Ivfi-rznvfli‘s'd- : - - , ~ ~ ' 1 ‘ _ fl. mutunmunmtumlumTfllilW M89 5 9- °! 3 3 1293 00625 9448 LEBMEY Michigan State Umlversty This is to certify that the dissertation entitled THE TUMOR-FORMING CAPACITY OF HUMAN FIBROBLASTS MALIGNANTLY TRANSFORMED _I_N_ VITRO AND I! VIVO presented by John Edward Dillberger has been accepted towards fulfillment of the requirements for PhD. degree in Pathology Q x k ‘(OA—WW‘ZL ’ Major professor Date November 10, 1989 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution THE TUMOR-FOthflNG CAPACITY OF HUMAN FIBROBLASTS MALIGNANTLY TRANSFORMED IN VYTRO AND IN VIVO By John Edward Dillberger A DISSERTATION ‘ Submitted to ‘ the Department of Pathology Michigan State University in partial fulfillment of the requirements for the degree of DocroR OF PHILOSOPHY 1989 ABSTRACT TIIE TUMOR-FORMING CAPACITY OF HUMAN FIBROBIASTS MALIGNANTLY TRANSFORMED IN VITRO AND IN VIVO By John Edward Dillberger To study the role of ras oncogenes in human tumorigenesis and tumor pathology, lexamined tumors produced in athymic nude mice by N-[fiq H—rits-v and K-w-transformed human cell strains, by human sarcoma-derived cell lines, and by a spontaneously- transformed human cell line. I also conducted experimental metastasis studies using ras- transformed cell strains. Injected subcutaneously, the gig-transformed cell strains made progressively-growmgv invasive sarcomas that were classified by microscopic features as myxoid sarcomas, spindle cell sarcomas (with and without whorls), round cell sarcomas, pleomorphic sarcomas (with mononuclear or multinuclear giant cells) and malignant mesenchymomaS- One H-w-transformed cell strain made S-lOO-positive sarcomas, and two N-gs-transformed cell strains made desmin-positive sarcomas. The transfected m oncogene and the recipient cell line both influenced the type of tumor produced by a given cell strain. The sarcoma-derived cell lines and spontaneously-transformed cell line also made sarcomas of several morphologic types, some of which were S-lOO- and desmin- positive. The mqransformed cell strains did not metastasize spontaneously from primary tumors in the subcutis but were capable of experimental metastasis via the bloodstream or by implantation Metastatic ability was influenced by the transfected gs oncogene and the recipient cell line. Copyright by John Edward Dillberger 1989 This work is dedicated to the memory of Mr. Brian Lanier‘ whose death in 1987 reminded me that mere is at stake in cancer research than the satisfaction of my own intellectual curiosity. ACKNOWLEDGEMENTS I am grateful to Dr. McCormick for his guidance and support throughout my studies, and to Dr. Maher, Dr. Fry, Lonnie Milam, Clay Spencer, Steve Dietrich, and others in the Carcinogenesis Laboratory for their willingness to share their time, ideas, and skills. Dr. Leader gave me constant support and encouragement in my studies and this dissertation reflects his commitment to expanding the traditional boundaries of pathology and to training the next century’s pathologists. Dr. Koestner, other members of the faculty, the staff, and students in the Pathology Department were all generous with their time and talents, particularly Dr. Louden. Doug Porter of Ingham Medical Center spent uncounted hours preparing immunoperoxidase-stained sections. Beyond these direct contributions I wish to express my gratitude to three others who had a profound impact on my studies. First, I thank Dr. Norman Altman for providing the solid grounding in comparative pathology that served me so well in this research. Second, lthank Dr. Walter L. Brown, whose unflagging curiosity and lifelong love of learning have been a constant source of inspiration. And finally, I thank my wife, CiGi, whose love, support, encouragement, enthusiasm; and strength have never wavered, and who always seemed to know which of them I needed most at any moment. TABLE OF CONTENTS 1. LIST OF TABLES .......................................... 11. LIST OF FIGURES HI. LIST OF ABBREVIATIONS .................................. IV. INTRODUCTION .......................................... V. BACKGROUND AND LITERATURE REVIEW A. Oncogenes and Carcinogenesis ............................ B. The @ Gene Family . . .- ................................. C. Theoretical Mechanisms of @ Activation .................... D. @ Oncogenes in Animal Tumors .......................... E. @ Oncogenes in Human Tumors .......................... F. I_n M Transformation by @ Oncogenes .................... G. @ Oncogenes and Tumor Pathology ........................ H. @ Oncogenes and Metastasis ............................. 1. References ............................................. 17 28 VI. THE TUMOR-FORMING CAPACITY OF RAS—TRANSFORMED HUMAN FIBROBLASTS A. Abstract .............................................. B. Introduction ........................................... C. Experimental Design . . . .i ................................. D. Results and Discussion ................................... E. Methods .............................................. Vi 45 49 50 65 F. References ............................................ 69 G Tables ............................................... 72 H. Figures .............................................. 78 VII. TUMORS FORMED BY HUMAN SARCOMA-DERIVED CELL LINES AND A SPONTANEOUSLY-TRANSFORMED FIBROBLAST LINE A. Abstract ............................................. 94 B. Introduction .......................................... 96 C. Materials and Methods ................................... 97 D. Results ............................................... 101 E. Discussion ............................................ 106 F. References ............................................ 111 G. Tables ............................................... 115 H. Figures ............................................... 119 VIII. EXPERIMENTAL METASTASIS OF BAS—TRANSFORMED HUMAN FIBROBLASTS A. Abstract ............................................. 137 B. Introduction ........................................... 138 C. Experimental Design ..................................... 140 D. Results and Discussion ................................... 141 E. Methods .............................................. 150 F. References ............................................ 153 G. Tables ............................................... 156 H. Figures ............................................... 162 IX. APPENDIX: ATTEMPTS TO CAUSE HUMAN FIBROBLASTS TO ACQUIRE AN INFINITE LIFE SPAN BY TREATMENT WITH ETHYLNITROSOUREA OR 5-AZACYTIDINE A. Background and Literature Review ......................... 167 1. Carcinogenesis, Transformation, and Infinite Life Span . . .' ................................. 167 2. Control of Cell Life Span ............................ 171 3. Immortalization of Nonhuman Cells .................... 173 4. Immortalization of Human Cells ...................... 174 B. Introduction ........................................... 178 C. Materials and Methods .................................. 182 D. Results ............................................... 184 E. Discussion ........................................... 188 F. Summary ............................................. 191 G. References ............................................ 193 viii I k Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 1. Table 2. Table 3. Table 4. Table 1. Table 2. Table 3. LIST OF TABLES Origin of rig-transformed Human Cell Strains .................. 72 Tumors Produced by K-w-transformed MSU-1.1 Human Fibroblasts ................................... 73 Tumors Produced by N-fl-transformed MSU-1.1 Human Fibroblasts .................................. 74 Immunoperoxidase Staining of Tumors Produced by Nags-transformed MSU-1.1 Human Fibroblasts ............. 75 Tumors Produced by H-fi-transformed Human Fibroblasts ........................................ 76 Immunoperoxidase Staining of Tumors Produced by H-w-transformed Human Fibroblasts .................. 77 Origin of Sarcoma-derived and Spontaneously- transformed Cell Lines ................................ 115 Tumors Produced by Human Sarcoma-derived Cells or Spontaneously-transformed Human Fibroblasts .............. 116 Microscopic Features of Tumors Produced by Human Sarcoma-derived Cells or Spontaneously-transformed Human Fibroblasts .................................. 117 Immunoperoxidase Staining of Tumors Produced by Human Sarcoma-derived Cells or Spontaneously- transformed Human Fibroblasts ......................... 118 Origin of Cell Lines and Strains ............................. 156 Experimental Metastasis of ras-transformed MSU-1.1 Cells, Spontaneously-transformed MSU—1.1 Cells, and Sarcoma-derived Cells ................................ 157 Experimental Metastasis of H-w-transformed Human Fibroblasts ................................... 158 Table 4. Table 5. Table 6. Table 1. Table 2. Table 3. Table 4. Distribution of Metastatic Tumors Produced by w-transformed Cell Strains, a Spontaneously- transformed Cell Line, and a Sarcoma-derived Cell Line ...... 159 Distribution of Tumors Produced by Intravenous or Intracardial Injection of N-w-transformed MSU-1.1 Cell Strains .......... 160 Tumor Formation by N-r_a_s-transformed MSU-1.1 Cell Strains Injected Intramuscularly or Subcutaneously ........... 161 Contrast Between Proposed Mechanisms Limiting Cell Life Span ..................................... 172 Growth of Untreated Fibroblasts in Low Calcium Agar ........... 184 Growth of ENU- and S-AzaC-treated LGl Fibroblasts in Low Calcium Agar ................................ 186 Growth of ENU- and 5-AzaC-treated SL88 Fibroblasts in Low Calcium Agar ................................ 186 LIST OF FIGURES Figure 1. Sarcomas stained for vimentin intermediate filaments .......................................... Figure 2. Myxoid sarcoma produced by K-gs—transformed MSU-1.1 fibroblasts (cell strain K-fl-la) .................. Figure 3. Spindle cell sarcoma produced by K—fl-transformed MSU-1.1 fibroblasts (cell strain K-fl-Zb) .................. Figure 4. Spindle cell sarcoma produced by N-w-transformed MSU-1.1 fibroblasts (cell strain N-fl-8) ................... Figure 5. Spindle cell sarcoma produced by N-w-transformed MSU-1.1 fibroblasts (cell strain N-rg-3) .................. Figure 6. Round cell sarcoma produced by N-fl-transformed MSU-1.1 fibroblasts (cell strain N-fl-Z) .................. Figure 7. Round cell sarcoma produced by N-w-transformed MSU-1.1 fibroblasts (cell strain N-w-2) .................. Figure 8. Pleomorphic sarcoma with multinuclear giant cells produced by N-w-transformed MSU-1.1 fibroblasts (cell strain N-[ifi-3) .................................. Figure 9. Malignant mesenchymoma produced by N-fl-transformed MSU-1.1 fibroblasts (cell strain N-w-3) .................. Figure 10. Sarcomas produced by N-w-transformed fibroblasts stained for desmin intermediate filaments ................. Figure 11. Spindle cell sarcoma produced by H-fl-transformed MSU-1.1 fibroblasts (cell strain H-w-3) ................... Figure 12. Sarcoma produced by H-Qs-transformed fibroblasts stained for S-lOO ................................... Figure 13. Spindle cell sarcoma produced by H-ras—transformed MSU-1.1 fibroblasts (cell strain H-ras-2) ................... 79 80 88 9O Figure 14. Pleomorphic sarcoma with mononuclear giant cells produced by H-is-transformed KMST-6 fibroblasts .......... 91 Figure 15. Pleomorphic sarcoma with mononuclear giant cells produced by H-fl-transformed GM637 fibroblasts ........... 92 Figure 1. Sarcoma produced by SHAC cells .......................... 119 Figure 2. Sarcoma produced by SHAC cells .......................... 120 Figure 3. Sarcoma produced by SHAC cells stained by the immunoperoxidase method for desmin .................... 121 Figure 4. Sarcoma produced by HT1080 cells ......................... 122 Figure 5. Sarcoma produced by HT1080 cells ......................... 123 Figure 6. Sarcoma produced by 8387 cells ............................ 124 Figure 7. Sarcoma produced by 8387 cells ............................ 125 Figure 8. Sarcoma produced by HUT-14 cells ......................... 126 Figure 9. Sarcoma produced by HUT-14 cells ......................... 127 Figure 10. Sarcoma formed by HUT-14 cells stained by the immunoperoxidase method for S-100 ..................... 128 Figure 11. Sarcoma produced by NCI cells ........................... 129 l Figure 12. Sarcoma produced by NCI cells ........................... 130 l Figure 13. Sarcoma produced by SW-684 cells ........................ 131 ‘ Figure 14. Sarcoma produced by SW-684 cells ........................ 132 Figure 15. Sarcoma produced by VIPzF-T cells ........................ 133 Figure 16. Sarcoma produced by VIPzF-T cells ........................ 134 Figure 1. Myxoid sarcoma produced by K-m-transformed MSU-1.1 cells (cell strain K-m-mT) injected into the peritoneal cavity ..................................... 162 Figure 2. Spindle cell sarcoma produced by K-w-transformed MSU-1.1 cells (cell strain K-w-laT) injected into the tail vein ..................................... 163 Figure 3. Spindle cell sarcoma in lungs produced by N-m-transformed MSU-1.1 cells (cell strain N-m-3T) injected into the tail vein ....................... 164 I; -m_ Figure 4. Spindle cell sarcoma produced by N-w-transformed MSU—1.1 cells (cell strain N-E-3T) injected into the peritoneal cavity .................................. 165 Figure 1. Model of human fibroblast transformation .................... 170 xiii LIST OF ABBREVIATIONS BPDE = (i)-7B,8a-dihydroxy—9a,10a-epoxy-7,8,9,10— tetrahydrobenzo[a]pyrene BSA = bovine serum albumin DEN = diethylnitrosamine DMBA = dimethylbenzanthracene DNA = deoxyribonucleic acid EBV = Epstein-Barr virus EDTA = ethylenediaminetetraacetic acid ENU = ethylnitrosourea FCS = fetal calf serum GDP = guanosine diphosphate GTP = guanosine triphosphate MNNG = N—methyl-N’-nitro-N-nitrosoguanidine MNU = N-nitroso-N-methylurea SV40 = simian virus 40 4-NQO = 4-nitroquinoline 1-oxide 5-AzaC = S-azacytidine INTRODUCTION Over the past 200 years, pathologists have accumulated a vast body of information on human tumor pathology. Much of that information consists of correlations between the microscopic features of human tumors and the behavior of those tumors i_n yiv_o. These correlations allow pathologists to predict the behavior of a tumor (offer a prognosis) by its microscopic features (diagnosis). Recently, pathologists have begun to investigate the molecular basis for the microscopic features on which they rely (Cline, 1989). Techniques such as immunohistochemical stains, immunoprecipitation, and Western blotting have enabled investigators to detect and 'measure specific proteins within tumor cells, while Southern analysis, Northern analysis, and i_n sit_u hybridization have permitted them to detect specific genes and mRNA transcripts within cells. At the same time such techniques were becoming available to pathologists, i_n vitro studies on rodent cell transformation revealed that specific genes, called oncogenes, were involved in carcinogenesis (Cooper, 1982; Weinberg, 1982; Varmus, 1984; Bishop, 1985 and 1987; Nishimura and Sekiya, 1987). Oncogenes turned out to be altered or inappropriately expressed forms of normal cellular genes. The first oncogene identified was a w oncogene from a human bladder carcinoma. Using the techniques of molecular analysis, pathologists and other cancer researchers have analyzed many human tumors for oncogenes. The oncogenes they have found most often belong to the [g family (Shih and Weeks, 1984; Barbacid, 1987). The widespread occurrence of @ oncogenes in human tumors raises the question, is there a relationship between @ oncogenes and the microscopic features of human 2 tumors? Most studies in which rodent cells have been transformed by @ transfection or infection with a w-containing retrovirus have not addressed the question because they have focused on the role of as in the transformation process itself, and the investigators have viewed tumor formation by the w-transformed cells as an endpoint. The few investigators who have examined tumors produced by gs—transformed rodent cells seldom have attempted to correlate the histopathologic features of the tumors with the method of transformation. Attempts to do so have been complicated by the relatively high rate of spontaneous transformation in many rodent cell systems. An experimental system for investigating the role of @ oncogenes in human tumor pathology recently became available in the Carcinogenesis Laboratory at Michigan State University with the successful i_n vitro transformation of human fibroblasts by transfected ra_s oncogenes (McCormick et al., 1989). A series of tumorigenic gs—transformed human cell strains, created by transfection of infinite life span human fibroblasts with an H-@ (Hurlin Lal, 1989), N-@ (Wilson et_al., 1989b), or K-as (Fry gal, 1989) oncogene, was on hand when Ijoined the laboratory in 1986. The studies described in this dissertation utilitzed those w-transformed cell strains, as well as, two spontaneously-transformed cell strains (L551-3T and VIP:F-T), and a series of sarcoma-derived cell lines. This dissertation begins by reviewing current concepts of carcinogenesis and the role of oncogenes, with a detailed discussion of @ oncogenes emphasizing the relationship between [as oncogenes and human tumor pathology. The studies I conducted on the tumor-forming capacity of various cell lines and strains are described in the next three sections, which are prepared as manuscripts to be submitted for publication in Laboratog Investigation (Chapters VI and VIII) and The Journal of the National Cancer Institute (Chapter VII). These chapters are organized in formats that comply with the requirements of those journals. The Appendix 3 describes studies in which I attempted unsuccessfully to induce human fibroblasts to acquire an infinite life span i_n vitro by treating them with ENU or 5-AzaC. BACKGROUND AND LITERATURE REVIEW l‘ and C... ' Most evidence to date indicates that carcinogenesis is a multi-step process. This conclusion is supported by epidemiologic studies ofhuman cancer (Doll, 1978), experimental studies on tumor induction in animals (Boutwell, 1974; Farber and Sarma, 1987), and i_n m studies on cell transformation (Barrett and Ts’o, 1978; Thomassen e_t 11., 1985). Decades of research also suggest that much human cancer is the result of environmental mutagens or carcinogens, both natural and synthetic, to which all of us are exposed in the food we eat, the water we drink, or the air we breathe (Ames, 1979 and 1983; D011 and Peto, 1981; Committee on Diet, Nutrition, and Cancer, 1982; Abelson, 1983; Rogers and Longnecker, 1988). These agents have in common an ability to interact directly with DNA, or to become metabolically activated into substances that can interact with DNA. Presumably, human cancer arises as a consequence of the multiple mutation-like events produced by such interactions, coupled with transient, non-heritable epigenetic events (Cairns, 1981; Weinstein e_t a_l., 1984; Weinstein e_t a_l., 1985; Klein and Klein, 1985). But ifmutation-like events are involved in carcinogenesis, what are the specific targets for the DNA-damaging agents that cause them? In other words, which of the tens of thousands of human genes, if damaged, could move a cell along the path toward malignancy? The answer began to emerge a decade ago when Weinberg and his co-workers reported the development of an i_n vitro system for screening DNA from tumor cells for potential cancer-causing genes (Shih e_t a_l., 1979). Their system, which involved transfection of DNA into NIH 3T3 mouse fibroblasts, led to the identification of the first transforming gene, or oncogene. The oncogene they identified was a mutated H-@ gene from the human bladder carcinoma cell line T24 (Duesberg, 1985). Many more dominant-acting transforming genes have been identified during the ensuing ten years (reviewed in Cooper, 1982; Weinberg, 1982; Varmus, 1984; Bishop, 1985 and 1987; Nishimura and Sekiya, 1987). Oncogenes have been found not only in the DNA of spontaneous human tumors, but also in the DNA of tumors induced in animals by chemicals or radiation and in the genomes of acutely-transforming retroviruses. Molecular analysis reveals that oncogenes are closely related in sequence to normal cellular genes called proto-oncogenes; oncogenes are, in effect, mutant alleles of wild—type proto-oncogenes. Proto-oncogenes have been highly conserved during evolution, consistent with the central role they appear to play in regulating basic processes such as cell division, growth, and differentiation. Such a role also makes proto-oncogenes attractive candidates for targets of environmental carcinogens. The alteration of a proto-oncogene so that it acquires transforming ability is termed "activation", and oncogenes are simply "activated" prom-oncogenes. (Some researchers prefer to call the normal cellular gene an oncogene and the transforming allele an "activated oncogene".) Proto-oncogenes become activated as a result of changes in DNA that alter their expression or the structure of the protein they produce. At the molecular level, DNA damage may lead to point mutations, large deletions, duplications, or rearrangements in a proto-oncogene that in turn cause increased gene expression, inappropriate or unregulated gene expression, expression of a mutant protein product, or a combination of these effects. For example, @ genes normally become activated by point mutations at a few key sites in their protein coding sequence (Shih and Weeks, 1984; Barbacid, 1987), while w genes most often become activated by gene amplification, chromosomal rearrangements, or proviral insertions that increase and/or deregulate my; expression (Kelly and Siebenlist, 1985', Modjtahedi e_t a_l., 1985; Kelly, 1986; Yokota e_t a_l., 1988; Tsuda e; i1.,1988). In the last few years researchers have discovered other genes involved in carcinogenesis whose normal expression prevents or inhibits transformation (Noda _t _l., 1989). These genes have been dubbed anti-oncogenes or tumor suppressing genes (reviewed in Barbacid, 1987; Friend et fl, 1988). Like oncogenes, tumor-suppressing genes are dominant-acting and can be targets for DNA-damaging agents; however, it is damage that mactivates tumor-suppressing genes which contributes to carcinogenesis. As might be inferred, individuals who are heterozygous for a tumor-suppressing gene as a result of inheritance appear to be at increased risk of cancer. For example, one copy of the tumor suppressing gene Rb-l appears to. be inactivated by hereditary deletion in people predisposed to develop familial retinoblastoma (Cavenee e_t a_l., 1983; Lee e_t a_l., 1987; Friend e_t a_l., 1988). If a somatic mutation inactivates the other copy of Rb-l, tumor is likely to develop. At first glance, the ability of single oncogenes to transform NIH 3T3 fibroblasts appears to contradict the idea of multistep carcinogenesis, but this is an illusion. NIH 3T3 fibroblasts have been passaged extensively i_n vitro, undergone gross genetic changes (they are tetraploid), and acquired phenotypic characteristics of tumors cells such as an infinite life span (Land e_t al, 1983b). The idea that a single oncogene can transform normal cells into cancer cells has been rejected by most researchers. Instead, the picture that has emerged is almost identical to that proposed 16 years ago by Comings (1973); namely, that the transformation of normal cells into malignant tumor cells is the result of the activation 0f multiple oncogenes coupled with the inactivation of one or more tumor suppressing genes, and that the process is influenced by epigenetic factors as well (Land e_t rd, 1983a; Duesberg, 1985; Klein and Klein, 1985 and 1986; Weinstein e_t a_l., 1985; Van Den Hooff, 1986; Nowell, 1988; Friend e_t a_l., 1988; Seemayer and Cavanee, 1989). The ras Gene Family Of the several dozen different oncogenes identified to date, one family of genes shows up more often than any other: the @ genes (for reviews, see Shih and Weeks, 1984 and Barbacid, 1987). The first oncogene identified a decade ago was an H-rg oncogene from a human bladder carcinoma cell line, and @ oncogenes subsequently have been found in a wide variety of spontaneous and induced tumors of many species. The human §a_s gene family consists of three genes (H-fl-l, K-lfi-Z, and N-@) and two pseudogenes (Hyg- Zand K—E-l) (Chang et al., 1982b; Hall et al., 1983; McGrath et al., 1983). These genes are located on different chromosomes: H-@ is on chromosome 11, K-@ is on chromosome 12, and N-@ is on chromosome 1. Certain gs genes have been captured by acutely-transforming retroviruses: H-@ and K-ras are the transforming genes of Harvey and Kirsten murine sarcoma viruses, respectively (Ellis et al., 1981; Tsuchida et al., 1982), and this gene family takes its name from these viruses (@ is an acronym for rat sarcoma virus). The three @ genes share a similar structure of 4 exons that code for a 21 kD protein of 189 amino acids (p21ras) (Taparowsky et al., 1983). K-Lrg has two fourth exons and so can code for two distinct p21s (McGrath et_al., 1983). K-@ also has a fifth exon (exon 0) located upstream from the 4 exons that code for the p21 (McGrath 9141., 1983). A similar upstream exon (exon -1) has recently been found in H-@ (Cichutek and Duesberg, 