor and c-erbB appear to be de- rived from a single locus. in 37 cases of ALV-induced erythroblastosis examined, most oi the proviral integration sites were clustered within a few hundred bases upstream from the first c-erbB exon with homology to v-erbB (Raines et al.. 1985). Of those proviruses analyzed, all were inserted in the same tran- scriptional orientation as c-erbB. and elevated expression oi c-erbB-related RNA was consistently observed (Fung et al.. 1983; Raines et al.. 1985). While these studies re- vealed important similarities between the mechanisms whereby ALV induces erythroblastosis and B lymphomas. the activation oi c-erbB is not directly analogous to the ac- tivation oi c-myc. ' Whereas proviruses integrated near c-myc frequently carry deletions near or encompass the upstream or 5' LTR (Neel et al.. 1982; Neel et al.. 1981; Payne et al.. 1981; Fung et al.. 1981; Pachl et al.. 1983). most proviruses in- serted into the c-erbB locus appeared to be lull-length (Raines et al.. 1985). One such provirus was recovered by molecular cloning and was shown to be completely intact (Raines et al.. 1985). Furthermore. proviral insertion does not directly disrupt or alter the protein coding sequence oi the cm gene (Hayward et al.. 1981; Payne et al.. 1982; Fung et al.. 1981: Shih et al.. 1984). Thus. enhanced or in- appropriate expression of the intact c—myc protein may be directly oncogenic. In the case oi erythroblastosis, how- ever, proviral insertion disrupts the coding sequence of the EGF receptor gene. The fact that all of the proviral in— sertions observed in ALV-induced erythroblastosis map to a small region in the middle of the EGF receptor gene coinciding with the point of transduction oi cellular se- quences into AEV strongly suggests that specific trunca- tion of the EGF receptor gene is required ior oncogenesis (Raines et al.. 1985). in an attempt to define the mechanism oi c-erbB activa- tion in ALV-induced erythroblastosis, we have begun a detailed analysis of erbB-speciiic transcripts in leukemic samples. We find that two prominent size classes of erbB transcripts are expressed. The nucleotide sequence of orbs-specific cDNA clones indicates that at least one of these transcripts is initiated in the 5' LTR of the inserted 188 Cell 720 PU) Figure 1. Position and Structure of the Integrated ALV Provirus in Leu- kemic Samples 202 and 208 The diagram illustrates the c-erbB locus containing an integrated ALV provirus. The approximate proviral integration sites determined for leu- kemic samples 202 and 208 are indicated by arrows. The assignment oi integration sites was based upon blot hybridization of restriction digests of leukemic DNA using a 4.5 kb fragment of chicken DNA, which containsthefirst exonoic-erbBwith homologyiov-erbB(Raines et al.. 1985). This fragment. designated R 45 is indicated on the diagram. Cellular DNA (thin line) with indicated Sac i and Eco RI sites was mapped using digests oi cloned DNA isolated from a genomic library of normal chicken DNA. Solid black boxes represent the 5'-most exons of coma. as defined by homology to v-erbB The restriction map of ALV DNA, represented as a double line, is identical with that reported else- where (Raines et al.. 1985). R, Eco RI; 8, Sec l. provirus. This transcript reads through the polyadenyla- tion signal present in the 3' LTR and is processed via a se- ries of remarkable splicing reactions to yield viral gag and env sequences linked to erbB sequences. The predicted amino acid sequence deduced from this mRNA indicates that the resultant protein contains six amino acids oi the viral gag gene and 53 amino acids oi the env gene fused to sequences corresponding to the entire v-erbB gene. in addition. sequences encoding 34 amino acids homolo- gous to the carboxyl terminus of the human EGF receptor, which are absent in v-erbB, are encoded in the mRNA of the activated c-erbB gene. This result provides additional evidence that the c-erbB locus is identical with the EGF receptor gene. Results Proviral Integration Sites Proviral insertion sites near c-erbB were mapped in two leukemic samples (designated 202 and 208) by employing a strategy previously described for the analysis of 37 other cases of ALV-induced erythroblastosis (Raines et al.. 1985). in that study, all of the proviral insertions were con- fined to a 4.5 kb Eco RI fragment of cellular DNA. which contains the first exon of c-erbB with homology to v-erbB (Figure 1). To determine whether provirus insertion had occurred within this region in samples 202 and 208, a subclone con- taining the 4.5 kb fragment was used to probe Eco RI digests of leukemic DNA, as well as DNA derived from control (nonleukemic) tissue from the same birds (data not shown). This analysis indicated that the proviruses in the leukemic samples 202 and 208 were inserted within the 4.5 kb fragment and had integrated approximately 300 and 100 bases, respectively, upstream oi where v-erbB homol- ogy begins The integration sites are indicated in Figure 1. Additional mapping analysis (Raines et al.. 1985) re- 189 A e c o E F ' i I sun-47.0» g“80 “"3-5" 8-31 " 5*“ l 1 Figure 2. Northern Blot Analysis of c-erbB-related RNAs and Viral RNAs in Leukemic Samples 202 and 208 Total cellular RNA was extracted from frozen bone marrow samples as described in Experimental Procedures; polyadenylated RNA was pre- pared and fractionated on 1% denaturing formaldehyde agarose gels and was transferred to Gene Screen (NEN). Five micrograms poly- adenylated RNA from leukemic sam ple 202 (lanes A. C, and E) or sam- ple 208 (lanes B. D. and F) was analyzed in each lane. Nicktranslated probes used in hybridization included the 550 bp Barn HI (Venstrom and Bishop. 1982) restriction fragment of v-erbB (lanes A—D) and a 300 bp Eco Rl restriction fragment of RSV LTR se- quences (Eco RI 0 in Delorbe et al.. 1980) (lanes E and F). Hybridizations and washes were as described in Experimental Pro- cedures. The blot containing lanes A and B was exposed for 24 hr. The blots containing lanes C-F were exposed for 4 hr. Sizes of transcripts were determined relative to the mobility of "P-labeled RNAs of known molecular weight electrophoresed in parallel lanes. These transcripts were generated in vitro with SP-B polymerase (NEN) and ranged in size from 7.3 kb to 1.4 kb. vealed that both proviruses appeared to be intact. were oriented in the same transcriptional direction as c-erbB, and represented the only ALV provirus present in the respective leukemic cells (data not shown). c-erbB Related mRNAs Having demonstrated that the c-erbB locus was disrupted by proviral insertion, we proceeded to examine c-erbB ex- pression in these two leukemic samples. The sizes of ”DB-specific mRNAs in samples 202 and 208 were deter- mined by Northern blot analysis using a fragment of v-erbB as a probe. The probe hybridized to two prominent size classes of mRNA. 7.0 kb and 3.6 kb, in both samples (Figure 2. lanes A and B). In light of the position and orien- tation of the integrated provirus, parallel blots were hybrid- ized with an LTR-specific probe to determine whether ei- ther or both of the c-erbB mRNAs were transcribed from a viral promoter (Figure 2, lanes E and F). The LTR probe hybridized with two prominent mRNA species of about 8 kb and 3.1 kb. These sizes are consistent with the genomic length transcript and the subgenomic env mRNA of ALV. Since there is a single proviral insertion in both leu- kemic samples. this analysis indicated that the provirus in- tegrated within the c-erbB locus was actively transcribed 07308 Activation in ALV-Induced Erythroblastosis and provided additional evidence that this provirus was full-length. The viral transcripts appeared to be at least ten times more abundant than were transcripts detected with the v-erbB probe (Figure 2. cf. lanes C and D with lanes E and F). This fact, coupled with the close similarity in size between viral transcripts and erbB transcripts. made it im- possible to determine by Northern blot analysis if the erbB transcripts contained LTR sequences. Nucleotide Sequence of c-erbB cDNA Clones To facilitate a detailed analysis of the sequence content of c-erbB mRNAs, an oligo(dT)-primed cDNA library was prepared from mRNA derived from the leukemic sample 200 Double-stranded cDNA was tailed with :10 residues and was inserted into the dG-tailed Pst I site of pBR322. Ten thousand clones were screened with a ”P-labeled 055 kb Barn HI restriction fragment of v—erbB (Venstrom and Bishop. 1982). A single clone, which hybridized to the probe, was purified. This clone. designated pErb-i, con- tained a 15 kb insert. Restriction digestion and Southern blot analysis using specific fragments of v-erbB as probes (not shown) indicated that the pErb-l insert contained the entire 055 kb Barn fragment. about 700 nucleotides 5' and about 250 nucleotides 3’ to this fragment (Figure 3). The entire insert was sequenced using the dideoxy- nucleotide chain termination procedure (Figure 3). The resulting sequence confirmed the presence of v-erbB ho- mologous sequence. which extended approximately 450 nucleotides 5' to the Barn fragment before diverging com- pletely from published v-erbB sequences (Yamamoto et al., 1983). The sequence at the point of divergence coin- cided exactly with a putative splice acceptor site postu- lated by Debuire et al. (1984) to serve in the expression of v-erbB in AEVR, suggesting that sequences 5' to v-erbB homology were spliced to this c-erbB transcript. Since Southern blot analysis had indicated that the site of ALV proviral integration was immediately adjacent to the start of v-erbB homologous sequence (see above). we com- pared the unknown sequence with the published se- quence of Rous Sarcoma Virus (RSV). as a comparable sequence of ALV is not available (Schwartz et al., 1983). Surprisingly, two regions of homology between pErb-1 and RSV were revealed by this analysis; nucleotides 1—93 of pErb-i closely matched nucleotides 324-397 of RSV and nucleotides 94-252 of pErb-1 were homologous to nucleotides 5078-5236 of RSV (Schwartz at al., 1983). The first region of homology spans a portion of the viral 5' untranslated leader sequence. the gag translational ini- tiation codon, and five additional codons of the gag precursor. The second region of homology includes codons 7—59 of the env precursor (Schwartz et al., 1983). The nucleotide sequence of the gag-env junction in pErb-1 (AAGIGCA) was exactly that predicted it this mRNA were processed through the same splice donor and acceptor sites thought to be used in the generation of the sub- genomic viral env mRNA (Figure 3; Hackett et al., 1982). The sequence in pErb-1 corresponding to the env-erbB junction was GGGIGGC. which would be expected if the splice acceptor site (agIGGCC) noted by Debuire et al. (1984; see above) were used in the formation ofthis c-erbB 190 mRNA (Figure 3). Inspection of the RSV env sequence corresponding to the env-erbB junction revealed the se- quence GGGIgtagg, which shares considerable homol- ogy with a consensus splice donor site (Schwartz et al.. 1983; Mount, 1982). A similar sequence, GGIgtgag. is a splice donor site in the late leader of adenovirus (Mount. 1982). These observations indicate that pErb-i was de- rived from a transcript that was processed using a cryptic splice donor site in the viral env gene and a splice accep- tor site presumably used in the generation of normal c-erbB mRNAs (see Figure 5). Although this sequence data indicated that at least one activated c-erbB transcript contained viral sequences spliced to c-erbB sequences, the transcriptional initiation site of the mRNA corresponding to pErb-1 was not defined by this analysis. To address this question, we screened an additional 50,000 cDNA clones with erbB-specific frag- ments of pErb-i and obtained four clones containing erbB sequence. Restriction digestion and Southern blotting using fragments of pErb-1 as probes (not shown) estab- lished that one of these clones, pErb-3. contained a se- quence that spanned the entire length of pErb-l, as well as additional sequences both 5' and 3' to the boundaries of pErb—t (Figure 3). Sequencing of the first 300 nucleo- tides of pErb-3 revealed that this clone contained 233 nucleotides 5' to the beginning of pErb-1. These nucleo- tides were homologous to nucleotides 91—323 of RSV (Schwartz et al.. 1983) and included the last 11 bases of the viral U, sequence (Figure 3). This result indicates that leukemic cells contain transcripts with the viral LTR se- quence physically linked to the c-erbB sequence and pro- vides strong evidence that at least one of the activated c-erbB transcripts is initiated in the 5' LTR of the integrated provirus (see Figure 5). The transcript defined by pErb-1 and pErb-3 contains a single open reading frame that begins at the initiator ATG of gag. encodes 6 amino acids of gag, 53 amino acids of env. and 426 amino acids of c-erbB. To describe com- pletely the portion of this transcript coding for c-erbB. we analyzed an additional overlapping cDNA clone. pErb-S. Restriction mapping, Southern blotting, and partial nu- cleotide sequencing established that this clone contained approximately 1.6 kb of sequence 3' to the end of pErb-t (Figure 3). This region was sequenced, and the resultant sequence as well as the sequence derived from pErb—1 were compared with the published sequence of v-erbB (Yamamoto et al., 1983). This analysis revealed that v-erbB is remarkably conserved with respect to c-erbB. There were only six base changes between the sequence we determined and the sequence of v-erbB derived from AEV“. Of these. four were conservative. and the other two resulted in a single amino acid change, phenylalanine in v-erbB to asparagine in c-erbB at residue 596 of the acti- vated c-erbB protein (Figure 3). A more striking difference between c-erbB and v-erbB became evident when the carboxyl termini of these pro- teins were compared. The coding sequence of c-erbB did not terminate at the end of v-erbB homology. but instead included an additional 34 amino acids (Figure 3). These 34 amino acids were of particular interest. since both iso- Cell 722 191 Pgog env L I : pEfb'l .IIIIIIIIIIDI- use-elvdhelvle In In ID ID e. ID ID I- GI'I' GD " " " " " " " '. " "' " " " " "" ‘,lEl't"'E’ pErb-SF------ i eeesettcetttseteeccccsecstsstcstteggseetegtestcssseesepessgcgtssssetsetgtesteetecet“‘: “““ ,___ . ,, . ‘:-' lll fl setesteeeggggestpsggstteggegggeepeegctgestesteteggegggegctctestgsegggegsceesetescc16666666666666666666616666666 666 net :lu ele eel lie 1 s cgggeeegeeeecegesgestgeecegtcceeeseeggsstgettceegttgstctgsptgettscsgtspcseggtggetseeps A16 AA 6CC 61C A1A A 6 666 u 1* ‘t ' 2: *. ~‘ A’ 2"?" ele phe lee thr sly tyr pro sly ele thr ser lys lys esp ser lys lys lye are pro ele thr‘eer lys lys ele pro is 6CA 111 C16 AC1 66A 1AC CC1 666 6A6 ACA A6C AA6 AA6 6AC 1Cc AA6 AA6 AA6 CC6 CCA 6CA ACA 46C AA6 AA6 6AC CCC 6A6 616 ’IIIIiIV/IIIIIIIIIIIIIIIIIIIIIIII lys thr pro leu its pro thr erg vsl ssh tyr AA6 ACA CCC 116 C16 CCA AC6 A6A ll 611 AAC 1A1 A11 6v tie ll .I ‘Iu eel ies IIIIIIIIII IIIII u 661 II‘ [III 1 C '1 1C AYC A11 ‘61 61C C16 616 C16 161 A6 611 AC‘ 666 rIIIIIIIII/IIIIIIIIIIIIIIIIIIIIIII IIIIII ene» "‘ IIIIIIIIIIIA '6 I 6C CCA 6AC his eye lys Cyl ele his phe sea 1 pro his zys eel lys el sys pre ele IA, eel lee ly ele esh es thr CAC 16C A16 AA6 161 6CC CA1 111 A1A 6A1 661 CCC CAC 161 616 AAC 6CC 16C CCC 6C1 6 61C C16 61 6A6 AA1 6A ACC 616 A lee eel trp lys tyr ele esp ele esn ele eel eye In lee eye his pro esh eye thr erx :ly eye lys :1] pee 6‘1 lee C16 61C 166 AA6 1A1 6CA 6A1 6CC AA1 6C1 611 16C A6 C1C 16C CA1 CCA AAC 161 ACA C6 66 16C AAA 6A CCA 6 C11 666 ple ply eye pre eee sly ser lys thr pre ser lle ele ele ply eel eel sly ply lee lee eye lee eel eel eel :ly lee 6AA 66A 161 CCA AA1 66C 1C1 AAA AC1 CCA 1C1 A1C 6C6 6C1 661 611 61C 66A 66A C1C C16 16C C16 611 616 611 6 C1A 166 C ply tle ply 160 tyr leu ere ere ere his lle eel ere lys ere thr ‘es ere ere 16a 16v sin sin erp ele lee eel :1. 66C A1C 661 C11 1AC C16 C66 C6A C61 CA1 A1C 616 C66 AA6 CCC ACC C16 C6C A66 C16 C16 CAA 6A6 A66 6A6 C11 61C A 636 pre lee thr pre ser ply elu ele pre see In ele his ‘66 erg ile les lys ls thr lu phe lys lys eel 1‘s eel lee CCA C16 ACA CCC A61 666 6A6 6CA CCA AAC A6 6CC CAC C16 A6A A11 11A AA6 AA ACA AA 111 AAA AA6 616 A A 611 116 616 .1, see ,1, at. pee .1, IA! eel syr lys eiy lee srp its pro ele sly ele lys eel lye tie pee eel ele ile lys :1e 66‘ 161 66A 661 111 66C AC1 611 1A1 AA6 66A C11 166 A1C CCA 6AA 666 6AA A66 611 AAA A11 C61 611 6C1 A11 AAA A 666 lee erg gle ele the ser pre lys ele esh lys gle ile lee esp ele ele tyr eel let ele eer eel esp ssh pre his eel 116 A6A 6A6 661 ACA 1CC CCA AAA 6CC AAC AA6 6AA A1A C11 6A1 6AA 6CC 1A1 616 A16 6C1 A61 611 6AC AA1 CC1 CA1 616 [666 ¢ys erg lee lee gly tle sys lee thr ser the eel gle lee ile thr gin lee set are tyr ly sys lee lee esp syr lle 16C CCC 116 C16 66A A1C 16C C1C AC1 1CC AC1 616 CA6 C1C A1C ACC CA6 C11 A16 CC1 1A1 6C 16C C1C C11 6AC 1AC A1C 1166 or. .1. his lye esp esh tie gly ser gln tyr lee lee esn srp cys eel glA ile ele lye ly ees ssh syr lee is is 66A 6A6 CAC AA6 6AC AAC A11 66C 1CC CA6 1AC C11 C1C AAC 166 161 616 CA6 A11 6CA AA6 6A A16 AAC 1AC C16 6A6 AAA 1666 er erg lee eel his erg esp lee ele ele erg ese eel lee eel lys thr pre glh his eel lye lle shr esp he ly lee C6 66C C16 616 CAC C61 6AC C11 6C1 6CC A66 AAC 61C C11 611 AA6 AC1 CCA CAA CA1 616 AAA A1C ACA AA: 11 :3; (1‘ 133. ple lys lee lee sly ele no sin In ele 11' his ele elu e1: sly in vol on He in "e not ele lee is see ile 6CA AA6 C16 C11 666 6CA 6A1 6A6 AA6 6A6 1A1 CAC 6CA 6A6 66A 66C AA6 611 CC1 A11 AAA 166 A16 6CA 116 A6 1CA A11 1616 lee sts erg lle tyr thr his sin ser esp eel tre eer tyr sly eel thr eel srp :16 lee eet thr phe l: ear 1 s pp. "A CAC 66A A11 1A1 AC1 car can A61 661 st: 166 A61 1A1 est sts ACA s11 tee s 116 A16 see 111 3“ rec A‘A ccr ts“ up up My 116 pre ele en el- ile I" I" "l in ele in el: 0'- er 10- ere '- we we lie s e s» tle es 1A1 6AC 666 A16 CCC 6CA A61 6AA A1C 1CC 1CC 61C 116 6A6 AA6 66A 6A6 C6 116 CCC A6 CCA CCC A11 161 ACC A11 6A 1666 est syr set its est eel lye sys trp eet lls esp ele esp ser er pre lye phe er :1. lee ile ele ple phe her I s 616 1AC A16 A1C A16 61C AAA 16C 166 A16 A11 6A1 6CA 6AC A6C C6 CCC AA6 111 C6 6 C16 A11 6CA 6A6 11C 1CC AAA 1616 est ele ere esp ere ere are In in "l ”0 '0 eh I» si- are on his 1.. we see we the es see 1 s 66 s r A16 661 C61 6AC CC1 CCC C6C 1A1 C11 611 A1A A6 66A 6A1 6AA A66 A16 CAC 116 CC1 AAC CC1 ACA 6A 1CC 6x6 11 1‘1 1166 .p ya: lo. eet gle gle gle esp est gle esp tle eel esp ele esp ele tyr lee eel pre his le ly he see she C6: ACC C16 A16 6A6 6A6 6A6 6AC A16 6AA 6AC A11 616 6A1 6CA 6A1 6A6 1A1 C11 61C CCA CAC :66 6C 111 1:: AAC 666 1666 1 re see the ser erg shr pre lee lee ser eer lee see ele thr ser esn esh ser ele shr see eye tle esp er sea 1 :CC 1C1 ACA 1C1 C66 AC1 CC1 C11 C16 A61 16A 116 A6C 6C1 AC1 A6C AAC AA1 161 661 ACA AAC 16C A11 6A6 66: AA1 ‘6‘ 1666 gt. gly his pre eel erg gle 666 ser 6.6 '0' 6'0 0'6 16' 50' '0' 666 6'6 thr gly 66h ple lee gle gle 66f lle as CA6 666 CAC CC1 616 A66 6AA 6AC A6C 111 61C CA6 A66 1AC A6C 1CA 6A1 CCA ACA 66: AAC 11C 116 6A6 6A6 661 A1A 6A 6666 .g. .Iy phe lee pre ele pre gle tyr eel esa gln lee let 6'0 lys lys ere ser thr ele let eel ple eee le tle tye 6A1 6AC 11C C16 CC1 6C1 CCA 6A6 1A1 61A AAC CA6 (16 A16 CCC AA6 AAA CCA 1C1 AC1 6CC A16 61C CA6 AA1 AA 616 1AC 6666 see ile see lee the ele ile ser lys lee pee eet esp ser erg tyr gla ese ser his see thr ele eel esp esa pre AAC {iii A16 161 C16 ACA 6CA A1C 1CA AA6 C1C CCC A16 6AC 1CA A6A 1AC CA6 AA1 1CC CAC A6C ACA 661 616 6AC AAC CC1 6IlA ,1. y lee see the sea gle ser pee lee ele lys the eel phe gle ser ser pre tyr srp tle gle see 1, ese his 1. AAA 161 C16 AAC AC1 AAC CA6 1CC CCA C16 6CC AAA ACA 61C 11C 6A6 A6C 1C1 CCC 1A1 166 A1C CAA 1CA 26C AA1 CAC {AA 6666 in us lee esp ese no us in sin sin see she in ere en ele "if in are no sly lee lee lys eel pee ele ele A1A AA1 C16 6AC AA1 CC1 6AC 1AC CA6 CA6 6AC 111 11A CCA AA1 6AA ACA AAA CC1 AA1 661 C16 116 AAA 611 C61 66A 66A 6666 ‘. .,. ,,. .j. syr 1.. or. eel ele ele pre lys ser gle tyr lie ele ele ser ele the :AA AAC CCA 6A6 1AC 116 A66 61A 666 6CA CCA AAA A61 6AA 1AC A11 6AA 6C1 16A 66A 16A ssesegeggestssssctteseetee 6A)! ggceegsseeesspgtgeettessteestestpteepeeseeeetetgttsegssseeetspeeeeseseetgsetseeteeeeesecttsegceseeeetsteeetstgs 6666 assessestssseeeseetestttgsetcttttsssgcccettteeetstetegsteeeecccetespeteeesteegetetetettgeeetegescggeetstssstete 666) segeeeeeetetggetesetssetttcsttttseeetseteesetgeteegeegscetttlgeeetgeesttgteeesteteseststetesegtseeetlsstttstese 6166 etesptessstteteeetettceteesesettttttgeetsepetsstsetectpcttatstestsettestsetseeetseccestssseeeeeeeeeesettecteeet 6616 egsttseseeeeetcstseeeceeeeeeeetssttseeeesceeeeeeeststeeeeeetsecteeeeeteetccetstteecetseeseseeeeegsgeesetessepes 6 geesgggepettetstctetsecesseteeesseeettettetseseeeeectesecseeeseetseessecessecesetcesseestsessectestssteeeeessee 6661 gaggeeeeetesegssseceeteettsgsseetttttesssestsetcssttttetsettsteeeeeeeeesststttstctgetgsgttespstgtsgtegeestetsee 6666 pesgsssstettssteeeseeeestgsteeepetettssseteestgeeeeetteegtetceeeteesgstttesegestetegssgggtgtreeggeeetgs pely A 6611 Figure 3. Composite Restriction Map and Nucleotide Sequence of cDNA Clones Derived from Activated c-erbB mRNAs AschematicdiagramofacompositecDNAlsshownatthetopofthefigure. Untranslatedsequencesareshownasaline. andcoding sequences are boxed. The diagram shows the location of viral gag (stippled box) and env (hatched box) sequences followed by c-erbB sequences (open box). The line underneath the schematic designates the carboxyl region of c-erbB not present in v-erbB The nucleotide sequence and predicted amino acidsequenceoithiecomposltecDNAisalsoehown.ThiseequencewasderivedfromthreeoveriapplnchNAclonee.whichareshownechemati- cdlybelowthediagrem. Regionsoltheseclonesthatweresequencedareindicatedbysolid linesTheuntranslated regionsareshownin small c-erbB Activation in ALV-induced Erythroblastosis 723 l 9 2 tyr gin gin esp phe in pro esn glu thr lys pro esn gly leu leu lys eel pro ele ele glu esn pro giu Figure 4. Comparison of the Carbon-Terminal 1AC CA6 CAB 6AC 111 11A CCA AA1 6AA ACA AAA CC1 AA1 661 C1C "6 AAA 611 CC1 GCA 6CA 6AA AAC CCA GAG chicken CTCC GCGA CA YGGCTCA 1 she lys ele ile phe gly ser thr tyr leu erg eel ele ele pro lys ser glu tyr lie glu ele ser ele [no TM: 116 A66 61A 6CA 6CA CCA AAA A61 6AA 1AC A11 6AA 6C1 1CA 6CA 16A ccecegeggetttcttcttgcegtgeggcee chicken C A C 9 m. gees an ester. mi. chicken c-erbB sequence are shown. Dashes G C A 6C 11 6 ...... pro gin ser - one 91, lates of v-erbB lack not only the EGF binding domain but also the carboxy-terminal region of the EGF receptor (Yamamoto et al., 1983; Sealy et al., 1983; Ullrich et al., 1984). Recombinational events involved in the transduc- tion of erbB sequences into AEVR or AEVH presumably create this “double truncation.” in the present situation, however, since c-erbB is altered at only the 5' end. it would be expected that activated c-erbB transcripts would an- code the missing carboxy-terminal region if c-erbB were identical with the EGF receptor gene. The carboxy- terminal 34 amino acids of chicken c-erbB share 60% he- mology with the 32 carboxy-terminal amino acids of the human EGF receptor (Figure 4). Notably, the tyrosine cor- responding to tyrosine 1173 of the human EGF receptor is conserved in the chicken gene. This tyrosine is a major site of receptor autophosphorylation and may be involved in receptor regulation (Hunter, 1984; Downward et al., 1984a). The presence of these amino acids at the carboxyl terminus of c-erbB provides additional evidence that c-erbB is indeed the EGF receptor gene. The protein coding sequence of c-erbB terminated ap- proximately 900 bases upstream from the end of pErb-s. Complete nucleotide sequence analysis of the 3' untrans- lated region revealed no significant homology with the corresponding region of the human EGF receptor gene (Ullrich et al., 1984). A sequence that is homologous to but not identical with a consensus polyadenylation signal was present 14 bases upstream from a poly(A) tract (Figure 3). Assuming that pErb-i, pErb-3, and pErb—S are derived from the same mRNA, and that transcription of this mRNA is initiated in the 5' LTR (see above), the resultant c-erbB mRNA would contain 575 bases corresponding to viral se- quence, 1917 bases coding for c-erbB. and a 909 base 3' untranslated sequence. Thus, the total length of this mRNA would be 3401 nucleotides. With the addition of a poly(A) tail, this size would come very close to the 36 kb estimated for the smaller of the two c-erbB transcripts de- scribed above (see Figure 2). 