1986). These exons are transcribed and form part of the processed @ mRNA, bUt they are not translated; their function is unknown, but they may be involved in regulating translation of is mRNA. ' This suggests that changes within these non-coding exons might alter the level of @ expression. The protein product of the @ genes has been the focus of several investigations. Translation of @ mRNA gives rise to a precursor polypeptide (pro-p21ras) that migrates to the inner surface of the plasma membrane, where it is processed, phosphorylated, and acquires palrnitic acid residues (Shih and Weeks, 1984). The specific cellular function of p21ras is unclear, but it has been found to bind GDP and GTP, to have GTPase activity, and to exhibit an autokinase activity (Shih et al., 1982; Shih and Weeks, 1984; Sweet et al., 1984; Lacal and Aaronson, 1986; Lacal et al., 1986). These characteristics, along with the location of the protein on the inner surface of the plasma membrane and its recently reported homology with the alpha subunit of the G-protein transducin (Lochrie et al., 1985), suggest that p21ras functions as a signal transducer, modulating the transfer of messages from receptors on the cell membrane to intracytoplasmic effectors (Gilman, 1984; Kahn and Graf, 1986). In this regard, the observation that ras genes activated by point mutations frequently have reduced GTPase activity suggests one mechanism by which mutation might alter function, leading to perturbed signal transduction (Finkel et al., 1984; Gibbs et al., 1984; Sweet et al., 1984). But the picture is not a simple one since mutations that reduce binding and hydrolysis of GTP do not always increase the transforming ability of [g oncogenes (Lacal et al., 1986) and may actually decrease their transforming ability (Walter et al., 1986). Theoretical Mr.' ' of ras Activation Observed differences between @ oncogenes and proto-oncogenes have led to two mechanistic theories of @ activation. The quantitative theory holds that increased expression of a normal ras proto-oncogene is sufficient to activate the gene’s transforming Potential. Such enhanced expression occurs in retroviruses by linkage of the w gene to a viral transcriptional enhancer, and might occur in tumor cells by gene amplification, by chromosomal rearrangements that link @ to a more active promoter or bring it in proximity to enhancers, or by mutations within non-coding exons or introns that alter mRNA translation (Bishop, 1987). Support for the quantitative theory comes from experiments in which E proto-oncogenes linked to viral promoters acquire transforming capability (DeFeo et al., 1981; Chang et al., 1982a; Pulciani et al., 1985; Ricketts and Levinson, 1988), although other studies fail to demonstrate this effect (Tabin and Weinberg, 1985). Cohen and Levinson (1988) recently demonstrated that a mutation within the fourth intron of the T24 H-fl oncogene, which increases expression tenfold, also increases the transforming ability of this gene, although the mutation is not sufficient in itself to activate the H-@ proto-oncogene. There is some i_n M support for the quantitative theory of @ activation as well. For example, enhanced expression ofgs occurs in tumors induced by retroviruses that carry transduced [fl oncogenes and in some chemically-induced tumors (Quintanilla e_t a_l., 1986). Increased amounts of H—@ mRNA have been detected in several types of human tumors (Tatosyan e_t a_l., 1985). Increased expression of H-ras is common in human breast carcinomas (Hand e_t a_l., 1984; Lundy e_t a_l., 1986; Thor Lab 1986), but alterations in gene sequence are rare (Kraus e_t 3,1984; Rochlitz e_t a_l., 1988). Ohuchi e_t a_l. (1987) found increased amounts of H-flS mRNA and p21 in human gastric adenocarcinomas compared to adjacent normal gastric mucosa, but no increased expression of N—ras or K-@. In a Similar study, Noguchi gt a_l (1986) found increased amounts of H-ras p21 in the parietal cells of normal gastric epithelium as well as in most gastric adenocarcinomas. Increased expression of H-m has been detected in prostatic carcinomas (Rjinders e_t a_l., 1985; Viola e_t a_l., 1986) and bladder carcinomas (Viola e_t a_l., 1985). In both organs, benign proliferative lesions and normal tissue do not have increased expression of H-ras, and a survey of bladder carcinomas found structural mutations in H-@ in only 2 of 23 tumors (Fujita e_t a_l., 1984). Increased amounts of H-ras and K-@ mRNA and ra_s p21 have been found in colon tumors (Thor e_t a_l., 1984; Hand e_t a_l., 1984; Spandidos and Kerr, 1984; Gallick, e_t _a_l., 1985) but @ p21 content was increased only in carcinomas and not in adenomas. The other theory of r_a_s activation is the qualitative theory, which holds that activation results from changes in proto-oncogene structure that alter the ras protein product. Such changes might be point mutations that cause single amino acid substitutions or frameshifts, or large deletions or rearrangements that cause large changes in the @ protein. Both types of qualitative changes have been found in various viral and cellular oncogenes, although so far only point mutations have been found in gag oncogenes. Structurally altered @ genes have been isolated from a wide range of human tumors (Tabin Lal, 1982; Reddy et al., 1982; Santos et al., 1983; Capon et al., 1983a and 1983b; Shimizu Lal, 1983; Fujita e_t a_l., 1984; Suarez gt a_l., 1987; Neri e_t a_l., 1988 and 1989; Bar-Eli _t g, 1989). Experimental support for their significance comes from numerous studies with w oncogenes that acquired transforming capability through point mutations induced i_n vitr O at codons 12 (Fasano et al., 1984; Seeburg et al., 1984; Spandidos and Wilkie, 1984), 13 (Fasano et al., 1984), 59 (Fasano et al., 1984), 61 (Taparowsky et al., 1983; Yuasa et al., 1983; Fasano et al., 1984; Der et al., 1986), 63 (Fasano et al., 1984), or 116 (Walter et al., 1986). Activation also has been produced by small deletions or insertions in the region of codon 12 (Chipperfield et al., 1985). These two mechanisms of @ activation are not mutually exclusive. In fact, both mechanisms operate in retrovirus-induced transformation (Tabin and Weinberg, 1985), and their additivity has been demonstrated i_n vitro (Spandidos and Wilkie, 1984; Der et al., 1986; Ricketts and Levinson, 1988). Nor are these two mechanisms necessarily caused by different agents; either type of activation can be produced by agents that damage DNA. For example, we now know that the T24 H-w oncogene owes its potent transforming ability not only to a point mutation in codon 12 that alters the structure of the p21, but also to a point mutation in the last intron that increases expression of the altered protein (Cohen and Levinson, 1988). ras Oncogenes in Animal Tumors No [fi oncogenes have been found in normal cells from cancer patients whose tumor cells contain such genes (Cline, 1989), so the activation of [Q genes appears to be the result of somatic mutation. Activated @ genes have been found not only in a wide range of human cancers, but also in spontaneous tumors and tumors induced by chemical carcinogens and radiation in experimental animals (for lists, see Slamon et al., 1984; Vousden and Marshall, 1984; Nishimura and Sekiya, 1987). Leibovitch e_t a_l. (1986) studied expression of all 3 @ genes in 3 related lines of rat myoblasts and found that expression of K-@ positively correlates with malignant behavior. Not only is K-@ mRNA content increased in the most malignant cells, but they contain a new K-w transcript that is absent from the non—tumorigenic parent cells. Study of &s oncogenes from induced tumors has revealed some of the molecular mechanisms by which environmental carcinogens may contribute to cancer development. Several studies suggest that activation of g is involved at an early stage in carcinogenesis. For instance, when Zarbl et a_l. (1985) gave female rats a single dose of MNU, mammary Carcinomas were readily induced, and more than 80% of these tumors carry a specific G- ->A transition in codon 12 of H-w. Guerrero gt a_l. (1984) found a similar G-->A transition leading to activation of K-r_a§ in tumor cells from mice with lymphomas induced by gamma radiation. K-w activation also accompanies induction of kidney mesenchymal tumors by methyl(methoxymethyl)nitrosamine (Sukumar et al., 1986) and induction of lung tumors by tetranitromethane (Stowers gt a_l., 1987). Almost all mouse skin tumors initiated with DMBA have a specific A-->T transversion in codon 61 of H-gs, and this change is frequently found even in premalignant papillomas (Balmain and Pragnell, 1983; Bizub et al., 1986; Quintanilla et al., 1986). The frequency of this mutation varies with the initiating agent used rather than the promoter, suggesting that mutation occurs at the time of initiation. There is also an increase in H-ra_s expression at the mRNA level in papillomas compared to surrounding normal epidermis (Balmain gt a_l., 1984). Sinha e_t a_l. (1988) found N—fl oncogenes in rat liver tumors induced by aflatoxin, as well as in rat hepatocyte cell lines transformed in vitro by aflatoxin treatment. H-fl oncogenes are found in almost 100% of hepatocellular carcinomas in B6C3F1 mice, whether the tumors arise spontaneously (Reynolds et al., 1986) or are induced by carcinogens (Wiseman Lal, 1986; Maronpot _t l., 1987; Barbacid, 1987). Moreover, the same H-gs oncogene is found in 40% of adenomas, suggesting that activation is an early event in tumor development. In the case of spontaneous tumors, H-gfi is activated by random mutations in codon 61, but in tumors induced by hydroxy-acetylaminofluorine or vinyl chloride the activating lesion in codon 61 corresponds to that normally produced by the respective carcinogen; that is, C-->A transversions in the case of hydroxy-acetylaminofluorine and A- ->T transversions in the case of vinyl chloride. On the other hand, when Hsieh e_t a_l. (1987) examined liver tumors induced in Sprague-Dawley rats by initiation with DEN and promotion with phenobarbital they found no consistent change in expression of H-@, although they did not determine if there were structural mutations within H-r_a_§ or alterations in the other two @ genes. 12 ras Oncogenes in Human Tumors Experimental results implicating is activation as a frequent and early event in carcinogenesis are supported by at least some studies of spontaneous human tumors. For example, not only do more than one third of human colorectal tumors contain point- mutated K-w genes, but K-w oncogenes are found in a high percentage of adenomas as well as carcinomas, suggesting that mutation of m might occur early in colon carcinogenesis (Bos et al., 1987; Forrester e_t a_l., 1987). Almoguera e_t a_l. (1988) found point-mutated K- @ genes in a high percentage of pancreatic cancer, too. Spandidos and Kerr (1984) detected increased amounts of H-@ and K-@ mRNA in both benign colon polyps and colon carcinomas, and they concluded that increased @ expression was an early event in colon carcinogenesis. However, Thor e_t a_l. (1984) found normal amounts of p21H'ras in adenomas but increased amounts in carcinomas, and they concluded that increased gs expression was a late event in colon carcinogenesis. H-Lfi expression (p21 content) is increased in cells of dysplastic lesions of the urinary bladder as well as bladder carcinomas, suggesting that increased 5% expression may be an early event in the development of tumors in this location, too (Viola e_t a_l., 1985). On the other hand, Albino e_t _a_l. (1984) found a mutationally activated N-is oncogene in only one of five melanoma cell lines derived from separate metastases in a single patient, and they concluded that in this instance the mutation was not involved in the early stages of carcinogenesis. Neri e_t a_l. (1988) found that tumor cells from 6/33 patients with acute lymphoblastic leukemia contained mutationally-activated N-ga_s_ oncogenes. In five cases, activation is due to a G-->A transition in codon 12 or 13. However, in four patients only a fraction of the tumor cells had the N-@ oncogene, suggesting that N-ra_s mutation occurred late in tumor development (see Toksoz e_t a_l., 1989 for review). Neri e1 a_l. (1989) reached the same conclusion regarding N-gs activation in multiple myelomas. Gerosa e_t $1. (1989) studied w expression in human glial tumors. They found that N-Qs expression is increased in highly malignant glioblastomas and in cell lines derived from them, compared to less malignant gliomas or normal glial cells, implying that enhanced @ expression might be a late event in progression of brain tumors. Fan (1988) found a similar situation in a patient with metastatic prostate cancer, where only 25% of the primary tumor cells had increased p21""“"‘S content, but almost all the cells from a vertebral metastasis contained increased p21, suggesting that increased H-ras expression was a late event in this patient’s cancer development. Study of p21H'ras content in tumors from patients with metastatic breast cancer has revealed that w expression is increased in primary tumors from patients that have metastases compared to primary tumors from those who do not (Lundy e_t a_l., 1986), but that @ expression in metastatic tumors is variable and may be higher or lower than that of the primary tumor (Fromowitz e_t a_l., 1987). In none of the studies on @ expression was the possibility of point-mutations within fl investigated. The variability in @ expression or lack of is mutation in metastatic tumors does not rule out that gs oncogenes are involved in metastasis. Rather, gs oncogenes may assist cells in metastasizing but be unnecessary for tumor growth once a distant site is reached. Egan e_t a_l. (1987b) reached exactly this conclusion in experimental studies of metastasis by H-m-transformed NIH 3T3 fibroblasts. If @ expression is irrelevant to metastatic tumor growth, one might expect it to vary. After all, metastatic tumor cells are a clonal population, and as Heppner (1984) and Schirrmacher (1985) have pointed out, cloned tumor cells are often phenotypically unstable compared to the parent cell population, changing even from one generation to the next. In Vitro Transformation by ras Oncogenes Activated r_a_s genes were first identified by their ability to transform NIH 3T3 fibroblasts to focus formation. The initial oncogene so discovered, T24 H-E, derives its potent transforming ability from no less than three changes: a mutation in codon 12 that alters the structure of the protein (Reddy e_t_al., 1982) coupled with a mutation in the third intron (Cohen and Levinson, 1988) and an upstream enhancer region (Puga _et_al., 1985), both of which increase expression of. the altered p21. Since the discovery of T24 H-gs, investigators have found that NIH 3T3 fibroblasts can be transformed to various phenotypes by as genes from other tumors (Tainsky e; a_l., 1984) or from oncogenic retroviruses (Cichutek and Duesberg, 1986), @ genes mutated i_n \Ltrg (Santos e_t _a_l., 1983; Fasano e_t al., 1984; Chipperfield gt fl, 1985; Der e_t a_l., 1986; Lacal e_t a_l., 1986), and even normal @ proto-oncogenes expressed at a high level (Santos e_t a_l., 1983; Cichutek and Duesberg, 1986). The role of as in human carcinogenesis is difficult to ascertain from these experiments, however. Not only do NIH 3T3 fibroblasts seem exquisitely sensitive to transformation by @ oncogenes, but in most experiments there was also a low frequency of spontaneous transformation in untreated or sham-treated controls. Transfected Leg oncogenes and proto-oncogenes are capable of transforming primary rat cells (Land t al., 1983b; Cichutek and Duesberg, 1986) and immortal rat cell lines (Land e_t a_l., 1983b; Seeburg e_t al., 1984; Walter Qt a_l., 1986; Ricketts and Levinson, 1988; Cohen and Levinson, 1988), but since rat cells also frequently transform spontaneously in culture (Auersperg e_t a_l., 1987), the question remains what part as genes play in human cell transformation. Tumorigenic transformation of rodent cells by r_a_s oncogenes is well-documented. Infection of NIH 3T3 fibroblasts (Hamner Qt a_l., 1986), rat adrenocortical cells (Auersperg e_ta_l., 1981), and mouse bone tissue (Silbermann e_t a_l., 1987) with Kirsten murine sarcoma Virus makes cells tumorigenic. Roop e_t a_l. (1986) and Dotto e_t a_l. (1988) found that !____A infection of mouse keratinocytes with Harvey murine sarcoma virus had a similar effect. Mouse NIH 3T3 fibroblasts and rat cells transformed by transfection with as oncogenes are almost always tumorigenic (Land e_t a_l., 1983b; Spandidos and Wilkie, 1984; Jenkins _e_t 11., 1984; Bernstein and Weinberg, 1985; Thorgeirsson gt a_l., 1985; Bolscher e_t a_l., 1986; Pozzatti e_t al., 1986; Wallace _e_t a_l., 1986; Gao e_t a_l., 1987; Storer e_t a_l., 1988; Ananthaswamy 91 a_l., 1988; Ricketts and Levinson, 1988). So are mouse C3H 10T1/ 2 cells (Egan e_t _a_l., 1987a), mouse C127 cells (Muschel e_t a_l., 1985), and Chinese hamster lung fibroblasts (Spandidos and Wilkie, 1984; Muschel e_t ad, 1985; Wyllie gt a_l., 1987; Chadwick and Lagarde, 1988). Transfection of T24 H-LQ into mouse melanoma cell lines enhances their tumorigenicity (Price _e_t a_l., 1988). Established rodent cell lines may be especially susceptible to tumorigenic transformation by ;a_s because they have infinite life spans, and the ease with which primary mouse and rat fibroblasts transform to tumorigenicity following m transfection may reflect the high rate at which these cells spontaneously acquire infinite life spans. Attempts to transform human cells with [Q oncogenes have been successful only rarely. Sager gt a_l. (1983) found no evidence of transformation in human foreskin fibroblasts transfected with the H-;a_s oncogene from the human bladder carcinoma cell line B]. However, Sutherland e_t a_l. (1985) and Hurlin e_t a_l. (1987; Fry e_t a_l., 1988) successfully transformed human foreskin fibroblasts to focus formation and anchorage independence by transfection with T24 H-ffi, and Wilson _et a_l. (1989a) achieved similar results with the N- @ oncogene from the human leukemia cell line 8402. In each case, the transformed cells have finite life spans and are non-tumorigenic. Pater and Pater (1986) transformed human embryonic kidney cells immortalized by papilloma virus to anchorage independence by transfection with the E] H-gs oncogene, but apparently did not test the tumorigenicity of the transformed cells. 16 17 Evidence from various experimental systems supports the idea that gs oncogenes do play a role in tumorigenic transformation of human cells. For example, Tainsky e_t a_l. (1984) attributed enhancement of the tumorigenicity of the human teratocarcinoma cell line PA1 to a spontaneous mutation in N-m. Agnor e_t Q. (1988) saw similar results following transfection of human breast carcinoma cells with H-@. There are few reports of tumorigenic transformation of human cells by transfected ra_s oncogenes. Human bronchial epithelial cells transfected with v-H-is eventually developed into tumorigenic cells, but only after a long time in culture during which the cells acquired an infinite lifespan (Yoakum e_t al., 1985). Human fibroblasts (O’Brien e_t a_l., 1986) and keratinocytes (Rhim e_t al., 1985) immortalized by SV40 virus can be transformed into tumor cells by infecting them with Kirsten murine sarcoma virus. Seremetis e_t a_l. (1989) malignantly transformed human B- lymphocytes immortalized by EBV with a viral H-@ or tumor-derived N-w oncogene. Namba e_t a_l. (1986, 1988) transformed infinite life span, aneuploid KMST-6 human fibroblasts into tumorigenic cells by infecting them with Harvey murine sarcoma virus or transfecting them with a tumor-derived H-m oncogene. In our laboratory, diploid MSU- l.1 human fibroblasts, also having an infinite life span, were transformed into tumorigenic cells by transfection with v-K-ras (Fry e_t a_l., 1989), T24 H-fl (Hurlin e_t a_l., 1989), or a tumor-derived N-w oncogene (Wilson e_t a_l., 1989b). L‘s Oncogenes and Tumor Pathology The relationship between @ genes and pathologic features of tumors i_n vivo seldom has been explored. This is because tumor histology is not generally used as a measure of arclnogeniCity in experimental systems Since tumorigeniCity itself 15 taken as a direct ndlcatoi' Of malignancy. However, in clinical tumor pathology, histologic criteria form the hlef baSis for diagnostic and prognostic deCiSions. This reliance on histologic criteria rests 18 on extensive historical evidence for correlations between histologic features and the behavior of human cancer cells. In essence, the microscopic appearance of any tumor -- cytologic features, arrangement of cells, patterns of growth, antigen expression -- describes its biological behavior (Auersperg e_t a_l., 1987). Yet little is known about the molecular mechanisms that determine the microscopic features of tumors. In the case of epithelial cells, Warburton e_t a_l. (1986) reported that Higg- transformed mouse mammary epithelial cells produce "invasive carcinomas”, while untransfected cells make only "benign cystadenomas", but the histologic features of the tumors were not further described. Yoakum e_t a_l. (1985) transformed human bronchial epithelial cells by transfection with v-H-r_a_s and found that they produce poorly- differentiated malignant tumors whose epithelial nature is revealed only by the prominent intermediate-type intercellular junctions visible with the electron microscope and by positive staining for keratin. Roop e_t a_l. (1986) infected mouse keratinocytes with Harvey murine sarcoma virus and found that the cells produce benign papillomas in athymic nude mice. 0n the other hand, Dotto e_t a1. (1988) infected mouse keratinocytes with Harvey murine sarcoma virus and found that the cells produce carcinomas with various histologic patterns in Syngeneic mice. Tumors range from sheets of epithelioid cells to interwoven bands of Spindle cells, but all are keratin-positive and vimentin- and Factor VIII-negative. Rhim e_t a_l. (1985) infected human keratinocytes with Kirsten murine sarcoma virus and reported that the cells make invasive, rapidly progressive squamous cell carcinomas with characteristic keratin pearls. In the case of mesenchymal cells, Thorgeirsson e_t a_l. (1985) described tumors PTOduced by N—rg- or H-Lag-transformed NIH 3T3 fibroblasts as "invasive fibrosarcomas," although Greig e_t a_l. (1985) found that untransfected NIH 3T3 fibroblasts also produce mvas‘ve SPirldle cell sarcomas that are histologically indistinguishable from those produced 19 by @-transformed cells. The findings of Greig and his colleagues recall earlier experiments by Franks e_t a_l. (1971), who examined tumors produced by various mouse cell lines spontaneously transformed i_n m. The cell lines had been established from whole embryos and selected organs from mature mice of two strains. In all, the investigators studied 68 tumorigenic cell lines. They found that the tumors produced by the various cell lines were similar regardless of the organ or tissue from which the cells had been derived, and that all were invasive sarcomas. Franks and his coworkers identified three patterns of growth, which were always mixed together in the tumors: a fibrosarcomatous pattern that was often myxoid, a leiomyomatous pattern that sometimes contained multinuclear giant cells, and a pleomorphic epithelioid pattern that usually contained many multinuclear giant cells. The transition from one pattern to another was gradual instead of abrupt. All the tumors had scant collagen and elastin fibers and, with the exception of the myxoid pattern, mitoses were frequent. Egan e_t a_l. (1987a) found that transformation of mouse C3H 10T1/2 cells by T24 H-m results in morphologically transformed cells whose degree of morphologic transformation in culture is positively correlated with @ mRNA content, but they apparently did not examine the tumors microscopically. Land e_t a_l. (1983b) found that H-fl-transformed primary rat embryo fibroblasts PFOduced small, slow-growing "cartilaginous nodules," while H-w-transformed infinite life Span Rat-1 cells produced "rapidly-growing fibrosarcomas" but their report includes no histologic descriptions or photomicrographs of the tumors. Wyllie et 31. (1987) reported that rat fibroblasts transformed by T24 H-fl produce "aggressive fibrosarcomas" that grow raf’idly, infiltrate surrounding tissues, produce only sparse collagen, exhibit multifocal necrOSiS, and have a high mitotic rate. The same cells transformed by the normal H-m Prom-oncogene make similar tumors (although the tumors grow less rapidly), while u . . . ntransfected cells produce slow-growmg "low grade fibrosarcomas"w1th abundant collagen, 20 no necrosis, and few mitoses. Still, even these "low grade fibrosarcomas" did invade adjacent adipose tissue, and other investigators have found that untransfected or mock- transfected rat cells often produce invasive tumors (Van Roy e_t a_l., 1986; Gao e_t a_l., 1987). Storer e_t a_l. (1988) found that H-rfi-transformed rat embryo cells produce "highly malignant spindle cell tumors, compatible with fibrosarcoma," while the same cells transformed by both H-Las and c-M produce "malignant neoplasms with little apparent differentiation..., essentially giant cell tumors." Their photomicrograph of a giant cell tumor formed by Egg-transformed cells shows a tumor composed of large epithelioid cells with many mononuclear giant cells. The picture is strikingly similar to that of an "undifferentiated fibrosarcoma" produced by H-w-transformed KMST-6 human fibroblasts (Namba e_t a_l., 1986 and 1988). Wyllie e_t a_l. (1987) transformed Chinese hamster lung fibroblasts with I24 H-@ and found that the cells produce "aggressive fibrosarcomas" that invaded adjacent fat, skeletal muscle, and dermis. Auersperg _e_t a_l. (1981) transformed rat adrenal cortical cells by infection with Kirsten murine sarcoma virus. (Although these cells are derived from glandular tissue, they are mesenchymal in origin.) Of the 18 transformed cell lines these investigators tested for tUmorigenicity, 7 produced pleomorphic carcinomas, 6 produced sarcomas, 4 produced mixed tumors (carcinosarcomas) and-1 produced anaplastic tumors. All tumors grow fast and most invade surrounding tissue. In culture, the morphology of the cell lines varies: ”The lines grow as epithelioid cells and others as spindle-shaped cells, but the type of tumor l; Produced by any cell line is unrelated to its morphology in culture. In summary, except for the work of Auersperg e_t a_l. (1981) and Wyllie e_t a_l. (1987), no systematic study has been done to investigate the relationship between @ oncogenes and fume; Pathology. Recently, Fry e_t a_l. (1989), Hurlin 9t a_l. (1989), and Wilson e_t a_l. (1989b) s - . . . UCCeeded in transforming human fibroblasts With the K-m, H-@, and N-@ oncogenes, # 21 respectively. These Lag-transformed human fibroblasts are tumorigenic in athymic nude mice, and thus provide the first experimental system for studying the relationship between tumor pathology and specific @ oncogenes in human cells. ras Oncogenes and " ‘ ‘ The relationship between @ oncogenes and the twin hallmarks of malignancy, invasion and metastasis, has been the subject of numerous investigations in recent years (Mareel and Van Roy, 1986; Liotta, 1988), and was recently reviewed by Nicolson (1987). In a number of experimental systems, as oncogenes transform cells not just into tumorigenic cells, but into fully malignant tumor cells capable of invading surrounding tissue and metastasizing. However, the degree to which this transformation occurs depends upon the nature of the recipient cell and the level of expression of the @ gene. Many investigators have utilized mouse NIH 3T3 fibroblasts. After transfection with H-m, these cells form invasive tumors in athymic nude or histocompatible mice (Thorgeirsson e_t a_l., 1985; Bernstein and Weinberg, 1985; Ananthaswamy e_t a_l., 1988). Tumors can metastasize spontaneously in athymic nude mice (Thorgeirrson e_t a_l., 1985; Bernstein and Weinberg, 1985; Pozzatti e_t a_l., 1986; Egan e_t a_l., 1987b ; Ananthaswamy e_t 3.1-, 1988) but Wallace gt a_l. (1986) found that they don’t always do so, and Bernstein and Weinberg (1985) found that they never metastasize spontaneously in histocompatible mice. When injected intravenously, H-gig-transformed NIH 3T3 fibroblasts form experimental metastases in the lungs of athymic nude or histocompatible mice (Thorgeirsson e_t a_l., 1985; _l., MuSChel et a]. 