1 G A lumen Sequence of the Chicken c-eroB and Human EGF Receptor cDNAs Onlythose basesand amino acidsofthehu- man eequence that are not identical with the indicate gaps introduced in the human se- quence to maximize homology. The arrow designates the 3' end of v-erbB (Yamamoto et al., 1983) and c-erbB homology. A conserved tyrosine residue (Tyr 1173 in the human EGF receptor), a major autophosphorylation site in the human protein,lsboaad.3'untranslatedse- quences are shown in small letters. Discussion We have analyzed the expression of the c-erbB locus in ALV-induced erythroblastosis. Our results indicate that the c-erbB protein expressed in these leukemic samples is an amino-truncated form of the EGF receptor fused to portions of ALV gag and env proteins (Figure 5). These results establish a novel pathway for promoter insertion oncogenesis. which stands in contrast to the pathways used in the activation of c-myc in B lymphomas. (This pathway has previously been suggested as a possible mode of oncogene activation [Neel et al., 1981; Payne et al., 1981).) it seems likely that these distinct pathways re- flect the unique molecular requirements that must be ful- filled to activate either c-erbB or c-rwc. Available evidence suggests that the level of expression of cm is of prime importance in ALV-induced B lympho- mas. Thus, either enhancement of the normal c-myc pro- moter (Payne et al., 1982) or, more commonly, provision of a strong viral promoter appears to be sufficient to activate the cm gene (Hayward et al., 1981). The requirement for strong 3' LTR promoter function probably explains the high frequency of deletions observed in proviruses in- tegrated upstream from c-myc (Neel et al., 1981; Payne et al., 1981; Fung et al., 1981; Pachl et al., 1983). These dele- tions. which usually eliminate 5' viral promoter activity, may allow efficient use of promoter elements in the 3' LTR (Cullen et al., 1984). In contrast, our results indicate that the presence of the 5' LTR and additional internal viral sequences may be re- quired to bring about activation of c-erbB. Furthermore, the absolute level of expression of c-erbB may be less im- portant than the structure of the activated c-erbB protein. in this regard, the sequence content of activated c-erbB mRNAs raises some interesting questions. The presence of the gag sequence may not be surprising, since, by anal- ogy to v-erbB, this sequence may be required to provide a translational start site for expression of c-erbB However, letters,andtheviralgagsnderrvcodingsequencesareindicatedbyboxes.1he11nucleotidesthatarepartoithevlrai LTRUSsequencsarebrack- eted.The6bpdlfferencesbetweenthec-ero8sequencedeterminedhereandthepublishedeequenceofv-erbBareindicatedbelouthec-eroa sequence.andtheeingleaminoaclddifferencewithinthev-erbadomainlaboured.Thearrowmarkstheendofthev-eroBsequencaAheaahucleotide with homology b the consensus polyadenylation signal is underlined. The sequence of pErb-1 and pErb-3 corresponding to viral regions was not totany homologous to the published sequence of RSV. Most notably. pErb-i contained a 14 base direct repeat 5' to the initiator ATG. To confirm that this repeat was indeed of ALV origin. we sequenced an appropriate restrictionfragmentlsolatsdfromalclonecontalnlngan intactALVprovlrus(Rsinesetal., 1995). Thismalyaisindicatedthattheeequencein pErb-i oracllymflhedtheALVsequencflnotslwown). P. Pst l; 8. Sam HI;E. Eco RI. Cell 724 “E 9°, =E+Iw 699 env a hens- “ 6F -bindirig 2 I nose 6 : . 7/////////////////////////////'////7/////////////////////A EGF Recepfw Wv-UDB ”WWW/M 939:3“ Figure 5 Schematic Representation of c-erbB Activation by ALV. and Comparison of the Resulting c-eroB Protein with the EGF Receptor (a) Schematic diagram of an ALV provirus integrated upstream from c-erbB exons (hatched boxes). and the resulting c-erbB-containing transcript. Evidence presented in the text suggests that c-erbB- containing messages are initiated in the 5' LT R of the integrated provi- rusandcontainviralgagandenvsequencesfusedtoc-eroBse- quences. Regions of the RNA appearing in the processed transcript are shown as thick lines. while introns are shown as thin lines. The AUG codonpresumedtobsusedforinitiationoitranslationoic-erbB- containing transcripts, as well as that used in translation of viral pro- teins, is indicated. (b) Comparison of erbB and EGF receptor proteins. The diagram of the EGF receptor is adapted from a similar schematic of the human recep- tor (Hunter. 1994) and shows the three domains of the protein. Below is shown the presumed structure of the v-erbB protein if its mRNA is spliced as described by Debuire et al. (1994). This protein would con- tain 9 amino acids of viral gag. 605 amino acids of erbB, and 4 amino acids of viral env. Below this is shown the proposed structure of the ALV-activated c-erbB protein as deduced from the sequence of pErb-i and pErb-5 Like v-erbB. this protein would begin with 6 amino acids of viral gag. However, it would also contain 53 amino acids of viral env between the gag and the erbB sequence. in addition. the activated c-erdeoesnotterminatewithviralerwaminoacidsbutinsteadcon- tains 34 carboxybterminal amino acids homologous to those present in the human EGF receptor. the presence of the env sequence was unexpected. The six amino acids of gag and 53 amino acids of env com- prise 59 of the 62 amino acid translational signal se- quence responsible for directing the viral env protein to the cell surface (Hunter 91 al., 1994). While we do not know whether this sequence is cleaved after translation from the c-erbB protein, it would undoubtedly serve as a signal sequence to direct c-erbB mRNAs to membrane-bound ribosomes. It is tempting to speculate that this sequence is required for correct intracellular transport of the c-erbB protein and may thus be important for oncogenic activa- tion. Recent results of Hannink and Donoghue, (1994) in- dicate that the transforming activity of v-sis is dependent upon a viral signal sequence fused to the v~sls protein. Alternatively, the presence of the env sequence may simply reflect accidental RNA processing resulting from efficient use of the gag-env splicing sequences and the presence of a fortuitous, inframe. splice donor site in the envelope gene. In this regard, v-erbB does not contain env sequence and is apparently positioned in the cell mem- brane (Hayman and Beug, 1994; Privalsky and Bishop, 1994). Furthermore, our data do not exclude the presence of an mRNA or mRNAs formed by direct gag-erbB splic- ing. Appropriate transfection experiments may provide the 193 means to assess the importance, if any, of any sequences fused to c-erbB The finding that the activated c-erbB mRNA or mRNAs contain sequences that code for 34 amino acids homolo- gous to the 32 carboxyl-terminal amino acids of the human EGF receptor further strengthens the case for the identity of c-erbB and the EGF receptor. Furthermore, this result indicates that the carboxyl-truncation of c-erbB found in both AEVH and AEV“, which results in the removal of a major autophosphorylation site (Hunter, 1994; Downward et al., 1994a), is probably not necessary for oncogenic ac- tivation. However, the clustering of proviral integration sites, as well as the structure of both v-erbB isolates, may indicate that precise amino-truncation of the EGF receptor is required. Alternatively, the apparent precision of trunca- tion could result from the use of preferred integration sites by ALV. Transfection experiments designed to assess the transforming potential of appropriate truncations of EGF receptor cDNAs will be needed to resolve this question. Two lines of evidence suggest that the mode of activa- tion of c-erbB revealed by analysis of tumor 209 may be used in most or all cases of ALV-induced erythroblastosis. First, in all cases studied. the provirus is integrated up- stream and in the same transcriptional orientation as c-erbB (Raines et al., 1995). Furthermore. all of the proviruses apparently retain the 5' LTR and the bulk of vi- ral sequence (Raines et al., 1995). Second, Northern blot analysis indicates that c-erbB mRNAs are of the same size. regardless of the site of proviral integration (Figure 2; unpublished results). This would be expected if a com- mon splicing pathway were used in the generation of acti. vated c-erbB mRNAs. ' The requirement to maintain the 5', rather than the 3', LTR of ALV to activate c-erbB may help explain the rela- tively high frequency generation of erbB-containing trans- ducing viruses in ALV-induced erythroblastosis. Under appropriate conditions, up to 50% of leukemic samples release such viruses (Miles and Robinson, 1985; C. Moscovici and the authors’ unpublished observations). if the 5' LTR is maintained intact. a deletion of the 3' end of the provirus could result in the appropriate fusion of viral and erbB sequences such that an mRNA capable of being packaged would be produced. Recombination during re- verse transcription as suggested by Swanstrom et al. (1993) could subsequently give rise to a virus containing erbB sequences. The presence of correctly processed viral genomic and envelope mRNAs in the leukemic cells we have analyzed indicates that the viral polyadenylation signal present in the 3' LTR is intact. Thus, transcripts containing c-erbB se- quences must result from the processing of RNAs that have “read through" this polyadenylation signal. The rel- ative abundance of mRNAs containing erbB and viral sequences may. to a large extent, reflect the relative effi- clencies of splicing of the primary transcript versus polyadenylation at the viral site. Without knowing the sta- bilities of viral mRNAs and c-erbB mRNAs. it is difficult to estimate the frequency of “read through” transcription. However. the existence of mRNAs containing viral ‘suuons" spliced to c-erbB exons may have important implications gsrbB Activation in ALV-Induced Erythroblastosis with regard to eukaryotic gene expression. While there is convincing evidence that polyadenylation site selection is important in differential gene expression (i.e., immune- globulin synthesis (Alt et al., 1980] and calcitonin produc- tion [Amara et al., 1992]), our observations raise the possi- bility that stochastic processes could be involved in the generation of multiple gene products using a single promoter. The activated c-erbB gene is expressed in the form of at least two mRNAs with sizes of about 7 kb and 3.6 kb. We have not established the exact relationship between these mRNAs. They appear to be expressed at about the same level (see Figure 2), and Northern blot analysis using various probes derived from c-erbB cDNA clones did not reveal any differences In sequence content (not shown). The presence of two mRNAs with erbB coding se- quence appears analogous to the presence of two major transcripts of the human EGF receptor (Ullrich et al., 1994; Lin et al., 1984; Xu et al., 1984; Merlino at al., 1994) and the two normal c-erbB mRNAs described by Vennstrom and Bishop (1992; and our unpublished results). The structures of the overlapping cDNA clones pErb-1, pErb-3, and pErb-S indicate that the 3.6 kb mRNA can encode the entire activated c-erbB protein. Thus, it seems likely that the two c-erbB mRNAs could have similar 5' ends and dif- for by alternate splicing pathways or poly(A) addition sites such that the 7 kb mRNA carries a longer 3’ untranslated sequence. Isolation of cDNA clones containing se- quences specific to the 7 kb mRNA should resolve these questions. Experimental Procedures Induction of E Approximately to3 infectious units of RAVoi. a prototype ALV. was used to inoculate 1 day old line 15. x 15l. chicks This chicken line was de- veloped at the Regional Poultry Research Lab. East Lansing, MI. for its high susceptibility to erythroblastosis and low incidence of non- specific lesions. Over 95% of the inoculated birds developed erythro- blastosis. The diagnosis of the disease and the collection of leukemic samples were as previously described (Fung st al., 1993; Raines et al., 1995). In birds with severe erythroblastosis, such as samples 202 and 209. bone marrow samples are composed primarily of leukemic erythroblast (390%). Livers are also heavily infiltrated with leukemic erythroblasts 030% of the cells present). DNA and RNA Extractions Total cellular DNA was extracted from frozen tissue samples by homogenization, pronase digestion. and phenol attraction as previ- ously described (Fung et al., 1993). Toni cellular RNA was extracted from frozen bone marrow samples using guanidinium leothiocyanate as described (Maniatis at al., 1992). Blot Hybridization Southern blot analysis was carried out as described (Raines et al.. 1985). For Northern blot analysis, polyadenylated RNA was prepared by oligo(dT)-cellulose chromatography, fractionated on 1% denaturing formaldehyde agarose gels, transisned to Gene Screen (NEN). and hybridlzsdasdeecribed(NilaenandMaroney, 1994). Washeswereat 65‘C with 2x SSC and 0.1% SDS. cDNAClonlng PolyadenylatedRNAwaspreparedasdescrlbedabove.Oligo(dT)- primed double-stranded cDNA was synthesized using this RNA as tsmplate(Maniatisetal..1992)andwaesizeselectdona1%agsross gel.cDNAslsrgerthan500bpweretailedwltthresiduesuslngtsr- 194 minal transierase (Ratliff Biochemicals) and were annealed with de- tailed pBR322 (NEN) (Maniatis et al.. 1982). Recombinant piasrnids were used to transform E. coil H8101 (Hanahan, 1993). Screening was as described by Hanahan and Meselson (1990). Nucleotide sequences were obtained from overlapping M13 subclones in both orientations using the dideoxynucleotide chain-termination technique (Sanger et al., 1977). Acknowledgment Wethank I. PastanforprovidingclonechNA,‘l’.Callaghan forcom- municating unpublished result. E. C. Goodwin for computer analysis. M. E. Bleakleylor preparationofthe manuscript. R. Ransohoifiorhelp- ful discussions. and K. Davidovic for providing labeled SP-6 tran- scripts. This researchwassupportedby NIH granttoT. W. N., F. M. R.. and H. J. K.andbyagrantfromtheLeukemiaResearchFoundationto H. J. K. H. J. K. gratefully acknowledges support from the Faculty Re- search Award of the American Cancer Society; as does T. W. N. for support from a Presidential Vbung Investigator Award from the NSF. The costs of publication of this article were defrayed in part by the payment of page charges. 