1985; Bernstein and Weinberg, 1985; Wallace e_t a_l., 1986; Bradley _t —_9 1985; Pozzatti gt a_l, 1986; Egan e_t a_l., 1987a and 1987b; Garbisa gt a_l., 1987; Ananthaswamy 63411., 1988). Ananthaswamy e_t a_l. (1988) found a positive correlation between the level of r - . . . . . 3 exPFeSSIOn in NIH 3T3 fibroblasts and their ability to form spontaneous metastases, but 22 no correlation between @ expression and experimental metastasis. On the other hand, Egan gt a_l. (1987a) did find a positive correlation between @ expression and experimental metastasis in beige mice and histocompatible mice, and Greenberg e_t at. (1987) found a similar correlation in athymic nude mice. H-m-transformed NIH 3T3 fibroblasts invade chicken heart fragments (Bolscher e_t a_l., 1986) and human amnion (Thorgeirsson e_t a_l., 1985) 'm vitro and colonize the liver of chick embryos when injected into blood vessels of the chorioallantoic membrane in chicken eggs (Bondy e_t a_l., 1985; Hill gt a_l., 1988). Hill e_t a_l. (1988) found a positive correlation between the level of @ expression (p21 content) and the ability to colonize the chick embryo. NIH 3T3 fibroblasts transfected with N-r_a_s (Thorgeirsson e_t a_l., 1985) or infected with Kirsten murine sarcoma virus (Hamner e_t a_l., 1986; Bolscher e_t a_l., 1986) also form spontaneous and experimental metastases in athymic nude mice. Hamner and Starkey (1988) found that metastatic ability of cells infected with Kirsten murine sarcoma virus is negatively correlated with methylation of the v-K-w oncogene. The ability of r_ag oncogenes to convey metastatic ability on NIH 3T3 fibroblasts seems to depend on activating mutations, since transfection of the normal proto-oncogene does not make these cells metastatic (Muschel e_t al., 1985; Garbisa gt a_l., 1987). However, Bradley e_t a_l. (1986) rePorted that H-gs proto-oncogene could make NIH 3T3 fibroblasts metastatic, and even Untreated or sham-treated NIH 3T3 fibroblasts have proven capable of metastatic tumor f0”nation in some situations (Egan e_t a_l., 1987b). Transfection of @ oncogenes. into mouse cell lines other than NIH 3T3 fibroblasts Produces results that vary with the recipient cell line. Muschel e_t a_l. (1985) found that mouSe C127 cells transfected with v-H-w do not form experimental lung metastases in athymic nude mice, whereas Egan e_t a_l. (1987a) found that mouse C3H 10T1/2 cells t . . . rathected With ”[24 H-@ form experimental lung metastases and metastaSize 23 spontaneously, too. In ra_s-transformed C3H 10T1/2 cells there is a positive correlation between @ mRNA content and the frequency of metastasis. The relationship between r_zg oncogenes and metastasis has been studied in several mouse tumor cell lines. Collard _e_t a_l (1987) transfected non-invasive, non-metastatic BW5147 T-lymphoma cells with T24 H-@ and found that the cells then can invade hepatocyte cultures i_n m and form experimental lung metastases in syngeneic mice 'm m. Invasiveness i_n_ y_itr_o correlates positively with tag mRNA content. Warburton _t a_l. (1986) found that mouse mammary epithelial cells, which initially made benign tumors, produced invasive tumors following transfection with T24 H-w. Kerbel e_t a_l. (1987) saw an increase in the frequency of spontaneous metastasis by mouse mammary adenocarcinoma cells transfected with T24 H-@, and Eccles e_t a_l. (1985) saw an increase in the frequency of experimental lung metastasis, too. In contrast, Price gt a_l. (1988) found that mouse melanoma cells transfected with T24 H-r_a_s make more experimental lung metastases in athymic nude or syngeneic mice, but that there is no increase in the rate of spontaneous metastasis. Highly metastatic variants have arisen spontaneously in a mouse lymphoma cell line and a fibrosarcoma line. The metastatic lymphoma variant has an activated K-fl gene Which the parental cells lack (Vousden and Marshall, 1984; Vousden e_t a_l., 1986), but in the fibrosarcoma cells Alon e_t a_l. (1987) found a negative correlation between frequency of metastasis and content of K-r_a_§ mRNA and p21. Rat cells also have been frequent recipients of transfected @ genes. Several investigators found that the T24 H-QS gene converts primary rat cells into cells that Spontaneously metastasize and form experimental metastases in athymic nude mice (Muschel e_t a_l., 1985; Pozzatti e_t a_l., 1986; Garbisa gt a_l., 1987; Alvarez and DeClerck, 1988), and Wyllie e_t a_l. (1987) reported similar results with the rat fibroblast line 208F. However, Storer e_t g_l. (1988) were unable to produce experimental lung metastases with H- 24 fi-transfected primary rat cells, although they could do so with cells derived from subcutaneous tumors produced by gig-transfected cells or with cells transfected with both cm and H-@. One possible explanation for these findings is suggested by studies by Kawano e_t a_l. (1987) in which they found that the ability of various rat cell lines transformed with the v-@ oncogene to form spontaneous and experimental metastases depends on the manner of integration and level of transcription of the transfected fgs gene. Garbisa e_t a_l. (1987) and Pozzatti e_t a_l. (1986) each found that cotransfection with H-Qs and the adenovirus Ela gene inhibited metastasis compared to transfection with H-@ alone. Interpretation of transfection experiments in rat cells is difficult, however, in light of reports by Gao e_t a_l. (1987) and Van Roy e_t a_l. (1986) that untransfected primary rat cells can invade chicken heart muscle i_n vitro, make tumors in syngeneic rats and athymic nude mice, and form spontaneous and experimental metastases. A few rat tumor cell lines have been utilized as recipients of @ genes. Nicolson e_t a_l. (1988) transfected T24 H-LELE into a non-metastatic mammary adenocarcinoma cell clone, thereby converting the cells into ones that form spontaneous metastases in athymic nude mice. Similar results followed transfection of T24 H-fl into prostatic adenocarcinoma cells by Isaacs e_t a_l. (1988). Transfection of T24 H-gLs into Chinese hamster lung fibroblasts converts them into Cells that metastasize spontaneously and form experimental lung metastases in athymic nude mice (Spandidos and Wilkie, 1984; Muschel _e_t a_l., 1985; Wyllie e_t a_l., 1987; Chadwick and Lagarde, 1988), although Chadwick and Lagarde (1988) found that untransfected Chinese hamster cells do occasionally form experimental lung metastases as well. Few studies of gs genes and metastasis have involved non-rodent cells. Sauerbier 2t a_l. (1986) investigated the expression of gig genes in a frog renal carcinoma cell line that spontaneously metastasizes at 28°C but not at 18°C. They found that expression of K- 25 m (and several other proto-oncogenes) is increased at 28°C compared with 18°C, and thus K-gts expression correlates positively with metastatic behavior in these cells. Only one study has been reported that utilized human cells. Agnor e_t a_l. (1988) transfected mutant c-H- @ or v-H-m into human breast carcinoma cells and found that this had no effect on the frequency of spontaneous metastasis in athymic nude mice. What might be the molecular mechanisms by which gig oncogenes influence metastasis? Metastasis is not a phenotypic characteristic but a complex process that reflects a spectrum of phenotypes. To metastasize, tumor cells must detach from their neighbors, invade through a vessel wall, survive in the circulation, attach to a vessel wall, traverse a vessel wall again, and proliferate (Lagarde and Kerbel, 1985; Schirrmacher, 1985; Nowell, 1986; Sanchez gt a_l., 1986). There is evidence that @ activation endows cells with several characteristics that are relevant to this process. Detachment of tumor cells from their fellows appears to reflect changes in cell SUrface recognition molecules that decrease cell adhesiveness (Buckley, 1985). In particular, an increase in sialic acid residues in fucose-containing glycoproteins on the cell membrane decreases cell adhesiveness and enhances invasion of chick heart fragments i_n m and metastasis i_n \LVQ (Roos, 1984; Mareel e_t a_l., 1988). Increased sialylation of cell membrane glycoproteins accompanies infection of mouse MO cells with Kirsten murine sarcoma virus and transfection of NIH 3T3 fibroblasts with T24 H-L2_S (Bolscher e_t a_l., 1986). Once detached, tumor cells must invade tissue and penetrate blood vessel walls. The greatest barrier to their passage is theextracellular matrix, especially basement membranes. Transfection of @ oncogenes into tumor cells decreases fibronectin and collagen production (Warburton e_t a_l., 1986; Isaacs e_t a_l., 1988), which may be one mechanism by which as Oncogenes enhance invasion and metastasis. Probably more importantly, is transfection into mouse and rat cells leads to increased production by the cells of a number of proteases, 26 including collagenases, gelatinase, an'd Cathepsin L (Denhardt _e_t_ _a_l., 1987; Alvarez and DeClerck, 1988). In particular, Lg§ oncogenes stimulate cells to produce and secrete more Type IV collagenase which is specifically directed against the type of collagen that is the major component of basement membranes (Turpeenniemi-Hujanen e_t a_l., 1984; Thorgeirsson gt gl, 1985; Garbisa e_t gt, 1987). Liotta e_t a_l. (1980) demonstrated a positive correlation between metastatic ability and Type IV collagenase production. Once in the circulation, cells must survive host defenses, attach at a distant site, escape from a vessel, and proliferate. One of their biggest obstacles is the immune system, especially NK cells and macrophages. Johnson e_t a_l. (1985) and Trimble e_t a_l. (1986) reported that @ transfection makes cells more susceptible to killing by NK cells ig v_itm, while Thorgeirsson e_t a_l. (1985) and Greenberg gt a_l. (1987) found that @-transformed cells are not easier for NK cells or macrophages to kill. Studies regarding the effect of @ Oncogenes on the ability of cells to colonize and survive in a distant tissue also provide Conflicting results. Greenberg e_t a_l (1987) found that the ability of mouse C3H 10T1/2 cells injected intravenously to arrest in the lungs reflects their sensitivity to NK cells, and since m transfection does not make cells less sensitive to NK cells it also does not make cells more likely to colonize the lung. On the other hand, Hill e_t a_l. (1988) reported that @ transfection enhances the ability of cells injected intravenously to colonize the liver in chick embryos. Upon reaching a distant site, cells must proliferate if they are to form a tumor. Greenberg e_t a_l. 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Box 12137, Research Triangle Park, NC 27709 d Fred Hutchison Cancer Research Center, M616, 1124 Columbia Street, Seattle, WA 98104 e Ingham Medical Center, Lansing, MI 4891 f Carcinogenesis Laboratory, Michigan State University, East Lansing, MI 48824-1316 44 Send galley proofs to: Dr. J. E. Dillberger Merrell Dow Pharmaceuticals Inc. PO. Box 68470 Indianapolis, IN 46268-0470 Send requests for reprints to: Carcinogenesis Laboratory West Fee Hall Michigan State University East Lansing, MI 48824-1316 Running Title: Tumors by Raw-transformed Fibroblasts ABSTRACT Hurlin gt_al. (14) recently succeeded in malignantly transforming 3 infinite life span human fibroblast lines by transfection with an H-@ oncogene. Malignant transformation of one of these cell lines, MSU-1.1, also has been achieved by transfection with an N-@ (31) or a K-@ (9) oncogene. To find out how closely the experimental system mimicked ig Vifl carcinogenesis, tumors produced in athymic nude mice by these fi-transformed human fibroblasts were examined microscopically and compared to each other and to Spontaneous human soft-tissue sarcomas described in the literature. Comparisons were made of their growth rate, invasive or metastatic behavior, cytologic features, patterns of growth, and immunohistochemical staining reactions. All tumors were progressively- growing, invasive sarcomas, and most were vimentin-positive. Spontaneous metastasis from primary subcutaneous sarcomas was never observed. Sarcomas were classified by morphology as spindle cell sarcomas (with or without whorls), round cell sarcomas, myxoid Sarcomas, pleomorphic sarcomas (with mononuclear or multinuclear giant cells), and malignant mesenchymomas. Only K—gs-transformed fibroblasts made myxoid sarcomas. Only H-w—transformed fibroblasts made spindle cell sarcomas with a whorling pattern or Sarcomas that were S-lOO-positive. Only N-rgg—transformed fibroblasts made round cell Sarcomas, pleomorphic sarcomas with multinuclear giant cells, or sarcomas that were desmin-positive. These results suggest relationships between specific members of the @ gene family and various histopathologic phenotypic features used by pathologists to classify and assign prognoses to spontaneous human soft-tissue sarcomas. This model system for human cell transformation may prove valuable not only for investigating the molecular 45 46 mechanisms involved in carcinogenesis, but also for exploring the molecular basis behind mesenchymal cell differentiation and the histogenesis of soft tissue sarcomas. Key Words: H-m, K-flg, N-w, transformation, fibroblast, sarcoma INTRODUCTION In order to study human carcinogenesis McCormick and his colleagues have sought to develop an i_n vitro model system for the malignant transformation of human fibroblasts. Recently, they succeeded in malignantly transforming several infinite life span human fibroblast lines by transfection with @ oncogenes. The infinite life span cell lines were derived from cultures of diploid human fibroblasts that were treated repeatedly with cobalt- 60 gamma radiation (KMST-6) (21,22), infected with SV40 virus (GM637), or transfected with a v-mLc oncogene (MSU-1.1) (18). These cell lines are non-tumorigenic, but following transfection with a K-m (9), H-@ (14), or N-gg oncogene (31), clonally-derived strains of gs-transformed fibroblasts formed tumors in athymic nude mice. To find out how closely this i_n vitro transformation model mimicked i_n vivo carcinogenesis, ras-transformed fibroblasts in culture were compared to a spontaneously- transformed human fibroblast line and to cells derived from human sarcomas, some of which also contain ras oncogenes. I_n vitro, sarcoma-derived, spontaneously-transformed, and fi-transformed fibroblasts were morphologically transformed, formed foci on a cell monolayer, grew in an anchorage independent manner when suspended in soft agar, and had similar growth curves when cultured in medium that lacked exogenous growth factors (9,14,26,31). In the studies reported here, we investigated the tumor-forming capacity of m-transformed fibroblasts by injecting them subcutaneously into athymic nude mice and examining the tumors microscopically using histochemical and immunohistochemical stains. Specifically, we asked if the particular gg oncogene used to transform the cells influenced the microscopic features or behavior of the tumors they produced. To answer this question, We compared the cytologic features, patterns of growth, and immunohistochemical staining reactions of tumors produced by cell strains derived from a single infinite life span fibroblast 47 48 line (MSU-1.1) transformed by 3 different @ oncogenes, each in a different plasmid vector. We also asked if the nature of the infinite life span fibroblast line used as a recipient for the transfected @ oncogene influenced the type of tumor produced. To address this question, we compared tumors produced by cell strains derived from 3 different infinite life span fibroblast lines (MSU-1. 1, KMST-6, GM637) transformed by the same H-m oncogene. Our results indicate that the nature of the recipient fibroblast line and the specific @ oncogene used to tranform the cells did influence the type of tumor produced by gig- transformed human fibroblasts, although other factors such as the plasmid vector, site of integration, and host characteristics appeared to play a role as well. We expected the tumors to be fibrosarcomas, but many had features reminiscent of other human soft tissue sarcomas, including myxofibrosarcomas, malignant fibrous histiocytomas, neurofibrosarcomas, and myosarcomas. Current uncertainties in the histogenetic classification of human soft tissue sarcomas, and the lack of a specific histochemical or immunologic marker for fibroblastic tumors (discussed in 4,6,7,13,16,19) made it difficult to compare tumors.produced by gig-transformed fibroblasts to specific human sarcomas described in the literature. Rather than imply similarities in behavior between our experimental tumors and spontaneous human sarcomas by using names taken from surgical pathology (10), we chose instead to classify the experimental tumors by morphology as spindle cell sarcomas (with and without whorls), round cell sarcomas, myxoid Sarcomas, pleomorphic sarcomas (with mononuclear or multinuclear giant cells), and malignant mesenchymomas. EXPERIMENTAL DESIGN The w-transformed cell strains used in these studies were created by Fry L211. (9), Wilson L211. (31), and Hurlin mi. (14) and are listed in Table 1. Tumors were produced by injecting {gs-transformed cells subcutaneously into athymic nude mice. Tumor- bearing mice received complete necropsies that included microscopic examination of the tumor itself and major organs and regional lymph nodes for metastases. Samples of tumors were fixed in formalin and B-5, and examined microscopically using various histochemical and immunohistochemical stains. Because immunoperoxidase staining for a variety of antigens has proven useful in classifying soft tissue sarcomas, we screened the tumors produced by fl-transformed fibroblasts using antibodies directed against vimentin and desmin intermediate filaments, S-100 protein, alpha-l-antichymotrypsin, and factor VIII antigen. To investigate the role of the transfected @ oncogene on tumor pathology, tumors produced by various strains of ra_s-transformed MSU-1.1 fibroblasts were compared. To investigate the role of the recipient cell line on tumor pathology, tumors produced by Various H-w-transformed cell strains were compared. 49 RESULTS AND DISCUSSION General Observations With the exception of a single strain of K-gg-transformed fibroblasts the tumors produced by tag-transformed human fibroblasts were all judged malignant by their progressive rather than self-limited growth, invasive rather than expansive growth, central or multifocal necrosis, and cytologic features such as high cell-to-matrix or nucleus-to- Cytoplasm ratio, nuclear atypia, high mitotic index, and multiple large nucleoli. Except as otherwise noted, tumors were not encapsulated and did not compress surrounding tissues. Instead, tumors invaded adjacent adipose tissue and skeletal muscle as they grew. Individual adipocytes or skeletal muscle fibers could be found entrapped within most tumors (see below). Invasion also frequently occurred into the dermis with entrapment of adnexal structures. However, no tumor invaded bone, cartilage, or peripheral nerves, nor did any Inmor penetrate into the thoracic cavity. Spontaneous metastases were not found in any tumor-bearing mouse. That human fibroblasts could be malignantly transformed by transfection of a single activated oncogene does not imply that an activating mutation alone is sufficient for i_n m transformation, or that _ip M carcinogenesis is not a multistep process. On the contrary, in their experiments Fry L211. (9), Hurlin Lal. (14), and Wilson L31. (31) introduced gag Oncogenes into cells in constructions designed to cause constitutive high expression of the transfected gene. In fact, the transfected oncogene is not highly expressed in the K-@- transformed cell strains, although it is in the H-_ra_s- and N-gig-transformed cell strains (J. McCormick, personal communication). Thus, at least in the N-gg- and H-tgg-transformed cell strains, not only were the ras oncogenes making mutant p21s but they also were PTOdUCing large amounts of p21 in an unregulated manner in cells that presumably had 50 51 two copies of the corresponding normal gig proto-oncogene. In addition, the T24 H-gg oncogene has a point mutation in the third intron (3) that increases its transforming ability. To duplicate this process i_n M would require at least four steps: duplication of a gs gene, an activating point mutation in one copy, a third change leading to increased expression of the mutationally-activated gene, and a fourth change freeing expression from normal regulation. Most likely, whether a transformed cell has one or two normal gg alleles along with a mutationally-activated gig oncogene is unimportant, so duplication is unnecessary. Also, increased and unregulated @ expression may result from a single change, rather than requiring two separate events. Thus, i_n vitro transformation by m oncogenes might be duplicated i_n m by as few as two events. In addition, the cell lines into which @ oncogenes were transfected already had acquired an infinite life span in culture, and evidence from this laboratory (9,14,18,31) and several others ( 9,14,17,23,24,25,28,31) suggests that this is a requirement for malignant transformation. MSU-1.1 fibroblasts are also partially grth factor-independent, and this also may be important for full malignant transformation. Tumors produced by w-transformed fibroblasts were all judged to be sarcomas, and in most cases this was supported by positive staining for vimentin intermediate filaments (Fig. 1). Vimentin intermediate filaments are a general marker for mesenchymal cells, and Consequently we expected all tumors to stain positively. Positive staining for vimentin indicated that fixation and processing had been adequate to preserve antigenic determinants of tumor cells. Furthermore, because the antibody we utilized was specific for an epitope On human vimentin filaments and failed to react with murine mesenchymal cells, positive Staining for vimentin not only indicated the mesenchymal nature of the tumor cells, but also Confirmed that they were of human rather than murine origin. Where possible, only Vimentin-positive specimens were examined with the other antibodies. In two cases (cell 52 lines H-fi-Z and K-w-la), alternate specimens were unavailable, so vimentin-negative tissues were utilized. Transfected Oncogene Influences Tumor Patholoa K-ras-transformed