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The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35. 71-79. .I APPENDIX C: RAV—l INDUCED ERYTHROLEUKEMIC CELLS EXHIBIT A WEAKLY TRANSFORMED PHENOTYPE IN VITRO AND RELEASE C-ERB B-CONTAINING PROVIRUSES UNABLE TO TRANSFORM FIBROBLASTS H. Beug, M. S. Hayman, M. A. Raines, H. J. Kung. and B. Vennstrom J. of Virology (1986) 57:1127-1138 JOURNAL or ViltOLocv, Mar. 1986. p. 1127-1138 0022-538X/86/031127-12502.00/0 Copyright 0 1986. American Society for Microbiology Vol. 57. No. 3 Rous-Associated Virus 1-Induced Erythroleukemic Cells Exhibit a Weakly Transformed Phenotype In Vitro and Release c-erbB-Containing Retroviruses Unable to Transform Fibroblasts H. BEUG,“ M. J. HAYMAN}? M. B. RAINES,3 H. I. KUNG,3 AND B. VENNS'I'ROM1 European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany‘; Imperial Cancer Research Fund Laboratories. St. Bartholomew’s Hospital, Dominion House. London EC IA 78E. England); and Department a Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 Received 14 June 1985/Accepted 7 August 1985 Avian leukosis viruses induce erythroblastosis in chicks by integrating Into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous-associated virus l-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both diflerentiate and proliferate extensively in vitro. All 14 leukemias studied expressed high levels of erbB-related proteins that were 5 to 10 kilodaltons larger but otherwise very similar to the gp74"” protein of avian erythroblastosis virus E84 with respect to biosynthesis, glycosylation, and cell surface expression. Two leukemias contained and released retroviruses that transduced erbB. Chicken embryo fibroblasts fully infected with these viruses expressed high levels of erbB RNA and protein but retained a normal phenotype. Our results suggest that certain forms of c-erbB, activated by long terminal repeat insertion or viral transduction, are capable of inducing erythroleukemia but unable to transform fibroblasts. Leukemia-inducing avian retroviruses have been subdi- vided into two main groups (for a review, see reference 12). The acute leukemia viruses, which contain cell-derived oncogenes, rapidly transform specific types of hematopoietic cells in vivo and in vitro (6, 10, 15, 26). In contrast, the avian leukosis viruses (ALVs), which lack oncogenes. induce a wide variety of leukemias and other neoplasms in chickens but do not transform cells in vitro (22, 35). One important step in the development of avian B-cell lymphomas is the activation of c-myc transcription by inte- gration of an ALV provirus juxtaposed to the oncogene (18. 24). Similarly, erythroleukemic cells induced by ALVs in certain strains of inbred chickens carry an ALV provirus next to the c-erbB locus and express greatly enhanced levels of c-erbB RNA (9, 25a). Thus, both the broad oncogenic spectrum of ALVs and their long latency period could be explained by the hypothesis that these viruses can activate certain cellular oncogenes by integration either next to or within them (9). Although certain molecular events leading to the activa- tion of the c-myc gene by ALV promoter insertion have been elucidated. it is still unclear how this event converts a normal avian lymphoid precursor into a leukemic B- lymphoma cell. Further study of this question is difficult, since neither normal nor leukemic chicken B-lymphoblasts can be easily grown in culture and the available v-myc- containing avian retroviruses do not transform lymphoid cells in vitro. Several reasons suggest that ALV-induced chicken erythroleukemia might provide a system for studying how insertional activation of the c-erbB gene leads to erythroblast transformation. First, both normal and leukemic erythroid precursors can be grown and analyzed in vitro (5, 28. 29). ‘ Corresponding author. 1 Permanent address: Department of Microbiology. State Univer- sity of New York at Stony Brook, Stony Brook. NY 11794. 1127 Second, avian erythroid precursors can be transformed in vitro by retroviruses that contain the v-erbA and v-erbB oncogenes or the v-erbB gene alone (3b, 8, 14, 20). Erythroblasts transformed by v-erbB or other oncogenes of the src family proliferate independently of the erythroid difl‘erentiation hormone erythropoietin (Epo). but also un- dergo spontaneous differentiation into erythrocytes with a certain frequency (3b, 3c, 5. 20). v-erbA acts in concert with v-erbB by fully arresting the differentiation of the infected erythroid progenitors and enabling them to grow in standard tissue culture media (8. 13). Finally. leukemic cells contain- ing an activated c-erbB gene should express erbB-related proteins at their cell surface, permitting them to be distin- guished from uninfected precursors at the single-cell level (3. 17). Although molecular studies on numerous Rous-associated virus 1 (RAV-lrinduced leukemias have unraveled two possible mechanisms of c-erbB activation, i.e., provirus insertion and c-erbB transduction (9, 25a, 30a), no attempts have been made to characterize the leukemic cells in vitro to determine whether c-erbB activation leads to elevated ex- pression of erbB gene products and causes a truly leukemic phenotype (25). In this paper, we demonstrate that pure populations of RAV-l-induced erythroleukemic cells could be explanted and studied in vitro. These cells resemble hormone- independent erythroid progenitors and expressed erbB- related cell surface glycoproteins at high levels. Some leu- kemias contained new c-erbB-transducing retroviruses, which efficiently replicated in chicken embryo fibroblasts without transforming them. MATERIALS AND METHODS Viruses, chickens, and Induction of erythroleukemia. Cloned stocks of RAV-l (9) were obtained from L. Crittenden. East Lansing, Mich. Chickens of the inbred strain L15-1 were injected with RAV-l in East Lansing as described previously 196 1128 BEUG ET AL. (9). In a second series of experiments. L15-1 chicks were hatched from embryonated eggs shipped to Heidelberg and were injected via the leg vein with 0.1 ml of undiluted RAV-l supernatant (10° to 107 infectious units/ml). They were monitored for leukemia development by inspection of blood smears as described previously (20). During a preleukemic phase of 40 to 60 days, low numbers of partially mature and immature erythroid cells appeared in the peripheral blood of 8 of 10 chicks. Shortly afterwards, four chicks rapidly developed lethal erythroleukemia. in which the bufly coat- containing leukemic blasts represented up to 30 to 50% of the total blood cell volume. Purification of leukemic cells. Leukemic cells obtained from moribund chicks by heart puncture were washed twice in phosphate-buffered saline. suspended at 10 X 107 cells per ml in CFU-E medium without anemic serum (25). and centrifuged through a layer of Percoll (density, 1.072 g/cm3 [3]). The immature cells banding at the interphase repre- sented essentially pure leukemic cells. since more than 90% of them expressed markers of erythroid cells as well as erbB proteins at their surface, as revealed by fluorescent staining with erbB-specific antisera (3). Cells and cell culture. For in vitro culture experiments. purified leukemic cells were seeded at 10 X 10“ to 20 X 10" cells per ml in CFU-E medium with or without anemic serum and supplemented with 1 pg of insulin (Actrapid; Bayer. Leverkusen. Federal Republic of Germany) per ml. Two days later. erythrocytes and dead cells were removed by centrifugation through Ficoll (4). and cells were reseeded at 2 X 10" to 5 X 10" cells per ml in the same medium. Cultures were fed daily by the addition of fresh medium. and cell numbers were kept above 5 X 10’ cells per ml by reducing culture size. Cultures were considered as nongrowing when all immature cells initially present had difl‘erentiated into erythrocytes without apparent massive cell death. Prolifer- ating leukemic cultures were kept at cell densities between 1 X 10" and 4 X 10" cells per ml. Chicken embryo fibroblasts were prepared from 11-day- old SPAFAS embryos as described previously (11) and cultivated in standard growth medium (Dulbecco modified Eagle medium supplemented with 8% fetal calf serum. 2% chicken serum. and 10 mM HEPES [N-Z-hydroxy- ethylpiperazine-N'-2-ethanesulfonic acid]. pH 7.3). Normal bone marrow cells were prepared from 1- to 4-week-old chicks as described previously (11). The origin and cultiva- tion of erythroblasts transformed by avian erythroblastosis virus (AEV)-ES4 (LSCC HD3) and by AEV 813. which contains a previously undetected oncogene rather than v- erbB (3a). have been described earlier (1, 3b). Plasma clot colony assay. The plasma clot colony assay was performed essentially as described earlier (5). Briefly. 10‘ cells were seeded into difl'erentiation medium with or without anemic serum (3) supplemented with 20% Methocel in lscoves Dulbecco modified Eagle medium-2 mg of fibrin- ogen per ml—0.1 U of thrombin. After clot formation at 37°C and 5% C02. cultures were kept at 41°C and 2% C02 and processed either 3 or 6 days later as described previously (5). Immunoprecipitation analysis. Fresh and cultivated RAV- l-induced leukemic cells (10 X 10" to 20 x 10") were labeled with [”Slmethionine (250 uCi) or [3H1glucosamine (200 uCi) as described earlier (3). Preparation of lysates. immunopre- cipitation with rat antiserum to erbA plus erbB (erbA + B) proteins or specific for erbB protein, and analysis of im- munoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were done as de- scribed previously (16. 17). Treatment of cells with tu- 197 .l. VIROL. nicamycin during metabolic labeling was done as described previously (17). Analysis of DNA and RNA. DNA and RNA from cells and tissues was purified and analyzed essentially as described previously (8. 33). The first exon probe was made from a subcloned 4.5-kilobase (kb) EcoRI fragment that encodes the first exon of c—erbB hybridizing with v-erbB (33). The erbA + B probe was made from a subcloned 2.5-kb Pvull fragment from AEV (34). The erbB 3' probe represents sequences present in a 570-nucleotide BamHI-EcoRI fragment of v- erbB (34). The long terminal repeat (LTR) probe was made from a 1.5-kb EcoRI-BamHl fragment of AEV that contains parts of env. LTR. and 5'-untranslated sequences of AEV. Infection of chicken embryo fibroblasts with erbB- containing retroviruses released by leukemic cells. Cultivated RAV-l erythroblasts (BH02, BH03) (10 x 10“) were seeded into 60-mm dishes containing CFU-E medium plus insulin. After the addition of 10° chicken embryo fibroblasts freshly trypsinized from minced embryos (11). the cultures were incubated for 2 days at 39°C. Thereafter, the dishes were carefully rinsed with standard growth medium to remove nonadherent cells, and the adherent fibroblasts were propa- gated in standard growth medium with daily rinses to remove any nonadherent cells. This procedure effectively removed all leukemic erythroblasts, which do not survive in standard growth medium. After an additional five to seven passages in standard growth medium. fibroblasts were frozen in liquid nitrogen or used for analysis. Assays for fibroblast transformation parameters. Im- munofluorescent staining for actin cables and fibronectin protein network were done as described previously (23). Hexose uptake was measured as described previously (27). Undiluted. filtered supernatants from either leukemic cells or infected fibroblasts were tested for focus formation on chicken embryo fibroblasts as described previously (8). Immunofluorescence. Staining of viable leukemic erythroblasts with fluorescent erbB-specific antibodies and differentiation-specific antibodies was done as described previously (3. 