MSU-1.1 Fibroblasts Cell strains K-m-l and K-w-Q were isolated by Fry gt_al. (9) as independent foci that appeared in cultures of MSU-1.1 fibroblasts transfected with K-rgag. Tumors produced when these cells were injected subcutaneously into athymic nude mice are shown in Table 2. Tumors were classified as myxoid fibromas, myxoid sarcomas, or spindle cell sarcomas. Cell strain K-w-l produced tumors that grew slowly and ceased enlarging when they reached about 1 cm in diameter. Grossly, the tumors were flattened, firm, white nodular masses within the subcutis, but when cut the interior of the nodules was soft. Microscopically, the tumors had a thick fibrous capsule and were composed of small uniform Stellate and fusiform cells imbedded in an abundant myxoid matrix containing short wavy Collagen fibers. A few small capillaries were scattered throughout the tumor. Cells had a Scant amount of eosinophilic fibrillar cytoplasm and oval or reniform nuclei with finely granular chromatin and one or two small nucleoli. Mitoses were scarce (<1/hpf). Because the tumors did not grow progressively and were encapsulated with no evidence of invasion or cytologic features of malignancy, they were classified as myxoid fibromas. Cell strain K-gg—Z produced soft, flattened tumors that grew slowly but progressively. Grossly, the tumors were translucent and gelatinous. Microscopically, the tumors resembled the myxoid fibromas just described (Fig. 2), but they were not encapsulated and could be Seen invading skeletal muscle. The extracellular matrix was abundant and myxoid. With reticulin stains, numerous thin collagen fibers could be seen surrounding individual cells, 53 while thicker wavy fibers were scattered throughout the matrix. Mitoses were still scarce (<1/hpf). On the basis of their progressive and invasive growth, these tumors were classified as myxoid sarcomas. Myxoid sarcomas produced by K—gg-2 were negative when stained by the immunoperoxidase method for vimentin, desmin, S-100, alpha-1- antichymotrypsin, or Factor VIII. Fry gt_a_l. (9) assayed each focus-derived K-w-transformed cell strain for grth in soft agar, and isolated 3 anchorage-independent clones (cell strains K-gg-la, K—fl-Za, and K—m-Zb). Tumors produced by these cell strains are shown in Table 2. Cell strains K-fi-la and K-_ra_s-2a, derived from K—gs-l and K-gg-2 respectively, produced myxoid sarcomas similar to those already described; however, cell strain K-w-2b produced firm nodular tumors that appeared after a shorter latent period and grew more rapidly. Microscopically, tumors formed by K-rg-2b cells consisted of dense sheets of cells separated into multiple lobules by bands of collagenous stroma, with occasional areas of more loosely- arranged cells within abundant myxoid matrix. In some of these looser areas cells grew in a storiform pattern (Fig. 3). There was extensive central necrosis of individual lobules. Closely-packed cells were round, oblong, or polyhedral with indistinct cell borders and scant cytoplasm. Cells in less dense areas were spindle-shaped and moderately pleomorphic with fibrillar cytoplasm. Nuclei were round or oval with coarsely-clumped chromatin and one or W0 large nucleoli. Mitoses were uncommon (1-3/hpf). These tumors were classifed as Spindle cell sarcomas. Electron microscopic examination of myxoid sarcomas revealed elongated cells with delicate filamentous cytoplasmic projections arranged in a loose granular matrix that Contained bundles of collagen fibers. Cells had numerous polyribosomes; a few profiles of rough endoplasmic reticulum, a few mitochondria, and an occasional Golgi complex could 54 be visualized in most cells. There was no sign of basement membrane production, and no cell junctions of any kind were seen. N-ras-transformed MSU-1.1 Fibroblasts Wilson ggl. (31) isolated cell strains N-gig-2, N-tg-3, N-gs-S, and N-w-8 from four independent foci that arose in cultures of MSU-1.1 fibroblasts transfected with N-gs. Tumors produced by these cell strains following subcutaneous inoculation into nude mice are shown in Table 3. Tumors were classified as spindle cells sarcomas, round cell sarcomas, pleomorphic sarcomas with multinuclear giant cells, or malignant mesenchymomas. Spindle cell sarcomas were produced by all four N-gig—transformed cell strains and consisted of oblong and spindle-shaped cells arranged in interwoven bundles. Sometimes, Short bundles of cells were radially arrayed in a manner reminiscent of the storiform pattern associated with malignant fibrous histiocytomas and dermatofibrosarcoma protruberans. In Other tumors, cell bundles intersected at regular angles to form the herringbone pattern Commonly associated with human fibrosarcomas (Fig. 4). Such tumors had abundant collagen fibers that were distributed throughout the tumor and appeared to be the product of tumor cells. Still other tumors had less uniform cell bundles that intersected at various angles (Fig. 5). These tumors, which resembled leiomyosarcomas, had a prominent fibrovascular stroma running between bundles of tumor cells, and the hyperchromatic stromal cell nuclei accentuated the fascicular pattern of the tumor. Collagen fibers in these tumors were almost exclusively confined to the stroma, suggesting that they were the product of murine stromal cells rather than the tumor cells. Occasional spindle cell sarcomas contained large, branching vascular channels typical of those described in human liposarcomas and hemangiopericytomas (4). 55 Cells in spindle cell sarcomas varied from uniform to moderately pleomorphic, but were always closely-packed with scant intercellular matrix. They had a moderate amount of fibrillar eosinophilic cytoplasm with indistinct cytoplasmic margins. Nuclei were elongated, sometimes with blunt ends like those seen in cells of smooth muscle tumors and sometimes with tapered ends more typical of cells in fibroblastic tumors. Chromatin was coarsely-clumped along the nuclear membrane or into strands that spanned the nucleus, and there were one to three nucleoli. The rapid growth of these tumors was reflected in their high mitotic rate (5-10/hpf). Central necrosis was common and also may have been related to the rapid growth rate. Electron microscopic examination of spindle cell sarcomas revealed only oblong cells with numerous polyribosomes, a few profiles of endoplasmic reticulum, and a few mitochondria. Neither intercellular junctions nor bundles of Cytoplasmic filaments were observed. Round cell sarcomas were produced by cell strain N-gg—2 and consisted of dense Sheets of fairly uniform cells with no hint of a fascicular pattern (Figs. 6 and 7). Short thin Collagen fibers could be seen between tumor cells on silver-stained sections, but no thick Collagen fibers were found. Cells were round or polyhedral with indistinct cytoplamic borders. Nuclei were round, oval, or indented and clumping of chromatin along the nuclear membrane gave the nuclei a vesicular appearance. There were one to three small nucleoli. Mitotic activity was similar to the spindle cell sarcomas, and round cell sarcomas also often had necrotic centers. Cell strain N-gig-3 produced tumors with a distinctive appearance, consisting of very pleomorphic cells arranged in interwoven bundles that sometimes assumed a herringbone pattern. A consistent feature of these tumors was inflammation characterized by multifocal infiltration of neutrophils and lymphocytes, sometimes associated with necrosis of individual tumor cells. These tumors contained a mixture of small spindle cells with oblong nuclei, 56 rounded cells with a moderate amount of cytoplasm and oval or reniform nuclei, and mononuclear and multinuclear giant cells that had abundant granular eosinophilic cytoplasm (Fig. 8). Giant cells were often elongated. They did not resemble the Langerhans or foreign body type giant cells seen in malignant fibrous histiocytoma, or the osteoclastic giant cells seen in osteosarcomas and Ewing’s sarcomas. Although they were sometimes strap- like, they did not have the deeply eosinophilic cytoplasm or myofilaments found in giant cells of rhabdomyosarcomas. These tumors had a lower mitotic rate (1-3/hpf) than tumors produced by other N-tag-transformed cell strains, but bizarre multiplanar mitotic figues were Common. There tumors were classified as pleomorphic sarcomas with multinuclear giant cells. In several tumors produced by cell strain N-m-3, the pleomorphic sarcoma pattern occurred in association with one of the other patterns of growth. In each of these tumors the two morphologic patterns were sharply demarcated rather than blending together (Fig. 9). Tumors that exhibited two distinct microscopic morphologies were classified as malignant mesenchymomas. The production of mixed tumors by cell strain N -_I;2§-3 raised the possibility that this Strain might be a mixture of two stable cell populations, one that produced spindle cell sarcomas and one that produced pleomorphic sarcomas with multinuclear giant cells. Microscopic examination of N-ES cell cultures revealed scattered multinucleated cells. We attempted to obtain sublines of N-fl-ii that produced only one type of tumor by plating cells at cloning density (100 cells per 100 mm culture dish). When culture dishes were examined two weeks later, individual clones could be differentiated by the presence or absence of multinucleated cells. Ten clones of each type were selected for expansion into cell strains. Two cell strains that contained multinucleated giant cells (N-gg-3.1 and N- £§§-3.2) and tw0 cell strains that lacked such cells (N-gig-3.3 and N-gig-3.4) were injected 57 subcutaneously into 5 mice each. N-gig-3.1 produced malignant mesenchymomas in 2 mice and spindle cell sarcomas in 3 mice, while N—w-32 produced a pleomorphic sarcoma with multinuclear giant cells in one mouse and spindle cell sarcomas in 4 mice. N-gs-3.3 and N-w-SA produced only typical spindle cell sarcomas. These results suggested that N-gg- 3 was a mixture of two cell strains, one of which contained only monoculcear cells in culture and produced only spindle cell sarcomas in mice, while the other contained multinuclear cells in culture and sometimes produced pleomorphic sarcomas with multinuclear giant cells or mesnchymomas in mice. Sarcomas produced by N-w-Z and N-m—3 cells stained positively for desmin, including a spindle cell sarcoma, a round cell sarcoma, and a pleomorphic sarcoma with multinuclear giant cells (Table 4 and Fig. 10). Generally there was diffuse pale staining of the entire cytoplasm of tumor cells, compared to the stronger cytoplasmic staining of mouse skeletal muscle fibers and vascular smooth muscle cells within the same section. H-ras-transformed MSU-1.1 Fibroblasts Hurlin et al. (14) isolated cell strains H-gg-2, H-fl-Ei, and H-w-6 from three independent foci that appeared in cultures of MSU-1.1 fibroblasts transfected with H-_@. Tumors produced when these cells were injected subcutaneously into nude mice are shown in Table 5. Tumors were classified as spindle cell sarcomas (with and without whorls) and malignant mesenchymomas. Cell strains H-gs-3 and H-gig-6 produced spindle cell sarcomas with a herringbone pattern. The tumors differed from the spindle cell sarcomas produced by N-@- and K- w-transformed fibroblasts in three ways. First, scattered among the bundles of spindle cells WCFC rounded nests of closely-packed polyhedral and round cells that did not represent individual fascicles cut in cross-section (Fig. 11). These nests were especially distinct on 58 silver-stained sections where thin collagen fibers were readily seen surrounding tumor cells in fascicular areas, but were absent within nests of cells. Sometimes the cells in these areas were concentrically arranged, giving the appearance of whorls. Second, thick collagen fibers were more common between cells in these tumors than they were in sarcomas produced by N-m- or K-m-transformed cells. Third, the mitotic rate of the tumors was low (1-3/hpf) and necrosis was rarely a feature even of very large tumors. Spindle cell sarcomas produced by H-r_a_s-3 cells stained positively for S-100 antigen (Table 6 and Fig. 12). Staining was diffuse, cytoplasmic, and roughly as intense as that of peripheral nerve fibers on the same section. Approximately one third of the tumor cells were S-100-positive. Cell strain H-fl-Z produced sarcomas with a variety of microscopic appearances but With one consistent feature; namely, the formation of whorls that sometimes had a small blood vessel or necrotic focus in the center (Fig. 13). These whorls resembled those described in human hemangiopericytomas, meningiomas, and Schannomas. Again, silver Stains revealed a lack of pericellular thin collagen fibers within these whorled areas. In two tUmors there were large branching vascular channels, a feature also associated with hemangiopericytomas. The remainder of the tumors was a combination of loose myxoid areas, interwoven short fascicles of cells in a storiform pattern, and in one case an area of pleomorphic sarcoma with multinuclear giant cells that led us to classify the tumor as a I‘nalignant mesenchymoma. These tumors had a low mitotic rate (1-3/hpf). 59 Conclusions Investigation of the relationship between specific @ oncogenes and tumor pathology was complicated by the fact that each of the @ oncogenes used to transform MSU-1.1 fibroblasts was introduced in a different plasmid construction. Attempts to construct a series of plasmids that are identical except for the oncogene they carry are currently underway. Nevertheless, the tumors produced did vary in many microscopic features and in several instances a specific type of tumor was associated with a particular transforming gene: a) Myxoid sarcomas were produced only by K-gs-transformed MSU-1.1 fibroblasts. b) Spindle cell sarcomas with a whorling pattern were produced only by H-w-transformed MSU-1.1 fibroblasts. c) Round cell sarcomas were produced only by a single strain of N-fl-transformed MSU- 1.1 fibroblasts (N-gig-Z). d) Pleomorphic sarcomas with multinuclear giant cells, or mesenchymomas with such a component, were produced only by a single strain of N-gig-transformed MSU-1.1 fibroblasts (N-gs-3). e) Sarcomas CORtaining desmin intermediate filaments were produced only by N-gig— transformed MSU-1.1 fibroblasts. f) Sarcomas containing S-100 protein were produced only by a single strain of H-gg- transformed MSU-1.1 fibroblasts (Hm-6)- Although activation of a specific gs oncogene might cause cells to produce, for example, abundant myxoid matrix or desmin intermediate filaments, such phenotypic features are really the end result of alterations in whole patterns of gene expression. 60 Regulation of these differentiation programs is undoubtedly complex. Evidence to date suggests that gas gene products are components of signal transduction pathways, probably playing a role similar to G-proteins in modulating signal transmission via transmembrane receptors to intracellular secondary messengers (11,15). That the three @ genes have different sequences suggests that they may play analogous roles in different signal transduction pathways, or function only in certain cell types or at certain periods of development. In such a situation it is reasonable to expect that alterations in @ structure or level of expression would have pleiotropic effects, effects that vary depending upon which @ genes is altered and the nature of the alteration. These studies suggest that changes in K-@ expression may be involved in differentiation of mesenchymal cells toward a phenotype of myxoid matrix production, that altered expression of H-gs may be involved in the program of differentiation that leads cells to arrange themselves in whorls or express S—100 protein, and that altered expression of N-gs may be involved in differentiation programs leading cells to produce desmin filaments, cease collagen production and grow as spheres, or dissociate their nuclear and cytoplasmic division so that multinucleated cells are created. Our conclusion that the transfected gs oncogene does influence the type of tumor produced by rfi-transformed human fibroblasts contrasts with that of Thorgeirsson _et_2§. (30) and Greig et al. (12) who studied mouse NIH 3T3 fibroblasts transformed spontaneously or by transfection with an H-gs or N-w oncogene. They found that transformed mouse fibroblasts produced poorly-differentiated sarcomas without distinguishing features regardless of the means by which they were transformed. Differences in experimental design make it impossible to ascribe the discrepancy between their results and those we report here solely to the difference between human and mouse cells. 61 Recigient Cell Influences Tumor Patholog H-ras-transformed KMST-6 Fibroblasts KMST-6 fibroblasts transfected with H-La_s produced rapidly growing tumors following subcutaneous injection in nude mice (Table 5). Tumors typically had extensive central necrosis accompanied by suppurative inflammation, and mice bearing such tumors had splenomegally due to extramedullary myelopoiesis. Tumors were composed of very pleomorphic cells arranged in solid sheets and separated into small clusters by a scant stroma, an arrangement made prominent by infiltrating neutrophils that accumulated in the stroma and by congestion of stromal capillaries (Fig. 14). Collagen fibers were scarce and confined to the stroma; there was no evidence of collagen production by tumor cells. Cells were round or polyhedral with abundant eosinophilic cytoplasm and distinct cell borders. Nuclei were of various shapes and frequently indented, with coarsly-clumped chromatin and multiple large nucleoli. Mononuclear giant cells with deeply lobulated nuclei were conspicuous. Mitoses were common (5-10/hpf) and bizarre mitotic figures were found in every tumor. These tumors were classified as pleomorphic sarcomas with mononuclear giant cells. Sarcomas produced by H-gis-transformed KMST-6 fibroblasts contained scattered cells that stained positively for alpha-l-antichymotrypsin (Table 6), but this observation was difficult to interpret. As already mentioned, these tumors were usually necrotic and secondarily inflamed, and it was impossible to ascertain whether the alpha-1- antichymotrypsin-positive cells were tumor cells or host macrophages that had infiltrated the tumor. r__i- 62 H-ras-transformed GM637 Fibroblasts GM637 fibroblasts transfected with H-[fi produced tumors that were similar in microscopic appearance to those produced by @ transformed KMST-6 fibroblasts. Again, tumors developed rapidly following subcutaneous injection in nude mice. As with the KMST-6 tumors, necrosis and suppurative inflammation were prominent features. Unlike the KMST-6 tumors, however, pleomorphic tumor cells more often assumed a spindle shape and were arranged in short interwoven bundles with a prominent collagenous matrix (Fig. 15). These tumors also were classified as pleomorphic sarcomas with mononuclear giant cells. Sarcomas produced by H-m—transformed GM637 fibroblasts were vimentin-positive but negative for desmin, S-100, alphafl-antichymotrypsin, and Factor VIII. Conclusions The nature of the recipient cell line did affect the type of tumor produced following [as transfection, as illustrated by the differences we observed in tumors produced by MSU-1.1, KMST-6, and GM637 fibroblasts transformed by the identical H-[fi oncogene. All three H'fl-transformed cell strains express similar amounts of p21"'ras (14). Previous studies have shown that the nature of the recipient cell line also influences the tumor-forming capacity Of w—transformed mouse cells (20). However, Franks _et_al. (8) found that a series 0f sI’Ontarleously-transformed mouse fibroblast lines derived from different organs produced tumOFS With similar microscopic features suggesting that, in mice, the origin of a cell line does nOt always influence the type of tumor produced. Other Factors Influence Tumor Pathology The transfected oncogene and recipient cell line were not the only factors that mfluenced what type of tumor a gs-transformed human cell strain produced. Differences 1n the tumors produced by various lines of Nils-transformed MSU-1.1 fibroblasts suggested A 63 that factors such as the site of integration of the transfected m oncogene also might influence tumor type. Integration site could directly influence the expression of the oncogene itself, of adjacent normal cellular genes, or both. Our results are similar to those of Auersperg _et_al. (1) who found that transformed cell strains derived from a single line of rat adrenal cortical cells infected with Kirsten murine sarcoma virus produced several different types of tumors. Of 18 transformed cell strains they isolated i_n v_itr_o and tested for tumorigenicity, 7 produced pleomorphic carcinomas, 6 produced sarcomas, 4 produced mixed tumors (carcinosarcomas), and 1 produced anaplastic tumors. Host factors, too, appeared to influence the type of tumor produced, as was the case with cell strain N-w-Z that produced both round cell sarcomas and spindle cell sarcomas, or cell strain N-m—Ev that produced giant cell sarcomas, spindle cell sarcomas, or mixed tumors. We were able to establish cell strains derived from N—rag-3 that produced only Spindle cell sarcomas, but not strains that produced only pleomorphic sarcomas. One Possible explanation for our observations is that variants capable of producing a Pleomorphic sarcoma appear within- the N-m-C‘) strain at some low frequency that is influenced by external factors. I_n M, such factors could include the age, sex, nutritional status, health status, and hormonal status of the host mouse. Another external factor that could influence the type of tumor produced is the microenvironment to which the cells are exposed, For example, the microscopic appearance of tumors produced by a particular as; transformed cell strain was found to differ when the cells were injected intraperitoneally instead 0f subcutaneously (5). Activated or inappropriately expressed @ oncogenes have been found in a wide Variety Of human tumors, including sarcomas (2,27). Taken together with the results of i_n m StUdies on transformation of rodent cells by m oncogenes, the prevalence of gs o - . . . . . ncogenes in human tumors strongly implicates @ oncogenes in human carcmogenesrs. 