6). To assay erbB protein expression at the surface of infected fibroblasts. cells were detached from the dish by EDTA treatment and stained in suspension as described above (17). RESULTS Characterization of RAV-l-induced erythroleukemia cells from leukemic animals and after culture in vitro. Purified leukemic cells from moribund, RAV-l-infected chicks (see above) were first characterized for their state of erythroid difi‘erentiation. The leukemic cells consisted almost exclu- sively of erythroid cells at various stages of maturation. about 60% of which resembled immature erythroblasts (Ta- ble 1). To test whether the RAV-l-induced erythroleukemic cells could be grown in tissue culture, we seeded the purified leukemic cells in CFU-E medium. since erythroid cells transformed by v-erbB alone proliferate well in this complex medium but quickly die in standard growth medium (8. 25). Three of eight leukemias were cultured successfully. In these cultures. total cell numbers decreased during the first 5 to 7 days. However. clumps of immature cells persisted among the increasing fraction of erythrocyte-like cells (Fig. 1B). Thereafter. the immature cells started to proliferate and continued to grow for up to 3 weeks (Fig. 18) with doubling times of about 24 h. In contrast. the leukemic cells from the other five animals tested survived in culture for the first 4 to 7 days. but then they all differentiated into erythrocytes. As VOL. 57. 1986 TABLE 1. Characterization of leukemic cells 96 Cells classified % Cells stained as‘: with": Ebl ER LR + E aEry aEbl aMbl Celltime Fresh leukemic cells 81-102 70 17 13 53 61 5 131-103 ND‘ ND ND 61 66 3 HM27237‘ 77 17 6 ND ND ND Peripheral blood 0 1 >95 >95 0 5 Leukemic cells after 10 days inCFU-E medium BHOZ 83 2 15 52 70 <1 81103 82 9 9 ND ND ND I-IM27237 95 2 3 ND ND ND AEV-ES4' >99 <0.1 <0.1 <1 99 <1 ‘ Cell types were defined by benzidine (at neutral pH) plus histological staining (5). Ebl, Erythroblasts; ER. early reticulocytes; LR. late reticulocytes; E. erythrocytes. " Characterization of these antisera has been described elsewhere (6). aEry. Antierythrocyte; aEbl. antierythroblast; aMbl. monoclonal antibody 51/2 directed against myeloblasts. ‘ ND. Not determined. " Smears prepared from frozen cells immediately afier thawing. ' tsl67 AEV clone E3 (3). expected, the leukemic cells fom all animals died after 2 to 3 days in standard growth medium. When characterized for differentiation parameters, the cultured RAV-l-induced erythroleukemic cells again con- sisted of a mixture of erythroblasts and more mature erythroid cells (Table 1; Fig 1C). Thus. both fresh and cultivated RAV-l-induced erythroleukemic cells resemble erythroblasts transformed by v-erbB or src (3b, 20). RAV-l-induced erythroleukemia cells proliferate and difl‘er- entiate independent of exogeneous Epo. The above findings prompted us to study in more detail how RAV-l eryth- roleukemic cells difler from normal, late erythroid progeni- tors (CFU-E cells) in their ability to self-renew and differ- entiate in vitro and in their dependence on exogeneously added Epo. Purified leukemic cells of chicks BHOZ and BI-I03 were seeded into plasma clot cultures in the presence or absence of anemic serum as a source of chicken Epo (5). Normal bone marrow cells were tested as controls. After 3 days of incubation, about 14% of the leukemia~derived colonies consisted entirely of immature erythroid cells (type III, Fig. 1A). a colony type which was absent from the controls. About 30% of the colonies contained partially mature erythroid cells (type 11, Fig. 1A). whereas more than half of the leukemia-derived erythroid colonies were indis- tinguishable from the normal CFU-E colonies (type I, Fig. 1A), obtained almost exclusively in the normal bone marrow controls (Table 2; data not shown). Table 2 also demonstrates that RAV-l-induced eryth- roleukemic cells grew into undifferentiated as well as differ- entiated colonies with similar efficiencies in the presence and absence of anemic serum, whereas normal CFU-E colonies were stimulated more than 20-fold by the addition of anemic serum. When the plasma clot cultures of RAV-l-induced eryth- roleukemic cells were incubated for 6 instead of 3 days before analysis, the mature and partially mature colonies had disintegrated. However, about 20% of the undifl‘erentiated. type III colonies seen at day 3 had grown into large colonies containing between 2.000 and 10,000 cells. About half of 198 RAV-l-INDUCED ERYTHROLEUKEMIC CELLS 1129 these colonies were completely undifferentiated, while the other half consisted of undifi‘erentiated cells as well as more differentiated cells. Taken together, these results indicate that the majority of the RAV-I-induced erythroleukemic cells resembled Epo-independent but otherwise normal CFU-E precursors. while a minority of the cells exhibited a sustained self-renewal capacity in vitro. RAV-l-induced erythroblasts express high levels of an erbB- related glycoprotein at their surface. To study whether the RAV-l-induced erythroblasts expressed erbB-related pro- teins. fresh leukemic cells from one chicken (BHOZ) were labeled with [”S]methionine and immunoprecipitated with anti-erb sera (16, 17). Antisera reactive either with both VoerbA and v-erbB or with v-erbB alone immunoprecipitated a group of 74- to 76-kilodalton (kDa) proteins (Fig. 2A. lanes 1 to 3) probably representing rough endoplasmic reticulum precursors of erbB-like proteins (16. 17). In contrast, anti- bodies to viral structural proteins or antibodies reacting with the gag or v-erbA domains of p75"""’""‘ did not immuno- precipitate the 74- to 76-kDa proteins (Fig. 2A, lanes 4 and 5; data not shown). When leukemic cells from 12 other RAV-l-infected chicks were analyzed, proteins of slightly difl‘ering sizes were immunoprecipitated by erbB-specific serum. Four of the leukemias contained 74- to 76-kDa proteins (Fig. 28, lane 4), whereas two groups of three animals each displayed smaller proteins of 72 to 74 kDa (lane 3) and 70 to 72 kDa (lanes 1 and 2), respectively. The leukemic cells of one animal expressed two distinct protein species of 69 to 71 kDa and 74 to 76 kDa (Fig. 2B, lane 5). In all cases in which the leukemic cells could be grown in culture, the proteins detected in the fresh leukemic cells were indistinguishable from those found in the cells from the same animal after 10 days of in vitro culture (Fig. 2C. lanes 1 and 2). Leukemic cells from chick BH02 were labeled with [3l-Ilglucosamine and immunoprecipitated to determine whether their erbB-related proteins were glycosylated. A protein of 83 kDa was detected which probably represented the mature cell surface form of the 74. to 76-kDa erbB- related proteins found in these cells after [”S]methionine labeling (Fig. 3A). When the cells were labeled with [”S]methionine in the presence of tunicamycin. a 72-kDa nonglycosylated form of the gp83""‘3 protein could be de- tected (Fig. 3B). These results indicate that the RAV-l erythroblasts expressed an erbB protein that was 9 to 10 kDa larger but otherwise closely related to the gp74"”” protein of AEV-E84 erythroblasts. This was confirmed by two- dimensional peptide-mapping studies and by immunoprecip- itation of erbB proteins from RAV-l-induced erythroblasts with an antiserum to a peptide of the human epidermal growth factor (EGF) receptor (21; data not shown). Finally. we tested whether fresh and cultivated eryth- roleukemic cells expressed erbB-related proteins at their surface. Virtually all immature erythroblast-like cells from both fresh and cultivated leukemic cells were strongly stained by erbB-specific antibodies (Fig. 4A; data not shown). while the mature cells apparently underwent down— regulation of erbB protein expression as seen in differenti- ated temperature-sensitive AEV erythroblasts (Fig. 4B) (3). Two RAV-I-induced leukemias contain retrovirus-trans- duced erbB genes. Previous studies have shown that in a large number of RAV-l-infected leukemic chicks the ALVs had integrated close to the first c-erbB exon homologous to v-erbB. leading to highly elevated levels of coerbB tran- scripts (9. 25a). As will be shown below. two of the ALV- induced leukemias studied here (BH02 and 81103. Table 1) 1130 BEUG ET AL. J. VIROL. FIG. 1. In vitro characterization of RAV-l-induced leukemic cells. (A) Purified leukemic cells of chicken 8H02 were seeded into plasma clot cultures withpm anemic serum. Three days later. clots were processed as described previously (5). Shown are photographs of mature (l). partially mature (II). and immature (Ill) colonies taken under blue light (5) to reveal staining for hemoglobin. (B) Photographs of leukemic cells from chicken 81-102 are shown after 4 and 14 days of in vitro culture. Note clumps of mature cells (arrow) and immature cells (arrowhead) visible after 4 days. (C) Leukemic cells from chicken BH02 were cytocentn'fuged onto slides and stained with neutral benzidine for hemoglobin (Hb) or stained with antierythrocyte serum (EryAg: 6) by indirect immunofluorescence. The culture consists of a mixture of immature and more mature cells. as revealed by both markers. contained unmodified c-erbB genes as well as retrovirus-like elements that had transduced exons but not introns of c- -.erbB These elements were present at multiple integration sites. suggesting an oligoclonal or polyclonal origin of the leukemias. The first 5' exon of the c-erbB gene with homology to v-erbB represents about 300 nucleotides encoded in a 4.5-kb (c-erbB 0: allele) or 2.3-Rb (c-erbB B allele) EcoRI fragment (25a). Since ALV LTRs contain EcoRI sites, integration of ALV into either of these fragments would interrupt them and thus generate two smaller fragments that can be detected by using the subcloned 4.5-kb EcoRI fragment of the c-erbB an allele as a probe (first exon probe). Chick 8H02 was homo- zygous for the an allele. and both alleles appeared to be intact in the 81102 leukemic cells (Fig. 5A). In addition. a single novel. weakly hybridizing. 3.8-kb fragment was detected with this probe. The same 3.8-kb fragment hybridized strongly with a v-erbA + 8 probe (which encodes all of v-erbB) and with a probe representative for the carboxy- terminal part of v-erbB (Fig. 58 and C). A second tumor- specific fragment of 2.2 kb was also detected with these probes. but it did not hybridize to the first exon probe. Finally. the additional erbB sequences in the leukemic cells were contained in a DNA segment similar in size to those in v-erbB. since double digestion with Apal and EcoRI (which cut at the extreme ends of the AEV-E84 v-erbB gene) generated a fragment of 1.7 kDa in the leukemic-cell DNA (Fig. 5D) which is of the same size as the corresponding VOL. 57. 1986 200 RAV-I-INDUCED ERYTHROLEUKEMIC CELLS 1131 TABLE 2. Efl‘ect of Epo on RAV-l-induced erythroleukemic cells Plasma clot colonies + Epo ' E1’0 Cell type analyzed Colonies/10’ % Type“: Colonies/10’ % Type: cells I u 111 cells 1 u ur Fresh 8H02 Leukemic cells 2.550 58 29 13 2,580 56 30 14 Normal bone marrow 1.150 92 8 0 - 50 80 20 0 AEV-884‘ TMTC‘ <01 1 99 TMTC <0.1 1 99 ‘ Types of colonies (I. differentiated colonies; II. partially differentiated or mixing colonies; III. colonies containing immature erythroblasts) are as shown in Fig. 1. " r3167 AEV clone E3 (3). ‘ TMTC. Too many to count. v-erbB fragment. Analysis of leukemic cells after 14 days of in vitro culture gave the same results as obtained with the fresh leukemic cells (data not shown). A parallel analysis performed on the erbB gene of the 81-103 leukemic cells (Table 1) gave similar results as above. except that these cells were heterozygous for the a and B erbB alleles and that the novel erbB fragment, which hybrid- ized weakly with the first exon probe but strongly with the erbA + B and erbB 3’ probes (Fig. 58 plus C). had a difi'erent size (3.4 kb) than in leukemia 81102. In conclusion. the above results show that both leukemic- cell DNAs contain 5' and 3' v-erbB sequences in a fragment of similar size as in v-erbB. suggesting that erbB is present in these leukemias as a transduced sequence lacking introns. An analysis of leukemic-cell DNA from both leukemias after digestion with SacI gave results supporting this hypothesis (data not shown). We then studied whether the leukemic cells were poly- clonal. as a result of spread of a c-erbB-containing retrovi- rus. or clonal, as a consequence of reverse transcription and subsequent reintegration of c-erbB mRNA. Leukemic-cell DNA was digested with restriction enzymes that do not :A B k C pr180-o - :89:! h . .