64 The role individual @ oncogenes play in influencing pathologic features of human tumors seldom has been studied, chiefly because a suitable experimental approach has been unavailable. But with the development of gs-transformed human cell strains by Fry L211. (9), Hurlin ml. (14), and Wilson L211. (31) it is now possible not only to study directly the role of ras oncogenes in malignant transformation of human cells, but also to investigate the relationships between specific @ oncogenes and various histopathologic features used by pathologists to classify and assign prognoses to spontaneous human soft tissue sarcomas. This experimental model of human carcinogenesis developed by McCormick and his colleagues will likely prove valuable for exploring the molecular basis behind mesenchymal cell differentiation and the histogenesis of soft tissue sarcomas. METHODS Cell Lines Creation of Qs-transformed cell strains by transfection of infinite life span human fibroblast line MSU—1.1 with plasmids containing a K-w, H-@, or N-m oncogene has been described elsewhere (9,14,31). MSU-1.1 fiboblasts were created in McCormick’s laboratory (18); they are a non-tumorigenic cell line derived from human foreskin fibroblasts transfected with a v-myg oncogene. MSU-1.1 fibroblasts carry and express both the v-myg gene and a neomycin resistance gene. Transfection of MSU-1.1 fibroblasts with a viral K- rzg oncogene, the H-@ oncogene from the human bladder carcinoma cell line T24, or the N-m oncogene from the human leukemia cell line 8402 (29) resulted in morphologically- transformed cells that could be identified as foci on a cell monolayer or as anchorage independent colonies in soft agar. Several clones of morphologically-transformed MSU- 1.1 fibroblasts that contain and express each of the three @ oncogenes were isolated, and these cell strains were utilized in the-studies reported here. Hurlin gal. (14) also transformed two other infinite life span fibroblast lines by transfection with T24 H-r_a_s. KMST—6 fibroblasts were obtained from Namba; they are derived from human embryo fibroblasts treated with 60Co gamma radiation (21,22). GM637 fibroblasts were obtained from the Camden, NJ; they are derived from adult dermal fllDFOblaSts infected with SV40 virus. Neither cell line is tumorigenic. Stable transformants were Obtained by transfection of KMST-6 or GM637 fibroblasts with a plasmid containing the H‘TLS oncogene linked to a neomycin resistance gene, followed by growth in selective medium containing G418 (14) 65 66 Assay for Tumorigenicigg in Nude Mice BALB/c athymic nude mice from a closed colony were utilized as tumor hosts in all experiments. Serum from heterozygous female breeders was tested regularly by Microbiological Associates (Rockville, MD) and was consistently free of antibodies to K virus, polyoma virus, murine hepatitis virus, ectromelia virus, GD VIII virus, murine adenovirus, lymphocytic choriomeningitis virus, minute virus of mice, Sendai virus, pneumonia virus of mice, reovirus type 3, EDIM virus, murine cytomegalovirus, Car bacillus, and Mycoplasma pulmonis. Mice were maintained in sterile isolators illuminated by fluorescent light, with a 12 hour/ 12 hour light/dark cycle, temperature of 70-75°F, and humidity of 30-50%. They were housed in plastic cages at up to five mice per cage. Cages had pine shaving bedding and individual water bottles. Mice were fed pelleted laboratory chow. Mice of both sexes were used as tumor hosts. Prior to injection, mice were X- irradiated at a dose of 150-300 rads/ mouse. From 1 x 106 to 5 x 106 transformed cells were injected into the subcutis on the dorsal midline of 6-10 week old mice. Afterwards, mice were examined weekly for tumor development. Mice were killed by CO? inhalation when tUmors were 1-3 cm in diameter, or sooner if the tumor became ulcerated or the mouse showed signs of illness such as listlessness or weight loss. Collection and Processing of Tissues Tumor-bearing mice received a complete necropsy that included gross and microscopic examination of at least the tumor itself, axillary, cervical, and mesenteric lymph nodes, thyroid gland, parathyroid glands, adrenal glands, salivary glands, trachea, lungs, heart, liver, spleen, pancreas, and kidneys. In some mice, the digestive tract, reproductive tr act, or brain were also examined. Specimens for routine microscopic examination were 67 fixed in neutral buffered 10% formalin. Sections of tumor for immunoperoxidase staining were fixed in freshly-prepared B-5 fixative for 2 hours, then transfered to 70% ethanol. Small pieces of tumor (1 mm3) for electron microscopic examination were fixed in cold 4% glutaraldehyde for 2 hours, then transfered to sucrose/ phosphate buffer. Sections of fresh tumor were also collected in Eagle’s minimal essential medium. Fresh tumor tissue was minced and returned to culture. Since all r_as-transformed cell strains carry the M gene coding for G418 resistance, cells were refed with selctive medium containing G418 after 48 hours to kill any contaminating mouse cells. Tissues fixed in formalin and B-5 were processed by routine histologic methods and imbedded in paraffin blocks. Tumors were serially sectioned into 2-3 mm slices and all slices were imbedded, so that 3-10 sections of each tumor, representing different areas, wereexamined microscopically. Six micron sections were cut and stained with hematoxylin and eosin for general examination. Additional sections of formalin-fixed tissue were cut and stained with Gomori’s trichrome stain or silver stain to demonstrate thick and thin collagen fibers, or Alcian blue or Toluidine Blue to demonstrate mucopolysaccharides. In some cases frozen sections of formalin-fixed tissue were prepared and stained for lipid with Oil Red 0. Additional sections of B—S-fixed tissues were stained by an avidin—biotin immunoperoxidase method for vimentin, desmin, S—100, alpha-l-antichymotrypsin, and Factor VIII antigen. Slides were stained in batches of 18, each batch including a known POSitiVe human tissue control and a negative primary antibody control. Briefly, 3 micron sections were placed on poly-L—lysine coated slides and deparaffinized overnight at 60°C. Slides were de-Zenkerized, rinsed, and immersed in methanol for 10 minutes. Endogenous peroxidase activity was blocked by incubation for 10 minutes in H202, followed by 5 minutes in normal goat serum. Slides were then labelled with one of five primary antibodies: monoclonal mouse anti-Vimentin, monoclonal mouse anti-desmin, polyclonal rabbit anti-S- 68 100, polyclonal rabbit anti-alpha-1-antichymotrypsin, and polyclonal rabbit anti-Factor VIII (all from Lipshaw, Detroit, MI). After treatment with the appropriate linking antibody and labelling reagant, slides were exposed to chromogenic substrate for 5-20 minutes. Slides were counterstained with Mayer’s hematoxylin. 14. Hurlin, PJ., Maher, V.M., McCormick, J .J . Malignant transformation of human fibroblasts caused by expression of a transfected T24 HRAS oncogene. Proc Natl Acad Sci USA 86: 187-191 (1989) 15. Kahn, P., Graf, T. (eds). Oncogenes and Growth Control. Springer—Verlag, Berlin: 135- 208 (1986) 16. Katenkamp, D., Raikhlin, N.T. Stem cell concept and heterogeneity of malignant soft tissue tumor -- a challenge to'reconsider diagnostics and therapy? Exp Pathol 28: 3-11 (1985) 17. Land, H., Parada, L.F., Weinberg, R.A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304: 596-602 (1983b) 18. McCormick, J.J, Fry, D.G., Hurlin, PJ., Morgan, TL, Wilson, D.M., Maher, V.M. Malignant transformation of human fibroblasts by transfected oncogenes. In Proceedings of the Workshop on Cell Transformation Systems Relevant to Radiation Induced Cancer in Man. Chadwick, K.H. (ed) IOP Publishing Ltd. (in press, 1989). p—i \O . Molenaar, W.M., DeJong, B., Buist, J ., Idenburg, V.J.S., Seruca, R., Vos, A.M., Hoekstra, H.J. Chromosomal analysis and the classification of soft tissue sarcomas. Lab Invest 60: 266-274 (1989) 20. Muschel, R.J., Williams, J.E., Lowy, D.R., Liotta, L.A. Harvey gs induction of metastatic potential depends upon oncogene activation and the type of recipient cell. Am J Pathol 121: 1-8 (1985) 21. Namba, M. Neoplastic transformation of human diploid fibroblasts (KMST-6) by treatment with Co-60 gamma rays. I_n: Barrett JC, Tennant RC (eds), Carcinogenesis, vol-9, Raven Press, New York, 217-231 (1985) 22. Namba, M., Nishitani, K, Kimoto, T. Carcinognensis in tissue culture 29. Neoplastic transformation of a normal human diploid cell strain, WI-38, with Co-60 gamma rays. Japan J Exp Med 48: 303-311 (1978) 23. Newbold, R.F., Overell, R.W. Fibroblast immortality is a prerequisite for transformation by EJ c-Ha-w oncogene. Nature 304: 648-651 (1983) 24. Newbold, R.F., Overell, R.W., Connell, J .R. Induction of immortality is an early event in malignant transformation of mammalian cells by carcinogens. Nature 299: 633- 635 (1982) 25. Ruley, H.E. Adenovirus early region 1A enables Viral and cellular transforming genes to transform primary cells in culture. Nature 304: 602-606 (1983) 26. Schilz, R.J., Dillberger, J .E., Maher, V.M., McCormick, J .J . Overexpression of multiple growth factor genes by fibrosarcoma-derived and other transformed human fibroblasts in culture. Cancer Res (in press): (1989) 70 27. Shih, T.Y., Weeks, M.O. Oncogenes and cancer: The p21 gs genes. Cancer Invest 2: 109-123 (1984) 28. Smets, L.A. Cell transformation as a model for tumor induction and neoplastic growth. Biochim Biophys Acta 605: 93-111 (1980) 29. Souyri, M., Koehne, C.F., O’Donnell, P.V., Aldrich, T.H., Furth, M.E., Fleissner, E. Biological effects of a murine retrovirus carrying an activated N-w gene of human origin. Virol 158: 69-78 (1987) 30. Thorgeirsson, U.P., Turpeenniemi—Hujanen, T., Williams, J .E., Westin, E.H., Heilman, C.A., Talmadge, J .E., Liotta, L.A. NIH/3T3 cells transfected with human tumor DNA containing activated ra_s oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol 5: 259-262 (1985) 31. Wilson, D.M., Yang, D., Dillberger, J .E., Dietrich, S.E., Maher, V.M., McCormick, J.J, Malignant transformation of an infinite life span, human fibroblast cell line by a transfected N-[fi oncogene. Cancer Res (submitted, 1989) 71 72 Table 1. Origin of ras-transformed Human Cell Strains Recipient Transforming Cell Line Oncogene Cell Strains Derived Reference MSU-1.1 K-r_as K-ras—l, K—w-la, K-fl-Z, 9 K-gs—Za, K-Qs-Zb N-gis N-gls-Z, N-ms, 31 N-ra_s-5, N-Qs-8 H-fl H-ng-2, H—gs—3, 14 H-gLs-6 KMST-6 H-Lfi K1-T1 14 GM637 H-[fi SV-Tl 14 Cell m K-w-2a K-w-2b _L Tumors new 2 2 73 Mean Latent Period 70 days 35 days 37 days 39 days 34 days Table 2. Tumors Produced by K-m—transformed MSU-1.1 Human Fibroblasts Tumor Classification Myxoid fibroma Myxoid sarcoma Myxoid sarcoma Myxoid sarcoma Spindle cell sarcoma 74 Table 3. Tumors Produced by N-w-transformed MSU-1.1 Human Fibroblasts Cell m N-l‘fi-Z N-m-S N-m-S Tumors Examined 4 10 Mean Latent Period 34 days 19 days 36 days 29 days Tumor Classification Round cell sarcoma (1/4) Spindle cell sarcoma (3 /4) Pleomorphic sarcoma with multinuclear giant cells (5/10) Malignant mesenchymoma (4/10) Spindle cell sarcoma (1 / 10) Spindle cell sarcoma Spindle cell sarcoma 75 Table 4. Immunoperoxidase Staining of Tumors Produced by Niels-transformed MSU-1.1 Human Fibroblastsa Cell m N-m—Z N-m—2 N-w-3 N-fl-S N-m-S Type of ”Pu—mg Spindle cell sarcoma Round cell sarcoma Pleomorphic sarcoma Spindle cell sarcoma Spindle cell sarcoma Alpha-l-anti Factor Vimentin Desmin S-100 chymotrypsin VIII + + - - - + + - - - + +b - - - + - - - - + - - - - aTwo tumors of each type were examined except for the round cell sarcoma produced by cell strain N-ras—2, where only a single tumor was examined, and pleomorphic sarcomas produced by N-ras-3, where four tumors were examined. For each tumor, 2-4 sections were stained and examined. t’I'hree of four tumors examined had desmin-positive cells, but none could be found in the fourth tumor. 76 Table 5. Tumors Produced by H-w-transformed Human Fibroblasts Cell Strain H-m—2 H-m—3 H-ra_s-6 Kl-Tl SV-Tl Tumors Examined 10 Mean Latent Period 30 days 30 days 34 days 25 days 106 days Tumor Classification Spindle cell sarcoma with whorls Spindle cell sarcoma Spindle cell sarcoma (4/5) Malignant mesenchymoma (1/5) Pleomorphic sarcoma with mononuclear giant cells Pleomorphic sarcoma with mononuclear giant cells 77 Table 6. Immunoperoxidase Staining of Tumors Produced by H-w-transformed Human Fibroblasts. Cell Strain H-w-Z H-Qs-3 H-fi-6 Kl-Tl SV-TI Type of Mo: Whorled sarcoma Spindle cell sarcoma Spindle cell sarcoma Pleomorphic sarcoma Pleomorphic sarcoma Vimentin Desmin S-100 Alpha-l-anti Factor chymotrypsin VIII 3 Scattered positive cells within the tumor may have been mouse macrophages. ILLUSTRATIONS Figure 1. Sarcomas stained for vimentin intermediate filaments. a) N-rfl-transformed MSU-1.1 fibroblasts (cell strain N-m—S). b) H-Las-transformed KMST.6 fibroblasts. c) H-m—transformed GM637 fibroblasts (all 500x). . 78 79 Figure 2. Myxoid sarcoma produced by K-Las—transformed MSU-1.1 fibroblasts (cell strain K-fila). Note abundant extracellular matrix rich in collagen fibers (HE, 500x). Figure 3. Spindle cell sarcoma produced by K—w-transformed MSU—1.1 fibroblasts (cell strain K-ras—Zb). Cells are arranged in short interwoven fascicles that create a storiform pattern. (_HE, 200x). 81 Figure 4. Spindle cell sarcoma produced by N-ra_s_- transformed MSU-1.1 fibroblasts (cell strain N-r_a_s- -8). Note moderate cellular pleomorphism and prominent herringbone pattern (HE, 200x). 82 strain N-fl-ii). The fascicular pattern is accentuated by stromal blood vessels that surround and delineate individual tumor cell bundles (HE, 200x). Figure 5. Spindle cell sarcoma produced by N—w-transformed MSU-1.1 fibroblasts (cell 83 Figure 6. Round cell sarcoma produced by N-w-transformed MSU-1.1 fibroblasts (cell strain N-gls-Z). Note absence of a fascicular pattern and numerous large vascular channels (HE, 200x). 84 Vie... an? . . ‘) lit!“ wt”? whaanb ON“. .. . . . ... . v. 5 \MW «34...... . QII’M 1’. in .t c». S ‘G.\ a.“ f... .. .. v. .A.'.!‘, . strain N-w—2). Note cross-sections of several skeletal muscle fibers (HE, Figure 7. Round cell sarcoma produced by N-ras-transformed MSU-1.1 fibroblasts (cell 500x). 85 Figut‘e 8. Pleomorphic sarcoma with multinuclear giant cells produced by N-fl-transformed MSU-1.1 fibroblasts (cell strain N-fl-ii). Note abnormal mitotic figure (HE, 500x). 86 Flgul‘e 9. Malignant mesenchymoma produced by Niels—transformed MSU-1.1 fibroblasts (cell strain N-w-3). There is a sharp demarcation between the monomorphic round cell pattern and pleomorphic multinucleated giant cell pattern. Note entrapped adipocytes and mammary gland duct (arrow) (HE, 200x). 87 F 1.gl-lre 10. Sarcomas produced by N-m—transformed-fibroblasts stained for desmin intermediate filaments. a) Cells of tumor formed by cell strain N—w-S are desmin-negative, but note staining of mouse skeletal muscle fibers and arteriolar smooth muscle. b) Desmin-positive round cell sarcoma (cell strain N-nfi-Z). c) Desmin-positive spindle cell sarcoma (cell strain N-fl-Z) (all 500x). 88 I. sap-ff ‘ “T ""V ..qa -~‘.~. - . . -- amt . fl,’,‘.f-"._ 6;:- -‘ .‘ p __ \1 ' ' V5 ’1';- w . er. '1 t ‘ Q as \- ' we . JR;- ) Figul‘e 11. Spindle cell sarcoma produced by H-w-transformed MSU-1.1 fibroblasts (cell strain H-m—3). Note rounded clusters of cells with pale cytoplasm that appear to be arranged concentrically (HE, 200x). 89 Figure 12. Sarcoma produced by H-pas—transformed fibroblasts stained for S-100. a) Mouse peripheral nerve at edge of tumor; compare intensity of S-100-positive reaction to diffuse cytoplasmic staining of tumor cells in b) Spindle cell sarcoma formed by cell strain H-m—6 (both 500x). 90 ‘1 q .. ‘ 5"-.. 3‘3‘7\ x A .. . ~. r ;’ J's:- ‘ K:\ >331: ’1‘\\¥ 3‘; fixl’k‘ -. . ' .' .’ g {- ‘2‘): § IN“ J 3'0“: VA fr," ‘ l t“. ‘ Wat?» I a a 1 m- ; . - .., . '. 1 r t =. ‘ _w. . ‘1“... fi' 0“ a; ‘3? “it“ 03.1. ’f» \ 310:". ygfi'x \L‘ 'W; “if; 91‘ .4... ’ ‘ll, Figure 13. Spindle cell sarcoma produced by H-w-transformed MSU-1.1 fibroblasts (cell strain H-w-Z). Note fairly uniform cells arranged in multiple whorls (HE, 200x). 91 a" ‘9’ '5 v ' g. 0 . _f 0 i3“. 4‘. ‘Q‘. 0" , ’s Fig‘Jre l4. Pleomorphic sarcoma with mononuclear giant cells produced by Hig- transformed KMST-6 fibroblasts. Cells are separated into small clusters by a delicate fibrovascular stroma. Note giant cell (HE, 200x). 92 FigUre 15. Pleomorphic sarcoma with mononuclear giant cells produced by H-@- transformed GM637 fibroblasts. Note pleomorphic spindle cells arranged in a storiform pattern (HE, 200x). TUMORS FORMED BY HUMAN SARCOMA-DERIVED CELL LINES AND A SPONTANEOUSLY-TRANSFORMED FIBROBLAST LINE John E. Dillbergera, Douglas A. Porterb, Veronica M. Maherc, and J. Justin McCormickC Carcinogenesis Laboratory, Michigan State University, East Lansing, MI 48824-1316 Authors’ current affiliations: aToxicology Department, Merrell Dow Research Institute, P.O. Box 68470, Indianapolis, IN 46268-0470 bIngham Medical Center, Lansing, MI 489 cCarcinogenesis Laboratory, Michigan State University, East Lansing, MI 48824-1316 Send Correspondence to: J. Justin McCormick Carcinogenesis Laboratory West Fee Hall Michigan State University East Lansing, MI 48824-1316 ABSTRACT Several transformed human cell strains that produce sarcomas in athymic nude mice have been developed m vitro by transfection of infinite life span human fibroblasts with @ oncogenes. To clarify how closely this i_n vitro model mimics i_n vivo carcinogenesis, we examined tumors produced in mice by six human sarcoma-derived cell lines, by a clonally- derived subline of one of them, and by a spontaneously-transformed human fibroblast line. One of the sarcoma-derived cell lines (SW-982) proved non-tumorigenic. Tumors produced by the other cell lines were examined microscopically and characterized by their cytologic features, patterns of growth, and immunohistochemical staining reactions. Tumors grew progressively and were invasive, but none metastasized from the subcutaneous injection site. All were classified as sarcomas by their microscopic appearance and positive staining for vimentin, but they differed. The spontaneously-transformed cell line (VIP:F-T) produced spindle cell sarcomas with a herringbone pattern and abundant collagen. Three sarcoma- derived cell lines (8387, SHAC, and NCI) and a subline of one of them (HUT-14) produced spindle cell sarcomas composed of short bundles of cells that intersected randomly, and tumors from all but one of these cell lines (SHAC) had abundant collagen. A fourth sarcoma-derived cell line (HT1080) produced round cell sarcomas without collagen, and a fifth (SW-684) produced a pleomorphic sarcoma with mononuclear giant cells and multifocal necrosis. Tumors produced by these transformed cell lines resembled spindle cell and round cell sarcomas produced by gs-transformed human fibroblasts in rate of growth, cytologic features, morphologic patterns of growth, and failure to spontaneously metastasize from the subcutis. In addition, one sarcoma-derived cell line (SHAC) produced tumors that expressed desmin and a subline of another sarcoma-derived cell line (HUT-l4) produced tumors that were S-100-positive, just as several strains of w-transformed human fibroblasts 94 do. These results demonstrate the phenotypic diversity of cell lines reportedly derived from tumors of the same histopathologic type, and support the relevance of i_n vitro transformation of human fibroblasts by transfected @ oncogenes as a model for studying human carcinogenesis. 95 INTRODUCTION Fry e_t a_l. (I), Hurlin e_t a_l. (2), and Wilson e_t a_l. (3) recently succeeded in transforming infinite life span human fibroblasts to tumorigenicity by transfection of @ oncogenes. The sarcomas produced in athymic nude mice by these cells w-transformed cell strains were characterized by cytologic features, patterns of growth, and immunohistochemical staining pattern into spindle cell sarcomas (with and without whorls), myxoid sarcomas, round cell sarcomas, pleomorphic sarcomas (with mononuclear or multinuclear giant cells), and malignant mesenchymomas (4). To clarify how closely i_n Elli—1‘0. transformation of human fibroblasts by @ oncogenes mimics i_n M carcinogenesis, we examined tumors produced by six cell lines reportedly derived from human fibrosarcomas, a clonally-derived subline of one of them, and a cell line spontaneously transformed i_n Litr_o and compared the results with what we and our colleagues found previously with tumors produced by the various pas-transformed cell strains. Two of the cell lines (HT1080 and SHAC) were known to contain activated N-is oncogenes (5,6). The human sarcoma-derived cell lines and spontaneously-transformed cell line were assessed for tumorigenicity by subcutaneous injection into athymic nude mice. Tumors produced by these cells were examined microscopically using chemical and immunohistochemical stains and characterized by the same criteria applied to classify the tumors produced by pas-transformed fibroblasts. The objective was to determine how tumors produced by sarcoma-derived or spontaneously-transformed human fibroblasts resembled those produced by w-transformed human fibroblasts. 96 MATERIALS AND METHODS Cell Lines The sarcoma-derived cell lines and their derivation are listed in Table 1. The SHAC cell line was derived from a gastric fibrosarcoma that arose in a 58-year old patient (6). The HT1080 cell line originally was reported to be derived from a fibrosarcoma that arose near the acetabulum of a 35-year old man who later died of metastatic disease (7), but a recent paper states that this line was derived from a metastatic tumor in the mediastinum (8). The 8387 cell line reportedly was derived from a fibrosarcoma in the leg of a 25-year old woman (9,10,11), but according to Fogh La], (12), the tumor actually may have been an osteosarcoma rather than a fibroSarcoma. The HuT-14 cell line was reported to be derived from a focus of transformed cells arising after treatment of a normal human fibroblast cell line (KD) with 4-nitroquinoline-1-oxide (13). The designation HUT-l4 was not used in that paper, but was used in subsequent studies of these cells (14,15). Molecular analysis has revealed that HUT-14 cells did not arise from the KD cell line (16) but instead are derived from the 8387 cell line (17). The SW-982 and SW-684 cell lines were derived from fibrosarcomas in separate patients. SW-982 cells were taken from an axillary tumor in a 25-year old woman (12; J orgen Fogh, personal communication), while the SW-684 cells were obtained from a recurrent tumor at an unspecified site in a 68-year old man (18; J orgen Fogh, personal communication). The NCI cell line reportedly was derived from a fibrosarcoma, but details are unknown (C. Cooper, personal communication). The VIP:F- T cell line was derived from a primary culture of skin fibroblasts from an adult woman with a malignant melanoma (19). Morphologically transformed cells appeared in untreated 10- week old cultures, and these cells gave rise to 97 the VIP:F-T cell line. VIP:F-T cells are fibroblastic rather than melanocytic. It was not possible to verify the origin of this cell line using molecular analysis, since the parental cell line was not available (16). Assay for Tumorigenicity in Nude Mice BALB/c athymic nude mice from a closed colony were utilized as tumor hosts. Serum from heterozygous female breeders was tested regularly by Microbiological Associates (Rockville, MD), and was consistently free of antibodies to K virus, polyoma virus, murine hepatitis virus, ectromelia Virus, GD VII Virus, murine adenovirus, lymphocytic choriomeningitis virus, minute virus of mice, Sendai virus, pneumonia virus of mice, reovirus type 3, EDIM virus, murine cytomegalovirus, Car bacillus, and Mycoplasma pulmonis. Mice were maintained in accordance with Michigan State University guidelines in isolators illuminated by fluorescent light, with a 12 hour/ 12 hour light / dark cycle, temperature of 70- 75°F, and humidity of 30-50%. They were housed in plastic cages at up to five mice per cage. Cages had pine shaving bedding and individual water bottles. Mice were fed pelleted laboratory chow. Mice of both sexes were used as tumor hosts. From 1 x 106 to 5 x 106 sarcoma- derived or spontaneously-transformed human fibroblasts were injected into the subcutis on the dorsal midline of 6-10 week old mice. Afterwards, mice were examined weekly for tumor development. Mice were killed by C02 inhalation when tumors were 2-3 cm in diameter, or sooner if the tumor became ulcerated or the mouse showed signs of illness such as listlessness or weight loss. If no tumor developed within 8 months of injection, the cell line was considered non-tumorigenic. 98 Collection and Processing of Tissues Mice received a complete necropsy that included gross and microscopic examination of the tumor itself, axillary, cervical, and mesenteric lymph nodes, thyroid gland, parathyroid glands, adrenal glands, salivary glands, trachea, lungs, heart, liver, spleen, pancreas, and kidneys. Specimens for routine microscopic examination were fixed in neutral buffered 10% formalin. Sections of tumor for immunoperoxidase staining were fixed in freshly-prepared B-5 fixative for 2 hours, then transferred to 70% ethanol. Tissues were processed by routine histologic methods and imbedded in paraffin blocks. Tumors were serially sectioned into 2-3 mm thick slices and the entire tumor was imbedded, so that 3-10 tumor sections representing different areas were examined microscopically. Six micron sections were cut and stained with hematoxylin and eosin for general examination. Additional sections were cut and stained with Gomori’s trichrome stain or silver stain to demonstrate thick and thin collagen fibers. In some cases, frozen sections of formalin-fixed tissue were prepared and stained for lipid with Oil Red O. Additional sections of B-5-fixed tissue were cut and stained by an immunoperoxidase method for vimentin, desmin, S-100, alpha-l-antichymotrypsin, and Factor VIII antigen. Slides were stained in batches of 20, each batch including 18 tumors, a positive human tissue control, and a negative antibody control. Briefly, 3 micron sections were placed on poly- L-lysine coated slides and incubated overnight at 60°C. Slides were de-Zenkerized, rinsed, and immersed in methanol for 10 minutes. Endogenous peroxidase activity was blocked by incubation for 10 minutes in H202, followed by 5 minutes in normal goat serum. Slides were then labelled with one of five primary antibodies: monoclonal mouse anti-vimentin, monoclonal mouse anti-desmin, polyclonal rabbit anti-S-100, polyclonal rabbit anti-alpha- l-antichymotrypsin, and polyclonal rabbit anti-Factor VIII (all from Lipshaw, Detroit, MI). After treatment with the appropriate linking antibody and labelling reagent, slides were 99 exposed to chromogenic substrate for 5-20 minutes. Slides were counterstained with Mayer’s hematoxylin. 100 RESULTS General Observations The results of the assay of the cell lines for tumorigencity are summarized in Table 2. The SW-982 cell line did not produce tumors in any mice injected. This cell line also failed to produce tumors in previous experiments in our laboratory (20). The SW-684 cell line produced a tumor in only one of three mice. The other cell lines produced tumors in all mice injected. The microscopic characteristics of these tumors are described below and summarized in Table 3. Tumors grew as nodular masses within the subcutis. Microscopically, they were not encapsulated and did not compress surrounding tissues as they grew. Instead, enlarging tumors invaded adjacent adipose tissue and skeletal muscle as they grew, entrapping individual adipocytes and muscle fibers within the tumor mass. Tumors did not invade bone or cartilage, nor penetrate through the thoracic wall in any mouse, although this may reflect the relatively early stage in tumor growth at which mice were killed rather than an inability of tumors to invade these tissues. Spontaneous metastases were not found in any tumor- bearing mouse. This also may reflect the relatively short time the tumors were allowed to grow. We stained tumors produced by six of the seven cell lines using an immunoperoxidase method and, as expected, all tumors contained cells that stained positively for vimentin intermediate filaments (Table 4). In fact, such staining was taken to indicate that processing and fixation had been sufficient to preserve tissue antigens. Positive staining for vimentin also gave evidence that the tumors were composed of human cells rather than mouse cells transformed i_n vivo, because the monoclonal antibody used was specific for an epitope on human vimentin filaments. 