- .:g:}gp }70-76 pr?6\ gait-4.. h" \ 9'50“ a. ‘ . -. , \9955 “‘5” 45 a "5 ~30“ -30 -30 3327" ‘n a. - I. -14“ -14 ~14 1 2 3 4 5 1 2 3' 4 5 1 2 3 FIG. 2. RAV-l-induced erythroleukemias express erbB-related proteins. (A) Purified leukemic cells from chick 81-102 were labeled with [”Slmethionine, and extracts were immunoprecipitated with anti-erbA + 8 serum (17) (lane 1). the same serum preincubated with virus lysate (2) (lane 2). anti-erbB serum (16) (lane 3). or anti-gag-erbA serum without (lane 4) or with (lane 5) competing virus. The positions of erbB-related proteins (gp74, gp76. probably representing rough endoplasmic reticulum precursors) are indicated. Small numerals indicate molecular weight markers (x103), (B) Leukemic cells from chicks 81103 (lanes 1 and 2). i-IM29393 (lane 3). HM27235 (lane 4). and HM8641 (lane 5) were immunoprecipitated with anti-erbA +8 serum without (lane 1) or with (lanes 2 to 5) competing virus. (C) Leukemic cells from chick 81102 were cultivated for 14 days and then labeled and immunoprecipitated with anti-erbA + 8 serum without (lane 1) or with (lane 2) competing virus. The positions of erbB-related proteins (gp76) and of the AEV-ES4-encoded proteins (lane 3) p75""‘WM (p75) and gp74"” precursors (gp66. gp68) are indicated. 1132 BEUG ET AL. 201 .l. VIROL. B fauoz AEV-E84 A . I'D-coo» , L33 arm—r ”76:5 L ‘43:.- 92a a h—GQ 71 - - 68 p 69 2:: $9366 ‘p 62 ._45 355 3H V + 7_ + .. Met Glu ' FIG. 3. Characterization of erbB-related proteins from RAV- 1- induced erythroleukemia. (A) Cultivated leukemic cells from chick BH02 were labeled with [”S]methionine (”8 Met) or [’nglucosamine (’H Glu) and immunoprecipitated with erbB-specific antibodies. An apparently mature form (gp83) Of erbB-related protein was immunoprecipitated from the glucosamine- labeled cells. Numbers are descnbed in the legend to Fig. 2. (8) Cultivated 8H02 cells and AEV- ES4-transformed erythroblasts (AEV- E84) were labeled with [”S]methionine In the presence (+) or absence (— ) of tunicamycin (17) and immunoprecipitated with erbB-specific antibodies. Nonglycosylated erbB protein precursors of 71 kDa (p71) and 62 kDa (p62) were immunoprecipitated from the tunicamycin- -treated BH02 and AEV- E84 erythroblasts. respectively. 0 ', "Ti. ' L- . i "a .. Lot A... in. «H.211. FIG. 4. erbB-related proteins of RAV-I-induced leukemias are expressed at the cell surface. Purified leukemic cells from chick 8H02 were double labeled with anti-erbB senIm (erbB) plus antierythroblast serum (A; EblAg) or antierythrocyte serum (8; EryAg) (6) in indirect immunofluorescence as described earlier (3). Photographs of the same cells viewed with bright-field illumination (left panels). fluorescein isothyocyanate-conjugated fluorescence (erbB. middle panels). or rhodamine fluorescence (EblAg. Ery-Ag; right panels) are shown. Erythroblasts (Ebl) and late reticulocytes (LR) are marked with arrow 0 2 RAV-l-INDUCED ERYTHROLEUKEMIC CELLS 1133 VOL. 57. 1986 A B C D E 2 3 B a 2 3 B a 2 a 2 3 B a. 2 3 (3 Kb _. Kb 20 -’ ‘- CD _ 11.5- .-a 7.3 4,5- - ." 32. .- —.1- ;825 - -22 - 2.0— - a — -; - 1 3 1.3- —--- _1'_1 0.5- - -- .- 1“ exon erb A+B erbB 3' erbA+8 erbB 3' L‘ II I Kpnl Hind lll FIG. 5. Identification of transduced erbB sequences in two RAV-l-induced leukemias. DNA was purified from the in vitro-cultivated leukemias BH02 and BH03 and subjected to analysis by the Southern transfer technique (29a). The restriction patterns of the erb sequences in the leukemic cells were compared with those of normal chicken DNAs homozygous for either the a or 8 allele of erbB. The arrows indicate novel erbB-specific fragments. (A) The DNAs were cut with EcoRI and hybridized with a probe representative for the first exon of c-erbB homologous to v-erbB. as described in the text. (8 and C) The EcoRl-digested DNAs were hybridized with probes representative for v—erbA + B and the v-erbB 3'-specific probe, respectively. (D) DNA from 8H02 was digested with Apal and EcoRI and hybridized with the v-erbA + B probe. (E) The DNAs were cut with Kpnl or Hindlll and hybridized with the v-erbB 3‘-specific probe. The sizes of the major c-erbB-specific fragments are indicated to the left and right. cleave within v-erbB and that cut once (Kpnl) or twice (Hindlll) in helper virus DNA. NO discrete extra erbB fragments were detected in the DNA from either leukemia after digestion with Kpnl (Fig. 5E), as would have been expected if these leukemias had been of clonal origin. The polyclonal origin of the BH02 leukemic cells was confirmed by Hindlll digestion, which showed a smear of erbB- hybridizing fragments with sizes between 5 and 7.5 kb (Fig. 5E). With BH03 leukemic cells. however. a discrete frag— ment of 7 kb was seen after Hindlll digestion. suggesting that this erbB-transducing element contained two internal HindIlI sites. Last, the other 11 RAV-l-induced leukemias were ana- lyzed for the presence of transduced c-erbB sequences and for a possible correlation of this event to a particular type of erbB protein (Fig. 2). No such correlation could be found (Table 3), because proteins Of several sizes were produced both from leukemias exhibiting modified c-erbB alleles and from those containing transduced erbB sequences. Interest- ingly. one leukemia contained both an insertional activation and a transduction of erbB; consequently. these leukemic cells expressed erbB proteins of two different sizes (Fig. 2; Table 3). RAV-I-induced leukemias with transduced erbB sequences express multiple species of erbB RNA at elevated levels. Polyadenylated RNA isolated from fresh BH02 leukemic cells as well as from in vitro-cultivated BH02 and BH03 erythroblasts was subjected to Northern analysis with an erbA + B probe and an LTR probe. One major 6.0-kb RNA hybridizing with both erb and LTR probes was seen in the fresh BH02 leukemic cells (Fig. 6A. 20. After 14 days of in vitro culture. however. two additional erb- and LTR-positive RNAs of 7.8 and 5.1 kb were seen (Fig. 6A. 2c). As expected. both RNA samples contained 7.8-kb genomic and 2. 8— kb subgenomic RNAs of RAV- 1 virus. Similarly the cultivated BH03 cells contained several RNA species posi- tive with both erb and LTR probes (4. 5. 3. 6. and 3. 2 kb [Fig. 6A. 3c]). Similar results were obtained with a v- e-r-bB specific probe (data not shown). This suggests that in vitro culture led to the selection of subpopulations of leukemic cells that contain erbB-transducing elements of similar size but have different modes Of erbB transcription. Next. we compared the level of erbB RNA transcription in RAV-l leukemic cells with that in AEV-transformed erythroblasts and in normal chicken embryo cells. The level of erbB RNA in BH02 cells was about fivefold lower than in AEV-transformed erythroblasts, but still 100- to 200-fold higher than the levels of the 12- and 9—kb c-erbB mRNAs in chicken embryo cell RNA (Fig. 6B). To rule out the possibility that high levels of c-erbB transcription are a common event in transformed erythro- blasts, the RNA of erythroblasts transformed by oncogenes other than erbB was examined. Erythroblasts transformed by 813 virus (3a) contained no c-erbB RNA (Fig. 6). Erythroblasts transformed by E26 virus (25) contained low erbB RNA levels similar to those found in normal chicken embryo RNA. whereas RNA from normal bone marrow cells (containing erythroid precursors similar to the RAV-l- induced leukemic cells) was negative for c-erbB RNA (data not shown). However. c-erbA RNAs were found in all three samples, at levels comparable to those found in normal chicken embryonic cells (not shown). 1134 BEUG ET AL. TABLE 3. c- -erbB activation and c-erbB protein expression in RAV- l-induced erythroleukemra Size of c-erbB extra 8:: Leukemia Mode of c-erbB activation‘ allele EcoRI protein affected fragments (kDa) (kb) HM27235 Proviral insertion a 3.14; 1.66 76 activation HM8640 Proviral insertion a 3.10; 1.70 76 activation HM8638 Proviral insertion a 3.32; 1.48 74 activation HM27442 Proviral insertion u 3.06; 1.74 74 activau 'On HM29393 Proviral insertion u 3.23; 1.57 74 activation HM27248 Proviral insertion 8 1.77; 0.83 76 activation HM27444 Proviral insertion 8 1.60; 1.00 74 activation I-IM8641 Two populations of cells; 8 3.12; 1.68 76 and 70 both insertion activation and transduced erbB HM8663 Transduced erbB NR” 76 HM8669 Transduced erbB NR 72 HM8660 Transduced erbB NR 72 81102 Transduced erbB NR 76 131-103 Transduced erbB NR 72 203 ‘ Analysis of leukemic-cell DNA was performed as described previoust (9. a . ‘ NR. Not relevant. RAV-l-induced leukemias 81102 and 81103 produce infec- tious erbB-containing retroviruses that do not transform fibro- blasts. To test whether the two leukemias containing transduced erbB sequences (8H02 and 81103) produce in- fectious, erbB-containing retroviruses, the leukemic cells were cocultivated with primary chicken embryo fibroblasts (see Materials and Methods). Analysis of these fibroblasts of erbB-specific RNA after cocultivation revealed that they expressed erbB RNAs of similar sizes as seen in the original erythroleukemic cells, although the relative abundance of the different RNA species seemed to be different in fibro- blasts and leukemic cells (Fig. 7A). This suggests that erbB-containing retroviruses had been transmitted to the fibroblasts in both cases. For the virus released from 81103 cells (referred to as ERR-2 virus below) this could be confirmed by protein analysis, since the infected fibroblasts expressed the expected 70- to 72-kDa erbB proteins at levels that were somewhat lower than in the leukemic cells (Fig. 78) but equivalent to levels seen in AEV-ES4-transformed fibroblasts (data not shown). In contrast, very little erbB- related protein could be immunoprecipitated from the fibro- blasts cocultivated with 81102 erythroblasts. To determine the proportion of virus-infected cells in the 81103 cocultivated culture, we tested the live fibroblasts for surface expression of erbB proteins with a fluorescent erbB- specific antiserum. Some 83% of the fibroblasts scored positive as compared with 86% in a control culture trans- formed with AEV-ES4 virus. The staining of the ER8-2- infected fibroblasts was stronger than that of the AEV- transformed control cells (data not shown), as was also observed with the corresponding erythroblasts (3). J. VIROL. 3., '21 9...», erb LTR erb LTR erb LTR 2° °"*’ emb $13 $13 l a. 0.1 1 1e 8 w ”9 U9 "9’ pg 4ug erbA+ ’ mus ' ____"__.1 FIG. 6. Leukemias 81102 and 81103 contain abundant novel RNAs that hybridize with erb and retroviral sequences. (A) RNA was isolated from the fresh (0 or cultivated (c) 8H02 and 8H03 leukemic cells and analde by the Northern transfer technique. Hybridization was done with either a v—erbA + 8 probe (erb) or with a labeled DNA fragment from AEV-containing LTR sequences (8) The abundance of erb-specific RNAs from AEV-ES4-transfor1'ned erythroblasts (AEV). 81103 leukemic cells. chicken embryonic cells (emb). and 813 virus-transfonned erythroblasts (813) was analyzed as described above. Two difi‘erent exposures of the 813 RNA are shown. Hybridizing fragment sizes are shown in kilobase pairs in both pan ls By using both immunoprecipitation and immunofluores- cence analysis, transmission of ER8-2 virus to fresh chicken embryo fibroblasts could also be demonstrated with filtered tissue culture supernatants from both leukemic cells and the ERB-Z-producing fibroblasts generated by cocultivation (data not shown). The ERB-Z-infected cells retained a normal morphology (Fig. 7C). despite the fact that essentially all the cells 2 0 4 VOL. 57, 1986 RAV-l-INDUCED ERYTHROLEUKEMIC CELLS 1135 Aemb BH02 131103 12-‘ .... -C “-1 . l a 30.! ‘ tar-w ebT 'fi'b’ ab] firs" ; I erbA-+8 B £1192 43693 $1769an . M . gp709rbB" -- —45 -30 ebl flb ebl fIb FIG. 7. Characterization of chicken embryo fibroblasts infected with erbB-containing virus from RAV-l-induced erythroleukemia. (A) Polyadenylated RNA from BH02 and 81103 leukemic erythroblasts (ebl) and from fibroblasts after cocultivation with the leukemic cells (fib) was analyzed by the Northern uansfer technique with an erbA + 8 probe. Similar erbB RNA species are expressed In both cell types which are clearly different from c-erbB RNA ( and 12 kb) or c-erbA RNA (3 and 4. 5 kb) present in normal chicken embryo RNA (emb). The BH03 RNA was subjected to autoradiography for 12 h and the BH02 RNA was autoradiographed for 48 h. Numbers on the left are in kilobases. (B) Leukemic erythroblasts (ebl) or fibroblasts afier cocultivation with leukemic cells (fib) were labeled with [”Slmethionine and immunoprecipi- tated with erb-specific antibody. The same 70- to 72- kDa erbB-related proteins (gp70""‘) are shown In 8H03 erythroblasts and In fibroblasts afier cocultivation. In contrast, little. if any. 76-kDa erbB protein (gp76"”)' Is detected' In fibroblasts cocultivated with BH02 erythroblasts Numbers on the right are in kilodaltons (C) Fibroblasts cocultivated with BH03 leukemic cells (ERB- 2) or infected with RAV- 1 were assayed for their morphology by phase-contrast microscopy (top) or for expression of actin filament cables (middle) and fibronectin protein network (bottom) by double~label immunofluorescence as described earlier (23. 27). 1136 BEUG ET AL. expressed the erbB protein at their cell surface. In addition, virus harvested from the ERB-Z virus-producing B1103 leukemic cells or from the ERB-Z-infected fibroblasts failed to induce foci in AEV focus assays. We therefore studied whether ER8-2-infected fibroblasts differed from normal cells in any other parameter of transformation (27). The cells expressed actin cables (88% positive) to a similar extent as helper virus-infected control cells (92% positive) (Fig. 7C). In addition, ER8-2 cells expressed a normal fibronectin protein network (Fig. 7C) and exhibited a low rate of hexose uptake (26,000 cpm/10° cells as compared with 33,000 cpm/lO" cells found with helper virus-infected control cells). AEV-ES4-transformed fibroblasts tested in parallel exhibited a typical. spindle-like morphology (25), no actin cables (13% of cells positive). a weak disordered fibronectin protein network (27), and elevated hexose uptake (80.600 cpm/10" cells). These results suggest that the ER8-2 virus contains a transduced c-erbB oncogene that does not transform fibroblasts. DISCUSSION RAV-l-induced leukemic cells resemble hormone-indepen- dent, late erythroid progenitors. We demonstrated that puri- fied RAV-l-induced erythroleukemic cells exhibit a pheno- type distinct from those Of normal erythroid progenitors (CFU-E) and erythroblasts transformed by v-erbB. The leukemic cells. similar to v-erbB-transformed erythroblasts. were independent of exogeneous Epo for survival and dif- ferentiation, whereas normal CFU-E cells are hormone dependent for these processes. However, most of the leuke- mic cells differentiated into erythrocytes in a manner similar to normal CFU-E cells, while only a minority of these exhibited the sustained self-renewal characteristic of v-erbB- transformed erythroblasts. The above phenotype of RAV-l-induced erythroleukemic cells is in accord with the idea that an activated c-erbB gene is less eflicient in inducing erythroid precursors to self-renew in vitro than v-erbB. Alternatively, RAV-l-induced leukemic cells might mainly proliferate in the bone marrow, whereas progeny cells in the peripheral blood would already be committed to terminal differentiation. It is also possible that in vitro cultivation selects for a minor population of leukemic cells with an increased self-renewal ability. c-erb-transducing retroviruses and their possible origin. Fung et al. (9) previously observed a processed or transduced form of the c-erbB gene found in an RAV-l- induced leukemia. The majority of the RAV-l-induced leu- kemias studied here contained the expected c-erbB alleles activated by helper virus integration. However, two of the leukemias analyzed in detail (B1102. BH03) harbored c-erbB- transducing retroviruses which could be transmitted to other «fills and were integrated in the host DNA at many different mics. suggesting an oligoclonal or polyclonal origin of these leukemias. Most likely, such c-erbB-transducing retrovi- ruses arose as a secondary event by acquisition of the activated c-erbB sequences by the helper virus (30). Once generated, such retroviruses will continuously infect and transform new precursors, leading to rapid replacement of the primary clonal leukemia by a virus-induced polyclonal cell population. This notion is supported by our observation that the leukemic cells from one leukemia (11M8641) con- tained both a modified c-erbB allele and transduced erb sequences. Owing to the small number of leukemias that can be grown into mass cultures. we do not know whether polyclonal 205 J. VIaOL. leukemias containing transduced erbB sequences difl‘er in their in vitro properties from those containing c-erbB alleles. Generation of such c-erbB-containing retroviruses, how- ever, does not seem to grossly change the structure of the respective c-erbB proteins, since both types of leukemias expressed proteins of similar sizes (Table 3). Mechanism of erbB-induced transformation of erythroid and fibroblastic cells. It was recently shown that v-erbB is homologous to part of the human EGF receptor (7, 32). RAV-l integration into the c-erbB/EGF receptor gene appar- ently leads to increased expression of a truncated receptor molecule which lacks the ligand-binding N-terminal part but retains the C-terminal intracellular kinase domain (9, 25a). This is supported by the finding that the erbB RNA from an RAV-l-induced leukemia contains sequences highly homol- ogous to the ultimate C terminus of the human EGF receptor (21a). In contrast, the v-erbB genes of both AEV-H and AEV-884 lack this sequence (36; personal communication). In this paper, we showed that 13 RAV-l-induced leuke- mias expressed erbB-related cell surface glycoproteins that were 5 to 10 kDa larger than gp74""" but which otherwise resembled the latter in their biosynthetic pathway. In addi- tion, we have recently been able to show that the erbB proteins of the leukemias 81102 and 81103 possess a tyrosine kinase activity similar to gp74"°”. However, they exhibited phosphotyrosine peptide maps similar to those of the chicken EGF receptor but distinct from those of gp74"""" (21; I. Lax, H. Beug, and J. Schlessinger, EMBO J.. in press). It is tempting to speculate that the truncated EGF receptor molecule expressed in RAV-l-induced erythroleukemic cells causes leukemia by constitutively producing a signal which mimics the signal produced by the Epo receptor in normal erythroid precursors when exposed to Epo (3c). This idea is in accord with our. observation that RAV-l-induced erythroleukemic cells mostly resemble Epo-independent but otherwise normal CF U-E cells. To substantiate this notion, however, the response to EGF of erythroid progenitors containing an activated, complete c-erbB/EGF receptor gene would have to be tested by in vitro studies. The c-erbB-containing retroviruses isolated from the 81103 erythroblasts did not transform fibroblasts. although they expressed high levels of erbB RNAs and proteins. These findings are in agreement with in vivo studies by others (30a) and suggest that the newly activated erbB genes had not yet acquired the ability to transform fibroblasts. This raises the possibility that an amino-truncated EGF receptor molecule with an intact C-terminal domain can render erythroid progenitors hormone independent by constitu- tively replacing the signal of the Epo receptor, but cannot induce a transformed phenotype in fibroblasts. Preliminary attempts to study this question were hampered by our inability to obtain more line 15 chickens. which appear to contain a dominant locus for susceptibility to c-erbB- transducing retroviruses (H. L. Robinson, submitted for publication). Although our studies on SPAFAS chickens showed that transformed erythroblast clones generated with ERB-Z virus supernatants from 81103 leukemic cells or infected fibroblasts expressed the 70- to 72-kDa erbB protein of the ER8-2 virus, we were unable to determine whether this protein was responsible for erythroblast transformation: all erythroblast clones expressed a second. erbB-transducing and erythroblast-transforming virus that was present in the original leukemia at levels too low to allow biochemical detection (ERB-ZA virus; 11. Beug et al.. manuscript in preparation). VOL. 57. 1986 In this context. it is noteworthy that the v-erbB-containing erthyroblastosis viruses described previously caused eryth- roblastosis but not sarcomas when first isolated from a diseased chicken. Only after prolonged in vivo passage was a sarcomagenic potential acquired (19, 31). Since none of these earlier virus isolates is available anymore, c-erbB- transducing retroviruses from ALV-induced leukemias might be useful to identify discrete steps by which the normal EGF receptor is converted into an oncogenic pro- tein. ACKNOWLEDGMENTS We thank Harriet Robinson and R. Kris for communication of results before publication, Mats Jansson for performing focus assays. Lars Frykberg for the LTR probe. Patricia Kahn and Thomas Graf for helpful discussions, Sigrid Grieser and Gabi Doederlein for excellent technical assistance, and Birgit Blanasch and Anne Walter for patient typing. LITERATURECITED 1. Beug, 11., G. Doederlein, C. Freudensteln, and T. Graf. 1982. Erythroblast cell lines transformed by a temperature sensitive mutant of avian erythroblastosis virus: a model system to study erythroid cell difl'erentiation. J. Cell. Physiol. 115:295-309. 2. Beug. 11., T. Graf, and M. Hayman. 1981. Production and characterization of antisera for the erb-portion of p75. the presumptive transforming protein of avian erythroblastosis vi- rus. Virology 111:201-210. 3. Beug, 11., and M. Hayman. 1984. Temperature sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation. Cell 36:963-972. 3a.8eug, 11., M. J. Hayman, T. Graf, 8. 11. Benedict, A. M. Wallbank, and P. K. Vogt. 1985. 813, a rapidly oncogenic replication—defective avian retrovirus. Virology 145:141-153. 3b.8eug, 11., P. Kahn, G. Doderlein, M. Hayman, and T. Graf. 1985. Characterization of hematopoietic cells transformed in vitro by AEV-11. an erb-containing avian erythroblastosis virus, p. 290-297. In R. Neth. M. Greaves, and R. Gallo (ed.). Modern trends in human leukemia VI. vol. 29. Springer-Vedag. Berlin. 3c.Beug, 11., P. Kahn, B. VennstrOm, M. Hayman, and T. Graf. 1985. How do retroviral oncogenes induce transformation in avian erythroid cells? Proc. R. Soc. 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Frykberg, L., 8. Palmieri, 11. Beug, T. Graf, M. Hayman, and 8. Vennstrbm. 1983. Transforming capacities of avian eryth- roblastosis virus mutants deleted in the erbA or erbB onco- genes. Cell 32:227-238. 9. Fang, T., W. G. Lewis, 11.4. Kung. nd L. 8. Crittenden. 1983. Activation of the cellular oncogene c-erbB by LTR insertion: molecular basis for induction of erythroblastosis by avian leukosis virus. Cell 33:357-368. 10. Gazaolo, L., C. Moscovici, M. G. Moscovici, and J. Samarut. 1979. Response of hemopoietic cells to avian acute leukemia viruses: effects on the difl’erentiation of target cells. Cell 16: 627-638. 11. Graf, T. 1973. Two types of target cells for transformation with 206 RAV-l-IN DUC ED ERYTHROLEUKEMIC CELLS 1137 avian myelocytomatosis viruses. Virology 54:398-413. 12. Graf, T., and 11. Beug. 1978. Avian leukemia viruses: interac- tion with their target cells in vitro and in viva. Biochim. Biophys. Acta 516:269—299. 13. Graf, T., and H. Beug. 1983. Role of the v-erbA and voerbB oncogenes of avian erythroblastosis virus in erythroid cell transformation. Cell 34:7-9. 14. Graf, T., 8. Royer-Pokora, G. E. Schubert, and 11. Beug. 1976. Evidence for the multiple oncogenic potential of cloned leuke- mia virus: in vitro and in vivo studies with avian erythroblastosis virus. Virology 71:423-433. 15. Graf, T., and D. Stehelin. 1982. Avian leukemia viruses: onco- genes and genome structure. Biochim. Biophys. Acta 651: 245-271. 