101 102 M The SHAC cell line formed densely cellular tumors consisting of short interwoven bundles of cells that occasionally intersected in a manner reminiscent of a storiform pattern (Figure 1). Focal necrosis was not prominent, although apoptosis was regularly present. Trichrome and reticulin stains revealed that collagen fibers were scarce. Fibers tended to be thin and usually were located at the periphery of individual fascicles in association with stromal blood vessels, suggesting that they were the product of host cells rather than tumor cells. Tumors cells were closely packed with very little cytoplasm and indistinct cell borders (Figure 2). Nuclei were moderately pleomorphic but generally oval with evenly dispersed, finely granular chromatin and 1 or 2 small nucleoli that were visualized best in B-5-fixed tissue. Mitoses were moderately common (3-5/hpf). Tumors produced by SHAC cells contained scattered cells that stained positively for desmin (Figure 3). Desmin-positive cells had diffuse cytoplasmic staining that was slightly less intense than that of mouse skeletal muscle fibers and vascular smooth muscle cells in the same section. HT1080 Cells The HT1080 cell line formed tumors consisting of solid sheets of closely packed but distinct cells with a scant fibrovascular stroma (Figure 4). Focal necrosis was only occasionally present. At times the tumor cells were segregated into discrete small clusters by the stroma. This arrangement, coupled with the epithelioid appearance of the cells, made the tumors resemble carcinomas rather than sarcomas. Fascicle formation was never observed. Trichrome and reticulin stains revealed that collagen fibers were scarce and always associated with stromal elements; there was no evidence of collagen production by tumor cells. Tumor cells were very large with a moderate amount of basophilic cytoplasm (Figure 5). They were generally round or polyhedral and usually had distinct cell borders. 103 Each cell contained a single, centrally-located nucleus. Nuclei were somewhat pleomorphic but tended to be oval or indented (reniform). They contained a granular chromatin that was concentrated along the nuclear membrane, making it easy to see the 3 to 5 nucleoli within each nucleus. Mitoses were very common (5-10/hpf). m The 8387 cell line formed tumors that consisted of short interwoven bands of cells intersecting at irregular angles that sometimes produced a storiform pattern (Figure 6). Multifocal necrosis was a consistent feature of these tumors, and apoptosis was also common. Thick and thin collagen fibers were common and surrounded virtually every tumor cell. Tumor cells were spindle shaped or crescentic with fibrillar basophilic cytoplasm and cell borders that were fairly distinct against the eosinophilic collagenous matrix (Figure 7). Nuclei were large and moderately pleomorphic. They were generally round or oval with a finely granular chromatin that, while evenly dispersed, did not obscure the 1 to 3 large magenta nucleoli that each nucleus contained. The mitotic rate of these tumors was low (1- 3/hpf). HuT-l4 Cells As expected, the HuT-14 subline of the 8387 cell line produced tumors very similar to those of 8387 cells. The HUT-14 cell line produced tumors consisting of interwoven bundles of spindle cells that sometimes formed a storiform pattern (Figure 8). In contrast to tumors produced by 8387 cells, necrosis was not a common feature of tumors produced by HUT-14 cells. Also in contrast to tumors produced by 8387 cells, tumors produced by HUT-14 cells frequently had areas where cells were somewhat loosely arranged within a pale staining, foamy, myxoid matrix. Oil Red O staining for fat revealed only occasional 104 small lipid droplets within tumor cells; no signet ring cells or physaliferous cells were found. Collagen fibers were common in these tumors as they were in tumors formed by 8387 cells, and almost every tumor cell was surrounded by collagen. Tumor cells were spindle shaped with fibrillar cytoplasm (Figure 9). Nuclei were moderately pleomorphic, ranging from oval to elongated with a finely granular chromatin and 1 to 3 small nucleoli. Mitoses were not common (1-3/hpf).The tumors formed by HUT-14 cells stained positively for S-100 antigen (Figure 10). Staining was diffuse and cytoplasmic, but slightly less intense than that of mouse peripheral nerve fibers within the same section. NCI Cells The NCI cell line formed tumors consisting of interwoven fascicles of spindle cells (Figure 11). At low magnification the tumors resembled those produced by 8387/HUT—14 cells, but the cells that comprised NCI tumors were more pleomorphic. Focal necrosis was not common, but apoptosis was. Collagen fibers were common throughout the tumors. Tumor cells were variable in size and shape but tended to be spindle shaped with scant, fibrillar, eosinophilic cytoplasm (Figure 12). Nuclear pleomorphism was great. Nuclei varied from round with multiple indentations to oval or elongated. Chromatin was coarsely granular and clumped along the nuclear membrane. Nucleoli were large, multiple, and often bizarrely-shaped. Mitoses were not common (1-3/hpf). SW-684 Cells SW-684 cells produced a tumor in only one of three mice injected. In that mouse, the tumor consisted of cells arranged in short, interwoven fascicles mixed with solid sheets. In many areas the cells were separated into clusters by a delicate fibrovascular stroma. There were extensive coalescing areas of necrosis within the tumor (Figure 13). Cells were 105 large and extremely pleomorphic (Figure 14). They varied from round or oval to polyhedral in shape, with granular basophilic cytoplasm and a single large nucleus. Nuclei were round or oval, sometimes reniform, with coarsely clumped chromatin located mostly along the nuclear membrane. There were 1 to‘3 very large nucleoli in each nucleus. Mononuclear tumor giant cells were easy to find, and mitotic figues were moderately common (3-5/hpf). VIP:F-T Cells Of all cell lines tested, VIP:F-T cells formed tumors that most closely resembled human fibrosarcomas. Tumors consisted of long interwoven fascicles of large spindle cells that often intersected at regular angles to form a herringbone pattern (Figure 15). Multifocal necrosis was a prominent and consistent feature of these tumors. Trichrome and silver staining revealed that thick collagen fibers were common throughout the tumor and were associated with nearly every tumor cell. Thin collagen fibers were scarce and confined to the stroma. The cells had a small amount of fibrillar eosinophilic cytoplasm (Figure 16). Individual cell borders were difficult to discern on HE-stained sections because the cytoplasm could not be distinguished easily from the collagenous matrix between cells. However, on trichrome-stained sections the elongated shape of the cells was easy to see. Nuclei were also elongated and moderately pleomorphic; many had the blunt ends typical of leiomyosarcoma cells instead of the tapered ends usually associated with fibrosarcoma cells. Chromatin was confined to the nuclear membrane giving the nuclei a vesicular appearance. Narrow bands of chromatin often bridged the width of the nucleus or connected nucleoli to the nuclear membrane. Nucleoli were generally small and multiple. Mitoses were very common (5-10/hpf). DISCUSSION The tumors produced by the sarcoma-derived cell lines and the spontaneously- transformed cell line varied in their microscopic features but, with the exception of those produced by HT1080 cells, were partly or entirely spindle cell sarcomas with cells arranged in interwoven bands. On the other hand, the microscopic features of the tumors produced by each particular cell line were distinctive, consistent, and reproducible, so that it was easy to recognize which cell line had produced a particular tumor even without prior knowledge of which cell line had been injected. Such phenotypic variety among tumors produced by these cell lines is not unexpected. Fogh and Trempe (21) pointed out over a decade ago that cell lines derived from the same type of tumor, based on pathologists’ diagnoses, may have different characteristics, and that as a consequence, studies of many cell lines derived from tumors of similar histopathologic appearance was the best approach to investigating carcinogenesis. There are discrepancies in some of the published reports of the derivation of the sarcoma-derived cell lines used in the studies reported here. In view of the conflicting reports regarding the histopathologic diagnosis of the original tumor from which the HT1080 and 8387 cell lines were derived, and whether it was primary or metastatic, and current ambiguities in the classification of soft tissue sarcomas (discussed in 22-26) it is not surprising that cell lines supposedly derived from the same type of tumor differ in many characteristics, including the type of tumor they produce. Transformation of mouse cells in association with injected human tumor cells has been reported many times (27-35) and is a potential complication of assays for tumorigenicity in nude mice. Evidence that the tumors we studied were composed of human cells rather than mouse cells comes from our finding that they stained with antibody specific for human vimentin intermediate filaments. 106 107 The tumors were not expected to stain positively for desmin intermediate filaments or S-100 protein. Desmin appears to be expressed only in normal muscle tissue and in tumors derived from such cells, and‘its presence is routinely utilized by pathologists to distinguish muscle cell tumors from other types of sarcomas (36, 37). However, there have been rare reports of fibrosarcomas that stain positively for desmin (38, 39), and with the electron microscope myofilaments can be seen within cells of some fibrosarcomas (40), prompting investigators to dub such tumors myofibroblastomas (41). Recently, Skalli e_t a_l. (42) demonstrated that the semimalignant proliferative lesions of fibromatosis contain myofibroblasts that have both vimentin and desmin intermediate filaments. Furthermore, the SHAC cell line that produced tumors containing desmin-positive cells was derived from a gastric sarcoma, and smooth muscle tumors are the most common type of sarcoma in the human gastrointestinal tract (24). The expression of desmin intermediate filaments by the SHAC cell line may reflect a general tendency of gastrointestinal sarcomas toward myogenic, or at least myofibroblastic; differentiation. The positive staining for S-100 of tumors produced by Hut-14 cells is more difficult to explain. The 8387 cell line from which this subline was derived produced tumors that were S-100-negative. Clonally-derived populations are known to differ from their parent cell line and from each other in many characteristics, including growth characteristics in_vim (43, 44). The fact that virtually every cell within the tumors produced by HUT-14 cells expressed S-100 protein is consistent with the hypothesis that the HUT-14 cell line was derived from a single variant cell with an activated S-100 gene. Although the HUT-14 and 8387 cell lines produced tumors that were quite similar, necrosis was prominent in tumors formed by 8387 cells but almost never seen in tumors formed by HuT-14 cells. Also, tumors produced by HUT-14 cells grew much more rapidly than tumors produced by 8387 cells, suggesting that the two cell lines differed in other properties besides S-100 expression. S- 100 protein is expressed in a limited number of normal tissues and tumor types. Among 108 spindle cell tumors, those exhibiting chondrocytic, neurogenic, or melanocytic differentiation are generally S-lOO-positive, while fibrosarcomas are S-100-negative (45, 46). Tumors produced by HUT-l4 cells showed no evidence of cartilage matrix production by light microscopy and no evidence of Schwann cell or melanocytic differentiation by electron microscopy (unpublished observations), so the activation of S-100 did not appear to be part of a general program of non-fibroblast differentiation. More likely, S-100 expression resulted from activation of a single gene in this subline. The tumors produced by the sarcoma-derived cell lines and the spontaneously- transformed fibroblast line resembled those produced by human fibroblasts transformed i_n m by m oncogene transfection (4) in latency period, cytologic features, morphologic patterns of growth, and failure to mestastasize spontaneously from the subcutis. In addition, comparison of the tumors with each other and with published descriptions of spontaneous human soft tissue sarcomas revealed that: a) The HT1080 cell line, whose cells contain an N-gLs oncogene (5), made round cell sarcomas that closely resembled those produced by one line of MSU-1.1 fibroblasts transformed by an N-@ oncogene derived from the human leukemia cell line 8402. These tumors did not resemble fibroblastic tumors that arise spontaneously in humans. b) The SHAC cell line, whose cells contain an N-pas oncogene and an amplified c- myg gene (6), made spindle cell sarcomas in which some of the cells contained desmin intermediate filaments. Desmin-positive spindle cell sarcomas were also produced by two strains of N-m-transformed MSU-1.1 fibroblasts. MSU-1.1 fibroblasts contain a v-my_c oncogene, so N-as transformants have alterations in the same two oncogenes that are altered 109 in SHAC cells. These tumors resembled myofibroblastomas that arise spontaneously in humans. c) The HuT-14 subline made spindle cell sarcomas that were S-100-positive, and so did one line of MSU-1.1 fibroblasts transformed by the H-@ oncogene derived from the human bladder carcinoma cell line T24. These tumors resembled neurofibrosarcomas that arise spontaneously in humans. d) The VIP-F:T cell line made spindle cell sarcomas with prominent herringbone patterns and abundant collagen fibers, and these tumors resembled those produced by several lines of MSU-1.1 fibroblasts transformed by the H-rgs and N-grs oncogenes. These tumors resembled fibrosarcomas that arise spontaneously in humans. e) The SW-684 cell line made a pleomorphic sarcoma with mononuclear giant cells that resembled those produced by KMST-6 fibroblasts or GM637 fibroblasts transformed by the T24 H-flfi oncogene. These tumors resembled fibrosarcomas or malignant fibrous histiocytomas that arise spontaneously in humans. f) No sarcoma-derived or spontaneously-transformed cell line produced myxoid sarcomas similar to those produced by several lines of K-w-transformed MSU-1.1 fibroblasts, pleomorphic sarcomas with multinucleated giant cells similar to those produced by one line of N-gs-transformed MSU-1.1 fibroblasts, or spindle cell sarcomas with a whorling pattern similar to those produced by one line of H-rgg-transformed MSU-1.1 fibroblasts. These studies reveal that the. range of tumor types produced by m-transformed flbrOblasts in previous studies (4) was not an artifact of their i_n_ vitro creation. Cell lines F______‘__4 110 derived from human sarcomas or spontaneously-transformed m \Litr_o also produced a range of tumor types, including sarcomas that expressed desmin or S-100, supporting the idea that 'm m transformation by @ oncogene transfection is a relevant model for i_n m carcinogenesis. REFERENCES (1) Fry, D.G., Milam, L.D., Dillberger, J .E., Maher, V.M., McCormick, J .J . Malignant transformation of immortalized human fibroblasts by transfection with v-Ki-w. Oncogene Res (in press): (1989) (2) Hurlin, PJ., Fry, D.G., Maher, V.M., McCormick, J .J . Morphologic transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-w oncogene. Cancer Res 47: 5752-5757 (1987) (3) Wilson, D.M., Yang, D., Dillberger, J.E., Dietrich, S.E., Maher, V.M., McCormick, J .J . Malignant transformation of an infinite life span, human fibroblast cell line by a transfected N-Qs oncogene. 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J Natl Cancer Inst 58: 209-214 (1977) Mukherji, B., MacAlister, T.J., Guha, A., Gillies, C.G., Jeffers, D.C., Slocum, S.K. Spontaneous i_n vitro transformation of human fibroblasts. J Natl Cancer Inst 73: 583-593 (1984) Schilz, R.J., Dillberger, J.E., Maher, V.M., McCormick, J.J. Overexpression of multiple growth factor genes by fibrosarcoma-derived and other transformed human fibroblasts in culture. Cancer Res (in press): (1989) Fogh, J ., Trempe, G. New human tumor cell lines. In Human Tumor Cells in vitro, J. Fogh (ed). Plenum Press, New York, NY: 115-150 (1975) Hajdu, 8.]. Pathology of Soft Tissue Tumors. Lea & Febiger, Philadelphia, PA: 1- 164 (1979) Fine, G., Hajdu, S.I., Morton, D.L., Eilber, F.R., Suit, H.D., Weiss, S.W. Soft tissue Sarcomas. Classification and treatment (a symposium). Pathol Ann 17: 155-195 (1982) Das Gupta, T.K. Pathology of soft tissue sarcomas. Tumors of the Soft Tissues, T.K. Das Gupta (ed). Appleton-Century-Crafts, Norwalk, Connecticut: 22-183 ( 1983) 4___‘——‘ (25) (26) (27) (28) (299 (30) (31) (32) (33) (34) (35) (36) (37) (38) 113 Enjoji, M., Hashimoto, H. Diagnosis of soft tissue sarcomas. Pathol Res Pract 178: 215-226 (1984) Katenkamp, D., Raikhlin, N.T. Stem cell concept and heterogeneity of malignant soft tissue tumor -- a challenge to reconsider diagnostics and therapy? Exp Pathol 28:3-11(1985) Houghton, J.A., Taylor, D.M. Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in irnmune-deprived mice. Br J Cancer 37: 199-212 (1978) Tveit, K. M. Pihl, A. Do cell lines in v_i__tro reflect the properties of the tumours of origin? A study of lines derived from human melanoma xenografts. Br J Cancer 44: 775- 786 (1981) Goldenberg, D.M., Pavia, R.A. Malignant potential of murine stromal cells after transplantation of human tumors into nude mice. Science 212: 65-67 (1981) Goldenberg, D.M., Pavia, R.A. I_n M horizontal oncogenesis by a human tumor in nude mice. Proc Natl Acad Sci USA 79: 2389-2392 (1982) Beattie, G.M., Knowles, A.F., Jensen, F.C., Baird, S.M., Kaplan, N.O. Induction of sarcomas in athymic mice. Proc Natl Acad Sci USA 79: 3033-3036 (1982) Costa, J ., Yee, C., Tabson, A. h; vivo transformation of mesenchymal murine cells in immunosuppressed animals bearing human xenografts. In Immune-Deficient Animals. 4th Int. Workshop on Immune-Deficient Animals in Exp. Res., Chexbres 1982. Karger, Basel: 230-234 (1984) Stark, M., Schlipkoter, H.W. Xenotransplants of human carcinomas. In Immune-Deficient Animals. 4th Int. Workshop on Immune-Deficient Animals in Exp Res., Chexbres 1982. Karger, Basel: 250-255 (1984) Sparrow, S, Jones, M, Billington, S. ,,Stace B. The i_n v__ivo malignant transformation of mouse fibroblasts in the presence of human tumour xenografts. Br J Cancer 53: 793- 797 (1986) Gupta, V., Rajaraman, S., Gadson, P., Costanzi, J.J, Primary transfection as a mechanism for transformation of host cells by human tumor cells implanted in nude mice. Cancer Res 47: 5194-5201 (1987) Gabbiani, G., Kapanci, Y., Barazzone, P., Franke, W.W. lmmunochemical identification of intermediate-sized filaments in human neoplastic cells. A diagnostic aid for the surgical pathologist. Am J Pathol 104: 206-216 (1981) Brooks, J .J . Immunohistochemistry of soft tissue tumors: Progress and prospects. Hum Pathol 13: 969-974 (1982) Miettinen, M., Lehto, V- P, Badley, R.A., Virtanen, 1. Expression of intermediate filaments in soft- tissue sarcomas. Int J Cancer 30: 541- 546 (1982) #___‘.—4 (39) (40) (41) (42) (4(3) (4*4) (45) (46) 114 Osborn, M., Weber, K. Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 48: 372-394 (1983) Churg, A.M., Kahn, L.B. Myofibroblasts and related cells in malignant fibrous and fibrohistiocytic tumors. Hum Pathol 8: 205-218 (1977) Ghadially, F .N., McNaughton, J .D., LaLonde, J-M.A. Myofibroblastoma: A tumour of myofibroblasts. J Submicrosc £23501 15: 1055-1063 (1983) Skalli, O., Schurch, W., Seemayer, T., Lagace, R., Montandon, D., Pittet, B., Gabbiani, G. Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest 60: 275-285 (1989) - Heppner, G.H. Tumor heterogeneity. Cancer Res 44: 2259-2265 (1984) Schirrmacher, V. Cancer metastasis: Experimental approaches, theoretical concepts, and impacts for treatment strategies. Adv Cancer Res 43: 1-73 (1985) Kahn, H.J., Marks, A., Thom, H., Baumal, R. Role of antibody to $100 protein in diagnostic pathology. Am J Clin Pathol 79: 341-347 (1983) Cocchia, D., Lauriola, L., Stolfi, V.M., Tallini, G., Michetti, F. S-100 antigen labels neoplastic cells in liposarcoma and cartilaginous tumours. Virchows Arch |Pathol Anat] 402: 139-145 (1983) 115 Table 1. Origin of Sarcoma-derived and Spontaneously-transformed Cell Lines Cell Line SHAC HT1080 8387 HUT-l4 SW-684 SW-982 N CI VIP:F—T Derivation gastric fibrosarcoma fibrosarcoma (primary or metastatic?) in the hip fibrosarcoma (osteosarcoma?) subline of 8387 recurrent fibrosarcoma--site unspecified axillary fibrosarcoma fibrosarcoma--site unspecified skin fibroblast culture transformed i_n vitro References 6 7,8 9,10,11,12 13,14,15,17 18 12 C. Cooper, personal communication 19 116 Table 2. Tumors Produced by Human Sarcoma-derived Cells or Spontaneously-transformed Human Fibroblasts Mean Cell Tumors Produced/ . Latency Line Mice Injected Period Mogphologic Classification SHAC 5 /5 20 days Spindle cell sarcoma HT1080 4/4 21 days Round cell sarcoma 83 87 5/5 88 days Spindle cell sarcoma I—IuT-14 3 /3 28 days Spindle cell sarcoma NCI 3 /4 63 days Spindle cell sarcoma SW-982 0 /4 —————————— VIP:F-T 4/4 17 days Spindle cell sarcoma 38822: .eeEEoO Eepchw E08238 .ceEEeO 025% .eotmem £8 29% .ceEEeoeD 882602 Room .eeEEoocb Excess/w 188328 ..eeEEeO Hempeecafl :83 6088823 380th .eteom £8 29% 6088083 380th .eotmom tee—PH. E 29:5. 5 mmmetooz team eewu=eO Eesmm beenmctteg .me_o_om£ emecm 836mg was 385 6380883 8028 we “octet: .me‘emomfl team EopSt E 80825 $2288 team 8022 an 885:: .medemomfi team 38:» pmem 885: E teteofi £2283 team £8 23% £8 EEm .oEBeEeeE mzee efipeam £8 seesaw 28 been; 230 eeset seesaw £8 swim emu? =00 EmfiEeueE aazeem Fiwfl> vwo$>m HOZ 3 9:2 wwmw owoitm Uém 0E...— :00 BEBE pestemmeebtnm:eecficemm to £60 pe>tepé8eetem BEBE 3 peoepetm 39:51 me metamem ommeometouz .m 29d. 117 118 Table 4. Immunoperoxidase Staining of Tumors Produced by Human Sarcoma-derived Cells and Spontaneously-transformed Human Fibroblastsa Cell ' Alpha-l-anti Factor VIII E Vimentin Being M chmoLrypsin m SHAC + + b - — - HT1080 + — - - _ 8387 + - - - - HuT— 14 + - + c - - NCI + — - - _ VIP:F-T + - - - - aExcept for the SHAC line, two tumors of each type were stained, and 2-4 sections of each tumor were examined. Four tumors produced by SHAC cells were examined. Tumors produced by SW-684 cells were not examined. t’I‘here was diffuse cytoplasmic staining of scattered tumor cells. cThere was diffuse cytoplasmic staining of most tumor cells. ILLUSTRATIONS -'€ a“ at? (mgr-h .. 0“ 1. .ar .3 «3&1 Q. s \ w: . dig“ . . .. .1; ~~. "-- 1.41». .. tn } 3.1.1.192 (grayish 1%., "‘~~ly%:\;':zr7"‘ ' Ms ‘ e ‘ 1:54?“ ‘. , , -r‘lfiéhi'fic i. tiff; Nix".- >5- '1.“ Figure l. Sarcoma produced by SHAC cells. Cells are arranged in short interwoven fascicles (HE, 200x). 119 120 Figure 2. Sarcoma produced by SHAC cells. Closely packed cells with numerous mitotic figures (arrows) (B-5, 500x). 121 Figure 3. Sarcoma produced by SHAC cells stained by the immunoperoxidase method for desmin: a) Staining of murine skeletal muscle and arteriolar smooth muscle at periphery of tumor. Compare intensity of staining to that of tumor cells in b) and c). b) SHAC tumor with several strongly desmin-positive cells. c) SHAC tumor with mitosis of desmin-positive cell (all at 400x). 122 H. .W.. is . .V..I.~‘)rrr .v V. ..v a .....r “I -L .W. ..,}... r Epithelioid cells arranged in solid sheet Figure 4. Sarcoma produced by HT1080 cells. (HE, 200x). Figure 5. Sarcoma produced by HT1080 cells. Very large cells with oval and indented nuclei (HE, 500x). 124 Figure 6. Sarcoma produced by 8387 cells. Short interwoven bands of cells (B-5, 200x). 125 Figure 7. Sarcoma produced by 8387 cells. Moderately pleomorphic spindle cells (B-5, 500x). 126 Figure 8. Sarcoma produced by HUT-l4 cells. Cells arranged in short interwoven bands. Compare with Figure 6 (HE, 200x). 127 Figure 9. Sarcoma produced by HuT-14 cells. Note similarity between this tumor and Figure 7 (HE, 500x). ‘1 128 Figure 10. Sarcoma formed by HuT—14 cells stained by the immunoperoxidase method for S-100: a) Murine peripheral nerve. Compare intensity of S-IOO-positive reaction to that of tumor cells in b). b) Tumor with diffuse cytoplasmic staining of most cells (both at 400x). 129 Figure 11. Sarcoma produced by NCI cells. Thick collagen fibers and entrapped adipocytes are prominent (HE, 200x). 130 Figure 12. Sarcoma produced by NCI cells. Pleomorphic spindle cells (HE, 500x). 131 Figure 13. Sarcoma produced by SW-684 cells. Large area of necrosis in the tumor (HE, 200x). uh 132 Figure 14. Sarcoma produced by SW-684 cells. Large and pleomorphic cells, with tumor giant cell (arrow) (HE, 500x). A 133 (m... w - w . . a? . . . ...» .mw SW14 «H .. . J K a o . l.» ... _ ... pattern (HE, 200x). ...f / IV/ 1 . . s . .... . «.1 M. . . .v, ..n .. ... . . x . . \i \. . .., . P. s it 7../ , .. .. . . \ .. . . _. 3.1..» .71.