16. Hayman, M., and 11. Beug. 1984. Identification of a form of the avian erythroblastosis virus erbB gene product at the cell surface. Nature (London) 309:460—462. 17. Hayman, M., G. Ramay, K. Savin, G. Kitchener, T. Graf, and 11. Beug. 1983. Identification and characterization of the avian erythroblastosis virus erbB gene product as a membrane glyco- protein. Cell 32:579-588. 18. Hayward, W.. 8. Neel, and S. Astrin. 1981. Activation of a cellular one gene by promoter insertion in ALV-induced lymphoid leukosis. Nature (London) 290:475—480. 19. Illhara, 11., 11. Yamamoto, K. Arai, and T. Shimizu. 1983. Avian erythroblastosis virus isolated from chick erythroblastosis in- duced by lymphatic leukemia virus subgroup A. J. Natl. Cancer Inst. 70:891-897. 20. Kahn, P., 8. Adkins, 11. Beug, and T. Graf. 1984. src and fps containing avian sarcoma viruses transform chicken erythroid cells. Proc. Natl. Acad. Sci. USA 81:7122—7126. 21. Kris, R.. I. Lax, W. Gullick, M. Waterfield. A. Ullrich, M. Frldkin, md J. Schlessinger. 1985. Antibodies against a syn- thetic peptide as a probe for the kinase activity of the avian EGF-receptor and v-erbB protein. Cell 40:619-625. 21a.Nilsen, T. W.. P. A. Maroney, R. E. Goodwin, F. M. Rottman, L. 8. Crittenden, M. A. Raihes, and 11.4. Kung. 1985. c-erbB activation in ALV-induced erythroblastosis: novel RNA proc- essing and promoter insertion result in expression of an amino- truncated EGF receptor. Cell 41:719-726. 22. Okazaki, W.. 11. G. Purchase, and L. 8. Crittenden. 1982. Pathogenicity of avian leukosis viruses. Avian Dis. 26:553-559. 23. Palmieri, 8., 11. Beug, and T. Graf. 1982. Isolation and charac- terization of four new temperature-sensitive mutants of avian erythroblastosis virus (AEV). Virology 123:293-311. 24. Payne, G.. M. Bkhop, and 11. Varmus. 1982. Multiple arrange- ments of viral DNA and an activated host oncogene (c-myc) in bursal lymphomas. Nature (London) 295:209-214. 25. Radke, K.. 11. Beug, S. Kornfeld, and T. Graf. 1982. Transfor- mation of both erythroid and myeloid cells by 826, an avian leukemia virus that contains the myb-gene. Cell 31:643-653. 25a.Raines, M. A., W. G. Wynne, L. 8. Crittenden, and 11.4. Kung. 1985. c-erbB activation in avian leukosis virus-induced erythroblastosis: clustered integration sites and the arrangement of provirus in the c-erbB alleles. Proc. Natl. Acad. Sci. USA 82:287-291. 26. Roussel, M., S. Saule, G. Lagrou, C. Rommeru, 11. Beug, T. Graf, and D. Stehelin. 1979. 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Molecular cloning of the avian erythroblastosis virus genome and recovery of oncogenic virus by transfection of chicken cells. J. Virol. 36:575-585. Weiss, R., N. Teich. 11. Varmus, and J. Como (ed.). 1983. RNA tumor viruses. Cold Spring Harbor Laboratory. Cold Spring Harbor. N.Y. Yamamoto, T., T. 111hara, N. Mltalaml, S. Kauai, T. Obol, and K. Toyoshlma. 1983. The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35:71-78. APPENDIX D: MATERIALS AND METHODS LI ST OF REFERENCES APPENDIX D: MATERIALS AND METHODS A. thckens Several inbred chicken lines were used in these studies and were provided by the Regional Poultry Research Laboratory, in East Lansing, Michigan (for description of inbred chicken lines see Stone, 1975). Most of the erythroblastosis samples were collected from used line 151 or 151 X 1514 chickens. Both SPAFAS and line 151 X 1514 chickens were used for sarcoma induction and testing of the oncogenic potential of leukemia extracts containing transduced erb B genes (Chapter 5). B. Virus stgcks RAV-l and RAV-2 strains of ALV, and the ES-4 strain of AEV were goriginally obtained from Dr. P.K. Vogt. ALV virus strains were further purified by limit dilution prior to inoculation. Viral extracts of tumorous tissues were made by preparing a 1:10 (w/vol) homogenate of tissue in 10% tryptose phosphate broth followed by passage through a 0.2 micron filter. In most of our experiments approximately 103 or 104 infectious units of virus was injected into the peritoneum of one-day old chicks. AEV inoculations were identical except chicks were inoculated at two weeks of age. The newly generated erb B transducing viruses were injected into either 6 day-old embryos or 1 day-old chicks. 208 209 C. o to n e t o a to i dev o e t ectio kem c samples Erythroleukemia development was monitored by collecting blood samples at regular intervals. Hematocrits (the percent of packed red blood cells per total blood volume) and differential counts of stained blood smears were used to diagnose erythroblastosis. Blood smears were stained with May-Grumwald and Giemsa as recommended by Lucas et al. (1961). The number of erythroblasts and polychromatic erythrocytes were scored relative to 100 white blood cells. Leukemic birds were sacrificed based on the number of erythroid precursors in the bloodstream. Blood samples were obtained via heart puncture and were fractionated on discontinuous Percoll gradients. Gradients consisted of 5 ml steps of 1.06, 1.07, 1.08, 1.09 g/ml Percoll (Pharmacia). 10 ml of blood was used per gradient and centifuged at 2200 rpm in a swinging bucket rotor (RB-4). Fractionated cells were aspirated off with a pasteur pipet and washed with phosphate-buffered saline (PBS). Bone marrow samples were collected by aspirating the long bones with Dulbecco's Modified Eagle's Medium (DMEM) or by quick-freezing immediately in liquid nitrogen. Particulate matter was removed by filtration through cheesecloth prior and cells were washed with PBS. Bone marrow and fractionated blood cells were frozen to —70°C in media containing DMEM 15% Fetal bovine serum (FBS, GIBCO) and 20% dimethyl sulfoxide (DMSO, Sigma). Other leukemic and non-leukemic tissues .210 samples were excised and quick-frozen in liquid nitrogen and stored at -7o°c. A11 birds that were sacrificed, died or were terminated were necropsied. Any gross lesions indicative of erythroblastosis or other neoplasms were recorded. Histological sections of tissues were made to verify macroscopic evidence of leukemia. Bone marrow smears were made from the aspirated cell suspensions. D. V us and c c ture QT-6 and line 0 chicken embryo fibroblasts (CEF) were obtained from the Regional Poultry Research Laboratory in East Lansing, Michigan and were maintained in DMEM-high glucose (GIBCO) supplemented with 5% fetal bovine serum and 1% chicken serum, 10 u/ml penicillin, 0.5 mg/ml streptomycin, and l ug/ml amphotericin B. Cells were infected with virus by seeding in 60 mm dishes, within 5 hours after seeding, media was aspirated off and replaced by 1.0 ml of virus containing media. Polybrene (Sigma) was added to 2 ug/ml to enhance infection. Cells were incubated for two to three hours before adding fresh media and passed for two weeks prior to virus collection. Virus stocks were collected 8 to 12 hours after media change and stored at 70°C. Prior to use cell debris was removed from the virus stocks by centrifugation at 10,000 rpm for 15 min. Virus production was verified by pelleting virus and extracting viral RNA. Cell debris was removed from the virus containing media and layered onto a cushion of 20% sucrose, 10 mM Tris pH 7.5, 0.1 M NaCl (1 211. to 4 ml depending on the amount of media used). Virus is pelleted by centrifuging two hours at 25,000 rpm, 4°C. Viral pellets are resuspended in 0.4 m1 of 0.1M NaCl, 10 mM Tris pH 8.0, 5 mM EDTA, 50ug/m1 proteinase K (Boerhinger-Manheim), 40 ug/ml tRNA (Sigma) and incubated for 30 minutes at 37°C. Viral RNA is extracted twice with equal volume of phenol/chloroform (1:1 mixture), precipitated with two volumes of absolute ethanol, resuspended in 15 ul of water. The viral RNA is denatured by adding 5 ul formaldehyde and heating 15 minutes at 65°C, and neutralized by adding 130 ul of 20X SSC. The RNA can be directly spotted onto nitrocellulose (50 ul/spot) or serially diluted (usually 1:4 with 16X SSC). The filter is baked and hybridized similar to other DNA and RNA blots (see below). Soft agar colonies from virus infected cells were selected by seeding various dilutions of cells in growth media containing 0.3% bacto-agar (soft agar). A 2.0 ml suspension of cells was plated onto a 4.0 m1 hard base containing growth media supplemented with 0.6% bacto- agar. After one week the cultures were fed with an additional 2.0 ml of soft agar twice a week. After two to three weeks, discrete colonies were removed with a drawn out pasteur pipet and seeded into 35 mm tissue culture dishes. Uninfected CEF were added as cells started to senesce . QT-6 cells were transfected with 20 ug of total plasmid DNA using the calcium phosphate technique (Graham et al., 1973, Vennstrom et a1., 1980). Approximately 15 ug of test DNA was cotransfected with 2 to 3 ug of pSV2-neo ( approximately 3:1 molar ratio; Southern et al., 1982). 212 Cells were split 1:4 18 hours prior to transfection and the media was changed 4 to 8 hours before addition of DNA. To precipitate DNA, 0.1 volumes of 1.25 M CaC12 was added to a 40 ug/ml DNA solution containing 20 mM HEPES pH 7.10, 150 mM NaCL, 0.7 mM NaPOa and incubated for 30 minutes. 0.5 m1 of CaPOa/DNA mix was added per 100 mm dish. DNA was removed and fluid media replaced after 18 hours. Growth media containing 1.0 mg/ml of G418 (GIBCO) was added 36 hours after transfection. After two weeks in selective media the remaining cells were trypsinized, replated, and grown up for further analysis. E. DNA analysis DNA was extracted from cell suspensions and tissues by homogenization, cell, lysis, pronase digestion, and phenol-chloroform extraction as previously described (Radinsky et a1., 1985). RNA was removed by digestion with 50 ug/ ml of DNase-free RNase (Sigma) and subsequent dialysis against 10 mM Tris pH 8.0, 1.0 mM EDTA. DNAs were digested according to the recommendations of the suppliers. Digested DNAs were separated by electrophoresis on agarose gels, transferred to nitrocellulose, and hybridized to nick translated DNA probes as described by Raines et a1., (1985). Filters were washed in 0.2x SSC and 0.1% SDS at 68°C and autoradiographed (2X SSC - 0.3 M sodium chloride, 0.03 M sodium citrate). F. RNA analysis Total RNA was extracted by pulverizing frozen tissue to a fine powder and lysing using the guanidinium/hot phenol method (Maniatis et 213 al., 1982). Poly (A)+ RNA was selected by oligo d(T)-cellulose chromatography (Maniatis et a1., 1982). RNA was selected two times, aliquoted and stored in ethanol at -70°C. Northern analysis was performed similar to that described by Radinsky et a1., (1987). Hybridization probes for both southern and northern analysis were synthesized by nick translation of gel purified fragments, using 32P- deoxyribonucleotide triphosphates (32P-dNTPs) as label. G. 81 analysis Appropriate restriction enzyme sites were radiolabelled at their 5' ends by dephosphorylating with calf intestinal phosphatase and treating with polynucleotide kinase in the presence of gamma-32P ATP as described in Maniatis et a1. (1982). Fragments labeled at their 3' ends were synthesized by treatment of Klenow in the presence of 32P- dNTPs. Gel purified fragments were used for most of the labeling reactions and increased the yields of probe considerably. 0.02 to 1.0 pmole of probe and either 1 ug of poly (A)+ RNA or 30 ug of total cellular RNA were used in each hybridization reaction. Optimal hybridization temperatures were determined empirically and gave minimal reannealing of the probe. Hybridizations were done overnight under standard conditions (see Maniatis et a1., 1982 or Berk, et a1., 1977). Unhybridized probe was digested with 200 units of 81 nuclease (Sigma) and the resistant fragments precipitated and run on a 5% polyacrylamide, 7 M urea gel. 214 H. M ecul n Genomic DNA libraries were synthesized according to Maniatis et al., 1982. Partial digested Eco R1 or Mbo I genomic DNA was size- selected on sucrose gradients and used to ligate to either EMBL-4 or EMBL-3 bacteriophage vectors (Lehrach et a1., 1982). Recombinant phage were packaged and screened using the appropriate hybridization probes. Recombinant clones were restriction mapped by single and double digestion and by southern blot analysis. A cDNA library was made using the Gubler and Hoffman (1983) method. A synthetic oligonucleotide was used to prime first strand cDNA synthesis by reverse transcriptase instead of oligo dT. Except for this one difference the procedure is identical to that reported. 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