“? ..,. ..,. . . . . . .. .. . x . OI 21F.ll 0.1;).Ilfz “171/ D. I. . a /4 If 0.1 .6 . \ ...... . . . .. ..ll « . .z. . l . . . L ,. v . . . Rank“ all, Vivi- Llrrlntrl. . .. Figure 15. Sarcoma produced by VIP:F-T cells. Broad bands of cells in a herringbone 134 ... a I. e F I I. . . I; Figure 16. Sarcoma produced by VIP:F—T cells. Spindle cells with elongated nuclei (HE, 500x). EXPERIIVIENTAL METASTASIS OF RAS-TRANSFORMED HUMAN FIBROBLASTS John E. Dillbergera, Dennis G. Fryb, Daniel M. Wilsonc, Peter J. Hurlind, Lonnie Milame, Veronica M. Maherf, and J. Justin McCormickf Carcinogenesis Laboratory, Michigan State University, East Lansing, MI 48824-1316 Authors’ current affiliations: a'Toxicology Department, Merrell Dow Research Institute, PO. Box 68470, Indianapolis, IN 46268-0470 ‘ bDept. of Molecular Biology, Abbott Laboratories, Abbott Park, IL 60064 cChemical Industries Institute of Toxicology, PO. Box 12137, Research Triangle Park, NC 27709 dF red Hutchinson Cancer Research Center, M616, 1124 Columbia Street, Seattle, WA 98104 eCarcinogenesis Laboratory, Michigan State University, East Lansing, MI 48824-1316 Send galley proofs to: Dr. J. E. Dillberger Merrell Dow Pharmaceuticals Inc. PO. Box 68470 Indianapolis, IN 46268-0470 Send requests for reprints to: Carcinogenesis Laboratory West Fee Hall Michigan State University East Lansing, MI 48824-1316 Running Title: Experimental Metastasis 136 ABSTRACT Cell strains derived from infinite life span human fibroblasts transformed i_n vile by transfection with a K—Qs, H-Qs, or N-r_a_s oncogene were injected intravenously and intraperitoneally into athymic nude mice to see if @ oncogenes influence the metastatic capacity of malignantly transformed human fibroblasts. The m-transformed cells metastasized via the bloodstream or by implantation in the peritoneal cavity. Metastasis was influenced by the transfected @ oncogene and the recipient cell line. Metastatic tumors often arose in skeletal muscle, but this was because w-transformed cells spread from the injection site via lymphatics rather than because muscle was a more favorable tissue for tumor formation by @-transformed cells. The microscopic features of metastatic tumors almost always resembled those of primary tumors produced by the same r_as- transformed cell strains. These results confirm that Lzfi oncogenes can transform non- tumorigenic infinite life span human fibroblasts into tumorigenic cells capable of experimental metastasis, and provide direct evidence that m oncogenes may play a role in the metastatic process in human cancer. Key Words: H-m, K-grs, N-m, metastasis, fibroblast 137 INTRODUCTION Oncogenes of the as family are capable of malignantly transforming rodent fibroblasts, and McCormick and his colleagues recently succeeded in malignantly transforming infinite life span human fibroblasts with K—m, N-ras, or H-l‘fi oncogenes (11,15,33). The resultant cell strains produced progressively-growing, invasive sarcomas with a variety of microscopic appearances when injected subcutaneously into athymic nude mice (7). These sarcomas were characterized by cytologic features, patterns of growth, and a panel of immunoperoxidase stains, and found to be similar in many respects to tumors produced by human sarcoma-derived cell lines and a spontaneously-transformecl human fibroblast line (8). Transformation of rodent fibroblasts by m oncogenes has been reported to confer metastatic ability on cells, asjudged by both spontaneous metastasis in tumor-bearing mice and experimental metastasis, in which tumors form after intravenous inoculation of transformed cells (2,3,4,5,6,9,10,13,18,25,29,30,32,34). In prior studies with gs-transformed human cell strains, spontaneous metastasis from primary subcutaneous tumors was never observed (7). Whether this reflected an inability of these tumorigenic cells to metastasize or simply a lack of sufficient time for spontaneous metastasis to occur was unknown, since tumors appeared soon after injection and grew rapidly so that tumor-bearing mice seldom survived longer than 4 weeks. To determine if m-transformed human fibroblasts could metastasize, cells were injected into the bloodstream or peritoneal cavity of athymic nude mice. To test whether or not the transfected fi-oncogene influenced experimental metastasis, we compared metastatic tumor formation by cell strains derived from a single receipient fibroblast line transformed by three different @ oncogenes. To test whether or not the nature of the recipient cell line influenced experimental metastasis, we compared 138 metastatic tumor formation by cell strains derived from three different fibroblast lines transformed by transfection with the same H-w oncogene. To investigate whether or not the microscopic features of metastatic'tumors would resemble those of subcutaneous tumors produced by the same cell strain, we compared metastatic tumors to primary subcutaneous tumors produced by m-transformed cell strains in this study and previous studies (7). We found that ra_s oncogenes transformed infinite life span human fibroblasts not only into tumorigenic cells, but also into fully malignant tumor cells capable of experimental metastasis Via the circulation or implantation within body cavities. The transfected oncogene and the nature of the recipient cell line both influenced metastasis. 139 EXPERIMENTAL DESIGN Cell lines used in these studies are listed in Table 1. In initial experiments we evaluated the metastatic ability of ra_s-transformed fibroblast strains by injecting cells into the tail vein or peritoneal cavity of mice. As controls, we also injected mice with spontaneously-transformed human fibroblasts (cell line L551-3T) and with cells from the human sarcoma-derived cell line HT1080. Besides injecting transformed cells intravenously we also injected them subcutaneously in additional mice to confirm that the cells were viable and tumorigenic. After being injected with transformed cells, mice were examined weekly and maintained for six months or until metastatic tumors developed. All mice received complete necropsies that included microscopic examination of major organs for metastatic tumors. To investigate the influence of m oncogenes on metastasis, we compared metastasic tumor formation by the various m-transformed MSU-1.1 fibroblast strains. To investigate the influence of the recipient cell line on metastasis, we compared metastatic tumor formation by three cell strains derived from three different infinite life span fibroblast lines transformed by the same H-w oncogene. To investigate factors affecting the distribution of metastatic tumors, a single fl-transformed cell strain (N-w-3T) was injected intracardially, intravenously, intramuscularly, and subcutaneously. 140 RESULTS AND DISCUSSION General Observations All of the cell lines and cell strains tested produced tumors when injected subcutaneously or intraperitoneally, indicating that the inocula contained viable tumorigenic cells. Mice that received intraperitoneal inoculations developed multiple tumors within their abdominal cavities. Abdominal tumors not only grew as nodular masses on the parietal and visceral peritoneum (Figure 1), but also invaded underlying tissues in many mice (Figure 2). The number of mice that developed metastatic tumors following intravenous inoculations are shown in Tables 2 and 3. All three m oncogenes could confer upon infinite life span human fibroblasts the prime attribute of cancer cells, the ability to metastasize. Metastatic tumors developed at various sites, and we were uncertain at first whether we could compare the rate of tumor growth at these different sites because our endpoint for subcutaneous or intramuscular tumors was a diameter of 1 cm, but our endpoint for intrathoracic or intraabdominal tumors was debilitation or palpable tumor. In fact, these two endpoints turned out to be very similar, since by the time metastatic tumors in the abdomen or thorax reached 1 cm in diameter they either caused obvious dyspnea or were palpable through the body wall, and metastatic tumors that arose in skeletal muscles could be monitored directly. Effect of Transfected ras Oncogene on Metastasis The E oncogenes differed in their ability to confer metastatic properties upon the MSU-1.1 fibroblast line (Table 2). One of the mice injected with the N-Las-3T cell strain was killed 45 days after injection because it was losing weight. At necropsy we found a 141 142 severe malocclusion with overgrth of incisors that probably prevented the mouse from eating and accounted for the weight loss. No tumors were found in any tissues from this mouse, but the rest of the mice that received N-w-3T cells intravenously developed metastatic tumors, giving an overall frequency of metastasis of 90%. Results were similar with the sarcoma-derived cell line, HT1080, which carries an N-pa_s oncogene (14). One mouse that received HT1080 cells developed a bacterial otitis media and when we killed it 56 days after injection we found no tumors; however, the remaining mice that received HT1080 cells developed metastatic tumors. A smaller percentage of mice receiving N-fi- 5T cells (50%) developed tumors. Only one of the two H-w-transformed cell strains (H- m-2T) formed metastatic tumors at all, and it did so with a frequency of 40%. Although both K—m-transformed cell strains formed metastatic tumors, they did so with low frequencies (10% and 30%). The spontaneously-transformed MSU-1.1 fibroblast line, L551- 3T, also had a low frequency of experimental metastasis (27%). The m-transformed cell strains and control cell lines fell into 2 groups with respect to the time required for metastatic tumors to form following intravenous inoculation. One N-fl-transformed cell strain (N -@-3T), the spontaneously—transformed fibroblast line L551- 3T, and sarcoma-derived cell line HT1080 formed metastatic tumors in 9-12 weeks. The other N-w-transformed cell strain (N-La_S-5T), both K—m-transformed cell strains, and the metastatic H-m-transformed cell strain (H-gs-2T) took 17-20 weeks to form metastatic tumors. The relationship between E oncogenes and metastasis has been the subject of numerous investigations in recent years (16,17) and was recently reviewed by Nicolson (23). Most studies have involved transfection of @ oncogenes into rodent fibroblasts. Transfection of H-ra_s or N-gts oncogenes into mouse NIH 3T3 cells, mouse C3H 10T1/2 cells, rat fibroblasts, or Chinese hamster lung fibroblasts makes the cells capable of 143 spontaneous and experimental metastasis (2,3,4,6,9,10,18,25,28,30,32,34). Interpretation of these rodent cell transfection experiments is difficult, however, because often even untransfected or mock-transfected mouse and rat cells prove capable of spontaneous and experimental metastasis (6,10,12,25,31). We could find only one report of a metastasis study using gs-transfected human cells. Agnor e_t a_l. (1) transfected a cellular or viral H-Qs oncogene into human breast carcinoma cells and found that _rfi had no effect on the frequency of spontaneous metastasis. Our studies extend the observations made in rodent cell systems to human cells, providing. the first direct evidence that @ oncogenes may play a role in the metastatic process in human cancer. Our results suggest that the ability of @ oncogenes to induce a metastatic phenotype is influenced by several factors, one of which is the transforming gene itself. For example, although N-rgs and K-@ oncogenes each made MSU-1.1 fibroblasts metastatic, they were not equally efficient at doing so. These two oncogenes differ in at least two ways. First, the activating mutations are different: the 8402 N-@ oncogene has a single activating mutation in codon 12, while the K-@ oncogene has two such mutations in codons 13 and 59. Second, although the two genes belong to the same gene family they have somewhat different sequences and presumably have different normal physiologic functions. Investigation of the relationship between specific @ oncogenes and metastasis was complicated by the fact that each of the gas oncogenes used to transform MSU-1.1 fibroblasts was introduced in a different plasmid construction. Attempts to construct a series of plasmids that are identical except for the oncogene they carry are currently underway. 144 Effect of Recipient Cell Line on Metastasis The nature of the parent cell line influenced acquisition of metastatic ability, as illustrated by comparing metastatic tumor formation by the H-w-transformed MSU-1.1, KMST—6, and GM637 fibroblasts (Table 3). Like the H-fl-3T cell strain, H-rgs- transformed GM637 fibroblasts failed to form metastatic tumors when injected intravenously. However, 7 of 9 mice injected intravenously with H—fl-transformed KMST- 6 fibroblasts did develop metastatic tumors. An eighth mouse injected with H-@- transformed KMST-6 fibroblasts developed signs suggestive of pulmonary metastasis but died unexpectedly on day 55 and was too autolyzed to be necropsied. Metastatic tumors produced by H-w-transformed KMST—6 fibroblasts developed rapidly, with an average latency period of 65 days. Muschel e_t a_l. (18) got similar results following transfection of infinite life span mouse fibroblast lines with v-H-w. They found that H-m-transformed NIH 3T3 cells made experimental metastases in nude mice but H-gs-transformed C127 cells did not. Effect of Other Factors on " ‘ ‘ Differences in the frequency of metastasis by the two N-gzg-transformed MSU-1.1 cell strains or the two H-Las-transformed MSU-1.1 cell strains suggested that factors such as the site of integration of the transfected [g oncogene might have influenced metastasis. Integration site could directly affect the expression of the transfected oncogene itself, of adjacent normal cellular genes, or both. Host factors such as age, sex, nutritional status, health status, and hormonal status also could influence metastasis of ras-transformed cells. The role of host factors in experimental metastasis assays was recently discussed by Nowell (24) and Mareel and Van Roy (17). In addition, the particular method used to test for experimental metastasis can influence the results (17). Procedural differences such as 145 injection site, size of the inoculum, and environmental conditions under which mice are maintained can all affect experimental metastasis and make it difficult to compare studies. Distribution of Metastatic Tumors Table 4 lists the sites where metastatic tumors developed when cells were injected intravenously. Contrary to our expectations, few mice injected with the w-transformed MSU-1.1 cell strains developed tumors in their lungs. Instead, tumors formed most often in the skeletal muscles of the thighs, pelvis, abdominal wall, and lumbar region. One mouse had a tumor in its dorsal thoracic cavity that was attached to the mediastinum but did not involve the lungs. Another mouse had tumors in its abdominal cavity and retroperitoneum, but the lungs were free of tumors. In contrast, the Kl-Tl/T cell strain and the HT1080 cell line chiefly formed pulmonary tumors when injected intravenously. We considered at least two explanations for the unexpected distribution of metastatic tumors produced by the m-transformed MSU-1.1 cell strains. On the one hand, @- transformed cells injected into the tail vein may have traversed the pulmonary capillary bed, entered the systemic circulation, and escaped to form tumors at the various sites. In that case the predominance of intramuscular tumors reflected a predilection on the part of transformed cells to establish themselves in these sites. On the other hand, fl-transformed cells may have escaped from the tail vein at the injection site and entered the lymphatic system. Once there, cells could migrate along peripheral lymphatic vessels in an anterograde or retrograde fashion to form tumors in skeletal muscles of the caudal regions, or travel via the mesenteric and thoracic ducts to form tumors in abdominal organs, the retroperitoneum, and the dorsal thoracic cavity. In that case, the predominance of tumors in the skeletal muscles of the caudal regions simply reflected the pattern of lymphatic drainage from the injection site. 146 To distinguish between these possibilities, the original focus-derived cell strain N- fl-3 and the highly metastatic N-w-3T line derived from it were injected by four routes. Intravenous and intracardial inoculations were done to see if tumor distribution was related to injection site or if tumors would develop in the same locations regardless of where we introduced cells into the bloodstream. Intramuscular (caudal thigh) and subcutaneous inoculations were done to see if tumors would develop more frequently or more rapidly at intramuscular sites, which would suggest that muscle tissue provided a more favorable environment for tumor growth. Results of intracardial and intravenous injections appear in Table 5. Following intracardial inoculation of transformed cells, tumors developed most often within the thoracic wall, but also occurred in abdominal organs, on the peritoneum, and within muscles of the abdominal wall. None of the thoracic tumors involved the lungs. Tumors that occurred within abdominal muscles occurred in deep layers and projected as raised nodules on the peritoneum. One mouse had a small tumor on the endocardial surface of the left ventricle. In summary, the majority of tumors developed near the injection site following intracardial inoculation. The rest of the tumors developed where they would be expected to do so if the transformed cells had been deposited into the left ventricle. No tumors developed in skeletal muscles of the rear legs following intracardial inoculation. When we injected the N-m—3T cell strain into the tail vein in our first experiments, metastatic tumors formed in 9 of 10 mice but in our second series of experiments, metastatic tumors appeared in only 1 of 4 mice receiving the N-gs-3 cell strain and none of 5 mice that received N-flfi-ST cells. The low frequency of metastasis in the second instance may be an artifact, since three of the four mice in the second series of experiments died within 35 days of being injected. The latency periods of N-@-3T cells was 78 days in the first experiments and even longer in the second experiments. 147 The results of intramuscular and subcutaneous inoculations (Table 6) revealed that the N-ng-3 and N-l‘fi-3T cell strains'did not form tumors more rapidly or consistently in skeletal muscle than in the subcutis. Tumors developed over the same time period at both injection sites. At first glance it appeared that tumors developed more rapidly within muscle, since 1/ 15 as many transformed cells were injected intramuscularly as subcutaneously. However, this difference in initial cell numbers represented only 5-6 population doublings. These results were not consistent with the idea that muscle tissue offered a more favorable "soil" for tumor growth, but they did not rule out that transformed cells were targeted in some way to blood vessels within skeletal muscle. These results suggested that tumors could develop not only from pas-transformed cells introduced into the bloodstream, but also from extravasated cells that either formed tumors locally (in the case of thoracic tumors) or travelled via lymphatics to form regional tumors at distant sites (in the case of skeletal muscle tumors). Metastatic tumors arising from transformed cells that escaped into lymphatic vessels at the time of inoculation necessarily must have arisen from much fewer cells than those arising from transformed cells injected into the bloodstream, suggesting that m-transformed cells might actually metastasize more readily via lymphatic vessels than via the bloodstream. Why might this be so? One possibility is that w-transformed cells survived better in the slow-moving lymphatic system than in the more turbulent vascular system. Another possibility is that cells carried to the lungs were rapidly removed by NK cells and pulmonary macrophages, while cells within extracellular spaces or lymphatic vessels were less rapidly or efficiently cleared by the immune system. The apparent metastasis of extravasated transformed cells via lymphatics raises the question, why was such metastasis was never observed following subcutaneous or intramuscular inoculation, even though regional lymph nodes were examined routinely? 148 One possibility is that tumors developed too rapidly following subcutaneous inoculation to allow adequate time for spontaneous lymphatic metastasis to take place. This seems unlikely, since metastatic tumors developed nearly as rapidly following inoculations into the tail as primary tumors did following inoculations into the subcutis. A second possibility is that subcutaneous inoculation of a large number of w-transformed cells incited a host response qualitatively or quantitatively different from that elicited by a smaller number of transformed cells injected intravenously, and that host defenses therefore inhibited lymphatic spread of transformed cells in the case of subcutaneous inoculation. These studies point out a difference between most of the m-transformed MSU-1.1 fibroblasts, which rarely formed lung tumors, and the N-w-ST cell strain, the K1-T1 /T cell strain, and the sarcoma-derived HT1080 cell line, which almost always formed lung tumors. The HT1080 cell line generally made single pulmonary tumors, while the IE- transformed cell strains produced multiple pulmonary tumors. We cannot explain why the HT1080 cell line produced only single metastatic lung tumors, although Alvarez and DeClerk (2) recently made the same observation. Microscopic Appearance of Primary and Metastatic Tumors Metastatic tumors were morphologically indistinguishable from primary subcutaneous tumors, with two exceptions. One exception was the K—w-laT cell strain, which formed myxoid sarcomas when given subcutaneously or intraperitoneally (Fig. 1), but non-myxoid Spindle cell sarcomas when given intravenously (Fig. 2). The other exception was the N- Qa_S-3T cell strain, which formed only spindle cell sarcomas when inoculated intravenously (Fig. 3), intracardially, or into the peritoneal cavity (Fig. 4), but whose parent cell strain, N- EG forms chiefly pleomorphic sarcomas with multinuclear giant cells when injected into the subcutis (7). The Niels-3 cell strain also produces mixed tumors (malignant 149 mesenchymomas) and spindle cell sarcomas subcutaneously, and in previously-reported studies (7) we were able to clonally-derive substrains of N-m-3 i_n m that formed only spindle cell sarcomas subcutaneously. A similar selection process may have occurred i_n yiv_o to give rise to the N-Qg3T cell strain. These observations support the idea that the phenotype of transformed cells i_n yifl is the result of host/ cell interplay rather than an inherent property of the transformed cells themselves. As already discussed regarding metastasic ability, host factors and assay conditions such as injection site influence expression of a variety of phenotypic characteristics of transformed cells, including the pattern of tumor growth. We reached a similar conclusion in previous studies with the fits-transformed cell strains (7). METHODS Cell Lines and Strains The derivation of m-transformed human cell strains by Fry et al. (11), Hurlin e_t a_l. (15), and Wilson gal. (33) has been described elsewhere. Briefly, infinite life span human fibroblasts were transfected with a plasmid containing the 8402 N-rjp, T24 H-fls, or Viral K-rg oncogene linked to a neomycin resistance gene. Stable as transfectants of KMST—6 fibroblasts, which are derived from human embryo fibroblasts treated with 60Co gamma radiation (19,22), and GM637 fibroblasts, which are derived from adult dermal fibroblasts infected with SV40 virus, were isolated by growth in selective medium containing the antibiotic G418. In the case of MSU-1.1 fibroblasts, which are derived from newborn foreskin fibroblasts transfected with a v-M oncogene, a different selection procedure had to be used, because MSU-1.1 fibroblasts already contain a neomycin resistance gene. The fl-transfected MSU-1.1 cell strains were isolated as m0rphologically-transformed cells growing as foci on a cell monolayer or as anchorage independent colonies in soft agar. MSU-1.1, GM637, and KMST—6 fibroblasts are non-tumorigenic in nude mice, but they can be malignantly transformed by transfection with an oncogene belonging to the m family (11,15,20,21,33). Cells can be recovered from tumors by mincing the tumor and inoculating tissue into 75 mm tissue culture flasks containing McM medium (27) with G418. In these studies, both primary transfectants and tumor-derived E-transformed cells were uSed. In addition, we used two cell lines as controls. One was the sarcoma-derived cell line HT1080, obtained from the American Type Culture Collection. Rasheed e_t a_l. (26) derived HT1080 cells from a fibrosarcoma that arose in a 35-year old man who later died of metastatic disease. HT1080 cells contain an N-@ oncogene (14). As another control, we Used 3 spontaneously-transformed MSU-1.1 cell strain, L551—3T. 150 Assays for Experimental Metastasis in Athymic Nude Mice BALB/c athymic nude mice from a closed colony were used as recipients for transformed cells in all experiments. Serum from heterozygous female breeders was tested regularly by Microbiological Associates (Rockville, MD) and was consistently free of antibodies to K virus, polyoma virus, murine hepatitis virus, ectromelia virus, GD VH virus, murine adenovirus, lymphocytic choriomeningitis virus, minute virus of mice, Sendai virus, pneumonia virus of mice, reovirus type 3, EDIM virus, murine cytomegalovirus, Car bacillus, and Mycoplasma pulmonis. Mice were maintained in isolators illuminated by fluorescent light, with a 12 hour/ 12 hour light/dark cycle, temperature of 70-75°F, and humidity of 30- 50%. They were housed in plastic cages at up to five mice per cage. Cages had pine shaving bedding and individual water bottles. Mice were fed pelleted laboratory chow. We used 6- to 10-week old mice of both sexes in these experiments. For injection, we suspended cells in McM medium at a concentration of 10‘5 cells/ml. Mice received 0.2- 0.5 mls. (2-5 x 105 cells) by one of four routes. Intravenous injections were given into the tail vein of ether-anesthetized mice using a 25-gauge needle. Intracardiac injections were given by inserting a 25-gauge needle through the left lateral thorax of anesthetized mice and aspirating slightly to check for blood to insure that the needle tip was within a heart Chamber. Intramuscular injections were given into caudal thigh muscles, and intraperitoneal injections were given by inserting the needle on the ventral abdominal midline. Schutaneous injections consisted of 2-5 x 10‘5 cells injected between the shoulder blades. We observed mice biweekly for signs of tumor development, and killed them by CO2 inhalation when tumors reached 2-3 cm in diameter, or when the mice showed signs of illness such as weight loss, dyspnea, or lameness. Mice that did not develop tumors or become ill were observed for six months, then killed. 151 We calculated latency periods for tumor development following subcutaneous and intramuscular injections as the time taken for tumors to reach 1 cm in diameter. For intravenous, intracardial, and intraperitoneal injections, we calculated latency periods as the time between injection and the appearance of signs of illness or palpable tumors. Collection and Processing of Tissues Mice received a complete necropsy that included gross and microscopic examination of at least any tumors, cervical, lumbar, and mesenteric lymph nodes, thyroid gland, parathyroid glands, adrenal glands, salivary glands, trachea, lungs, heart, liver, spleen, pancreas, and kidneys. In some mice, portions of the musculoskeletal system and spinal cord were also examined. Specimens of all tissues were fixed in neutral buffered 10% formalin, processed by routine histologic methods, and imbedded in paraffin blocks. Six micron sections were cut and stained with hematoxylin and eosin for general microscopic examination. In particular, the lungs from mice that had received intravenous or intracardial injections were imbedded in their entirety, and multiple sections were examined for metastatic tumors. 152 REFERENCES 1. Agnor, C., Papageorge, A., Wilding, G., Gelmann, E. The effects of LasH transfection and estradiol treatment on MCF—7 cell tumor formation and metastasis. Proc Am Assoc Cancer Res 29: 451 (1988) 2. Alvarez, A., DeClerck, Y. Elevated gelatinase activity in tumor cells is associated with increased metastatic potential. Proc Am Assoc Cancer Res 29: 65 (1988) 3. Ananthaswamy, H.N., Price, J .E., Goldberg, L.H., Bales, E.S. Correlation between Ha-ng gene amplification and spontaneous metastasis in NIH 3T3 cells transfected with genomic DNA from human skin cancers. Proc Am Assoc Cancer Res 29: 466 (1988) 4. Bernstein, S.C., Weinberg, R.A. 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Nature 310: 469-475 (1984) Storer, R.D., Allen, H.L., Kraynak, A.R., Bradley, M.O. Rapid induction of an experimental metastatic phenotype in first passage rat embryo cells by cotransfection of EJ c-Ha-gis and c-M oncogenes. Oncogene 2: 141-147 (1988) Thorgeirsson, U.P., Turpeenniemi-Hujanen, T., Williams, J .E., Westin, E.H., Heilman, C.A., Talmadge, J.E., Liotta, L.A. NIH/3T3 cells transfected with human tumor DNA containing activated gs oncogenes express the metastatic phenotype in nude mice. Mol Cell Biol 5: 259-262 (1985) Van Roy, F.M., Messiaen, L., Liebaut, G., Gao, J., Dragonetti, C.H., Fiers, W.C., Mareel, M.M. Invasiveness and metastatic capability of rat fibroblast-like cells before and after transfection with irnmortalizing and transforming genes. Cancer E 46: 4787-4795 (1986) Wallace, J .S., Syms, A.J., Hayle, A.J., Fleming, K.A., Tarin, D. Investigation of whether transfection with the activated @ oncogene can induce metastatic behaviour. Proc Am Assoc Cancer Res 27: 59 (1986) Wilson, D.M., Yang, D., Dillberger, J.E., Dietrich, S.E., Maher, V.M., McCormick, J.J. Malignant transformation of an infinite life span, human fibroblast cell line by a transfected N-gLs oncogene. Cancer Res (submitted, 1989) Wyllie, A.H., Rose, K.A., Morris, R.G., Steel, C.M., Foster, E., Spandidos, D.A. Rodent fibroblast tumours expressing human m and [Q genes: Growth, metastasis and endogenous oncogene expression. Br J Cancer 56: 251-259 (1987) 156 Table 1. Origin of Cell Lines and Strainsa Recipient Transforming Cell Line ras Oncogene MSU-1.1 K—@ N -ras H-ras (spontaneous transformant) GM637 H—r_a§ KMST—6 H-_ra_s Injected Cells K-I‘fi- 1a K-fl-ZaT N-fl-3T N-w-ST H-w-ZT H-Es-3T L55I-3T SV-Tl / T K1-T1/T Reference 11 33 15 Milam: (personal communication) 15 15 aAll cell lines used in these experiments were derived from subcutaneous tumors produced by their respective w-transformed cell strain (7). 157 Table 2. Experimental Metastasis of w-transformed MSU-1.1 Cells, Spontaneously- transformed MSU-1.1 cells, and Sarcoma-derived Cellsa Cell Strain or Mice with Cell Line Tumors/Mice Rate of Mean I_ntheg I_nje_ct_e_d Metastasis Latency Period K-pas-laT 4/13 31% 133 days K-gs-ZaT 1/10 10% 138 days N-EST 9 /10 b 90% 78 days N-w-ST 5 / 10 50% 115 days H-m-3T 0/9 H-r_a_s-2T 4/9 44% 133 days L551—3T 3/11 27% 76 days HT1080 9/10 c ‘ 90% 85 days a. The data shown here represent the pooled results of two separate experiments. b. The tenth mouse, killed 45 days after injection because of weight loss, had no tumors. c. The tenth mouse, killed 56 days after injection because of a bacterial infection, had no tumors. 158 Table 3. Experimental Metastasis of H-m—transformed Human Fibroblastsa Cell Strain or Mice with Parental Cell Line Tumors/Mice Rate of Mean Latency Cell Line Injected Injected Metastasis Period MSU-1.1 H-g-3T 0/9 --- H-Q-2T 4/9 44% 133 days KMST-6 Kl-Tl/T 7/9b 78% 65 days GM637 SV-Tl / T 0/ 10 --- --- a. The data shown here represent the pooled results of two separate experiments. b. An tenth mouse that died with signs of metastatic disease 55 days after injection was unavailable for necropsy. 159 Table 4. Distribution of Metastatic Tumors Produced by m-transformed Cell Strains, a Spontaneously-transformed Cell Line, and a Sarcoma-derived Cell Line Cell Strain or Cell Line Injected K-Qs- laT K—fl-ZaT Mas-3T N-fiS-ST H-ra_s-2T Kl-Tl /T L551-3T HT1080 TM muscles of rear legs (3 /4) peritoneum, kidneys, pancreas, stomach, adrenals, lumbar vertebra (1/4) muscles of rear legs (1 / 1) muscle of rear legs (8/ 9) muscle of abdominal wall (3/9) dorsal thoracic cavity (not lung) (1/9) muscle of rear legs (1/5) lungs (single tumor) (4/5) muscle of rear legs (4/4) muscle of rear legs, back (1 /7) lungs (multiple tumors) (6/7) muscle of rear legs (3/3) lungs (single tumor) (6/9) muscle of rear legs (3/9) 160 Table 5. Distribution of Tumors Produced by Intravenous or Intracardial Inoculation of N-m-transformed MSU-1.1 Cell Strains Mice with Mean Cell Strain Route of Tumors/ Latency Injected Injection Mice Injected Period Tumor Location N-gs-3 IV 1/4 a 200 days lungs (multiple tumors) IC 2/4 b 148 days endocardium (1 /2) abdominal muscles, peritoneum, kidney, thoracic wall (1/2) N-w-3T IV 0/5 c --- --- IC 4/6 151 days thoracic wall (4/4) abdominal muscles, peritoneum, kidney, adrenals (1/4) Combined IV 1/9 200 days lungs (multiple results tumors) IC 6/10 150 days thoracic wall (5/6) abdominal muscles, peritoneum, kidney (2/6) adrenals (1 /6) endocardium (1/6) a. Two mice that died 6 days and 35 days after injection had no tumors. b. Two mice that died 29 days and 60 days after injection had no tumors. c. One mouse that died 4 days after injection had no tumors. 161 Table 6. Tumor Formation by N-w-transformed MSU-1.1 Cell Strains Injected Intramuscularly or Subcutaneously Cell Strain Route of No. cells Mice with Tumors/ Mean Latency I_njem Injection I_njm Mice Injected Pe_riog Nflse IM 105 4/4 49 days SQ 5 x 106 4/4 48 days Niger IM 105 3 /5 99 days SQ 5 x 106 5/6 a 86 days a. One mouse that died 63 days after injection had no tumor. ILLUSTRATIONS ‘ ...-a: KX‘ t . v. - wt- :3 \k‘ 0‘ 1 ‘4 ‘ WK.“ 1‘81 . .x. Figure l. Myxoid sarcoma produced by K-fl-transformed MSU-1.1 cells (cell strain K- w-laT) injected into the peritoneal cavity. Empty circles are entrapped adipocytes (HE, 200x). 162 A1 -. 163 Figure 2. Spindle cell sarcoma produced by K-fl-transformed MSU-1.1 cells (cell strain K-rfi-1aT) injected into the tail vein. Compare with myxoid sarcoma produced by the same cells injected into the peritoneal cavity (Figure 1). 164 L 951"" "7" t 3. I ~ Figure 3. Spindle cell sarcoma in lungs produced'by N-w-transformed MSU-1.1 cells (cell strain N-r_z§—3T) injected into the tail vein. Compare with pleomorphic sarcoma produced by the same cells injected subcutaneously (Figure 7) (HE, 200x). A- 165 Figure 4. Spindle cell sarcoma produced by N-rfi-transformed MSU-1.1 cells (cell strain N-pag-3T) injected into the peritoneal cavity. This tumor invaded the kidney; note renal tubules (HE, 200x). APPENDIX ATTEMPTS TO CAUSE HUMAN F IBROBLASTS TO ACQUIRE INFINITE LIFE SPAN BY TREATMENT WITH ETHYLNITROSOUREA OR 5-AZACYTIDINE BACKGROUND AND LITERATURE REVIEW Carcinogenesis, Transformation, and Cell Life Span Transformation is a term borrowed from microbiology, where it refers to the transfer of genetic information between two bacteria, with acquisition of a new phenotype by the recipient. An example of bacterial transformation would be the acquisition of antibiotic resistance by a bacterium that received a plasmid bearing a drug metabolizing gene. Transformation of eukaryotic cells is more broadly defined as the acquisition of a new phenotype resulting from some change in the genome of the cell (Ponten, 1976). Cell transformation may follow the receipt of new genetic information in the form of a transfected DNA fragment, viral or proviral genome, or an entire chromosome acquired during cell fusion. Transformation also may follow the loss of DNA or alterations in DNA sequence. Even a change as small as a single base substitution may transform cells. Obviously there are thousands of phenotypic characteristics that could be altered in transformed cells. Clues about which of them might be important in the process of carcinogenesis have come from the study of tumors and tumor-derived cell lines. For instance, many tumor-derived cells form dense, piled-up clusters of cells, called foci, when they grow on a monolayer of normal fibroblasts, while normal cells do not. This suggests that normal cells which have acquired the ability to form foci (been transformed to focus formation) as a result of some i_n m manipulation have gained a characteristic of tumor cells, and this reasoning has prompted investigators to study the molecular mechanisms 167 168 involved in focus formation and the agents which can induce that particular phenotype. For the same reason, investigators have studied transformation to other phenotypic endpoints, such as altered cell shape (morphologic'transformation), reduced growth factor requirements (transformation to grth factor independence), ability to grow in soft agar (transformation to anchorage independence), increased proliferative capacity (transformation to infinite life span, or immortalization), ability to form tumors in an appropriate host (transformation to tumorigenicity), and ability to form progressively-growing malignant tumors (malignant transformation). Several lines of evidence suggest that the acquisition of an infinite life span, commonly referred to as establishment or immortalization, is a prerequisite for full malignant transformation. Part of the evidence comes from studies of spontaneous and induced tumors; in contrast to normal cells, cells derived from tumors often have an infinite life span (Dobrynin, 1963; Ponten and Saksela, 1967; McAllister e_t a_l., 1969; Giard _t _l., 1973; Rheinwald and Beckett, 1981).' Cancer cells presumably escape from the normal mechanisms that limit cell proliferation in various ways, and the mechanism of immortalization may well differ for different cells (Hull, 1981). Nevertheless, the observation that infinite life span is a common characteristic of many cancer cells suggests that acquisition of an infinite life span may be fundamental to the process of carcinogenesis (Smets, 1980). Evidence of a connection between infinite life span and malignant transformation comes from several sources. Lee e_t al. (1989) studied two strains of mice and demonstrated a positive correlation between the frequency with which their liver cells give rise to infinite life span clones i_n m and the frequency of spontaneous liver tumor formation ip M. Acquisition of an infinite life span seems regularly to accompany tumorigenic transformation induced ip vitro by chemicals (Berwald and Sachs, 1965; Igel e_t a_l., 1975; Benedict _t a_l., 169 1975; Newbold e_t a_l., 1982), radiation (Borek, 1980), or viruses (Ponten, 1971; Shin e1 a_l., 1975; Petit _e_t a_l., 1983), as well as spontaneous transformation i_n 11m (Ponten, 1971; Barrett, 1980; Mukherji e_t a_l., 1984; Gorman e_t al., 1984; Kraemer e_t al., 1986). In addition, acquisition of an infinite life span appears to be an absolute requirement for tumorigenic transformation induced i_n_yiirp by oncogene transfection (Rassoulzadegan gt a_l., 1982; Land e_t a_l., 1983; Newbold and Overell, 1983; Ruley, 1983; Spandidos and Wilkie, 1984; Jenkins 9; a_l., 1984; Yoakum e_t al., 1985; Namba etal., 1988; Seremetis e_ta_l., 1989; Fry e_t a_l., 1989; Wilson e_t a_l., 1989; Hurlin e_t a_l., 1989). O’Brien pt a_l. (1986), Namba e_ta_l. (1988), and Klein (1979) all suggest that, at least with human cells, senescence is the key limiting factor in malignant transformation i_n m. Klein and Namba e_t a_l. even propose that the process of initiation in multi-stage carcinogenesis i_n y_iv_o creates preneoplastic cells with an infinite life span that subsequently undergo further changes to become tumorigenic. The acquisition of phenotypic characteristics is often temporally separated m yifl into early events (initiation) and late events (promotion and progression), but such distinctions are difficult to make i_n vitro. For example, Land e_t a_l. (1983) have demonstrated that, although an infinite life span is a prerequisite for malignant transformation of fibroblasts, it makes no difference whether the cells acquire this characteristic "early" or "late". On a theoretical level, multi-step models of carcinogenesis require that cells acquire an extended or infinite life span if tumors are to arise 1pm. To illustrate, consider a simple multistep model in which a normal cell must undergo two independent mutation-like events to become a malignant tumor cell. If the phenotypes associated with these events are designated A and B, then the normal cell is A'B' and its malignant progeny is A‘B”. Assuming that each mutation-like event occurs spontaneously once per 106 cells per generation, then the transformation process can be diagrammed as in Figure 1. 170 clonal clonal ’l’ 1” selection ; selection I ’9 ———> I 20 p.d. 30 p.d. 1,14 ’ Among 106 normal Among 106 A tumor of 109 AB" cells an AB' A+B' cells an AJ'B+ cells is variant arises. A+B+ variant formed (1 cm3). arises. Figure 1. Model of human fibroblast transformation. In this model, malignant transformation results from 2 mutation-like events (phenotypes A and B). Note that unless the A or B phenotype extends the cells’ life span beyond the normal 50 p.d., a tumor large enough to be life-threatening will not develop. If more than two mutation-like events are required, a tumor will not develop at all. (p.d. = population doublings) To derive 109 malignant cells from 106 normal cells requires the cells to undergo 50 population doublings (pd). However, human fibroblasts have an i_r_1 Egg life span of only 50- 70 pd (Hayflick, 1974) which is reduced by 10-20 pd when they are clonally selected (McCormick, unpublished studies), and their life span Q v_iv_o appears similar (Cristofalo and Stanulis-Praeger, 1982). Consequently, if the original A'B‘cells in this model are human fibroblasts then the malignantly transformed A"B+ cells that make up the resulting fibrosarcoma are very near the end of their life span. In other words, this 1-cm-diameter fibrosarcoma, which may be clinically undetectable and is far from life-threatening, is made up of very old cells. The tumor cannot grow much larger, let alone recur following surgical excision or grow at a metastatic site, unless the A+ or B" phenotype has conferred upon the cells an infinite life span. If more than two mutation-like events are required for malignant transformation, or if those events occur less often than once per 106 cells per generation, 171 then immortality becomes an absolute requirement for a fully malignant cell to arise at all. Carcinogens may increase the frequency of a particular mutation-like event, but will not appreciably change the number of doublings required even in the simple two-stage model. As a result, carcinogen induction does not abrogate the need for tumor cells to acquire unlimited proliferative potential. Control of Cell Life Span Although acquisition of an infinite life span seems central to the process of carcinogenesis, we know comparatively little about the mechanisms that govern cell life span. We do know, as already mentioned, that normal vertebrate cells have a finite life span _ip vitro (Hayflick, 1965 and 1974) and apparently also in yiyp (Cristofalo and Stanulis-Praeger, 1982). The process whereby cells accumulate deleterious changes as they age, culminating in loss of proliferative potential and / or cell death, is termed senescence (Yanishevsky and Stein, 1981; Macieira-Coelho and Azzarone, 1982; Stein La], 1985; Cristofalo La], 1986). The life span of cells i_n yitfl generally falls within a narrow range that is a function of the number of population doublings the cells have undergone, rather than chronological time (Kajik, 1979). Life span is determined by the interplay of genetic and environmental factors; senescence is therefore the combined result of both genetically-determined and stochastic events, rather than being strictly a deterministic process (Macieiro-Coelho, 1980). Cell fusion studies indicate that the phenotype of finite proliferative potential is dominant (Pereira-Smith and Smith, 1982 and 1983) but the molecular mechanisms that limit cell life span are obscure. Hypotheses usually center around the gradual accumulation of deleterious changes in DNA and proteins (error catastrophe hypotheses [Orgel, 1973; Laughrea, 1982]) or a predetermined, genetically-controlled event or series of events (genetic clock hypotheses [Hayflick, 1965]). The main features of these two theories are listed in Table 1. 172 Table 1. Contrast Between Proposed Mechanisms Limiting Cell Life Span Error Catastrophe Genetic Clock Life span limited by ' Life span limited by a “information loss" "genetic clock" Process is stochastic, due to Process is pre-programmed synthesis errors, wear-and- (planned obsolescence) tear Aging = accumulation of damage Aging = terminal differentiation Immortality = acquired ability to Immortality = failure to avoid / repair damage mortalize (program malfunction) Numerous types of "errors" have been observed in aging cells, including the accumulation of extrachromosomal DNA sequences (Schmookler-Reis e_t a_l., 1983), loss of repetitive DNA sequences (Schmookler-Reis and Goldstein, 1980), loss of DNA from the margins of chromosomes (Good, 1977), decreased DNA methylation (Wilson and Jones, 1983), loss of gene repression activity (Smith and Lumpkin, 1980), and loss of flexibility of the protein synthetic pathway to respond to growth factors (Ballard and Read, 1985). The various changes in DNA associated with aging of cells were recently reviewed (Maciera-Coelho, 1984). However, protein synthesis (translation) errors do not increase as cells age (Harley _e_t a_l., 1985) and the possibility exists that observed "errors" are only a secondary consequence of aging, rather than causally related to it (Thompson and Holliday, 1975). The genetic clock hypothesis proposes a pre-programmed signal that either inactivates key genes involved in proliferation or activates genes involved in differentiation, thereby "mortalizing" the cell (Shall and Stein, 1979). In this model, aging is usually considered a differentiation process and cell death is interpreted as terminal differentiation (Bell e_t a_l., 1978; Kantermann and Bayreuther, 1979). There is evidence for the production 173 of an intracellular protein that inhibits proliferation (Hull, 1981; Burmer, e_t a_l., 1982; Drescher-Lincoln and Smith, 1984; Lincoln pt a_l., 1984; Pereira—Smith gt a_l., 1985; Stein e_t al., 1985; Lumpkin _e_t a_l., 1986; Smith e_t a_l., 1987), as well as for inhibitory factors in serum that act intercellularly (Loo e_t al., 1987). There is also evidence that random activation of genes by treatment with 5-AzaC shortens cell life span (Holliday, 1986; Fairweather e_t a_l., 1987), although whether this represents an acceleration of a normal program of gene activation is unknown. The various hypotheses concerning the control of cell life span are not mutually exclusive; rather, cell proliferation is probably regulated at multiple points in the cell cycle, which Hull (1981) called restriction points. Passage through restriction points is regulated by specific molecules that may be stimulatory or inhibitory (Yanishevsky and Stein, 1981; Pardee, 1987). Immortalinitinn of ," ' Cells The ability to derive infinite life span cell lines i_n vitro differs widely, depending upon the species from which the cells are taken (Terzi and Hawkins, 1975: Macieira-Coelho e_t a_l., 1977). For example, spontaneous immortal variants regularly arise in mouse fibroblast cultures (Todaro and Green, 1963; Berwald and Sachs, 1965; Reznikoff e_t A, 1973) and numerous established cell lines exist such as the various 3T3 fibroblasts lines used in i_n vitro assays for transforming genes. In contrast, spontaneous infinite life span variants arise less commonly in cultures of rat and Syrian hamster fibroblasts, and almost never appear in chicken fibroblast cultures (Ponten, 1971). Despite extensive studies with human fibroblasts, spontaneously-arising infinite life span variants have been very rarely reported (Thielmann e_t a_l., 1983; Mukherji e_t a_l., 1984), as discussed by DiPaolo (1983). 174 Infinite life span non-human fibroblast lines have been generated by treatment with carcinogenic chemicals (Oshimura et _a_l., 1986), infection with viruses, and transfection with oncogenes derived from viruses or tumors (Houweling e_t al., 1980; Rassoulzadegan e_t a_l., 1982; Jenkins 9; a_l., 1984; Spandidos and Wilkie, 1984; Griffin and Karran, 1984; Connan e_t a_l., 1985; Shimada e_t al., 1987; Rovinski and Benchimol, 1988). Most of the time, the infinite life span lines exhibit other characteristics of transformed cells, such as anchorage independence, focus formation, altered morphology, or tumorigenicity in immunodeficient or histocompatible hosts, but there are exceptions. Rat kidney cells transfected with a fragment of human adenovirus type 5 DNA by Houweling gt a_l. (1980), rat embryo cells transfected with the gene for the polyoma virus large T antigen by Rassoulzadegan et a_l. (1982), and rat chondrocytes transfected with the mouse p53 gene by Jenkins e_t a_l. (1984) all acquired an infinite life span without showing changes in morphology or grth characteristics. Also, Newbold e_t a_l. (1982) immortalized hamster dermal fibroblasts by treating them with chemical carcinogens and the infinite life span cells did not exhibit other characteristics of transformed cells. In most of these examples the infinite life span cells gradually became "fully transformed" with passage i_n_ vitro. In contrast, when Griffin and Karran (1984) immortalized monkey kidney epithelial cells by transfection with a fragment of the Epstein-Barr virus genome, the cells remained stable in culture and did not progressively acquire other characteristics of transformed cells, perhaps reflecting greater genetic stability of primate cells compared to rodent cells. Immortalization of Human Cells Unlike rodent cells, human cells seem resistant to establishment i_n vitro as infinite life span cell lines. The sole exception seems to be human B-lymphocytes, which are readily immortalized by infection with Epstein-Barr virus (Spear, 1983). EBV-immortalized 175 lymphocytes do not show morphologic or karyotypic changes and are nontumorigenic in nude mice (Nilsson and Ponten, 1975; Nilsson e_t a_l., 1977). The mechanism of immortalization by EBV is unknown. Other viruses can irnmortalize human cells, but the connection between infection and the acquisition of an infinite life span is less direct. Human embryonic kidney cells can be immortalized by infection with human papovavirus, but with a very low frequency (Purchio and Fareed, 1979). Human keratinocytes (Rhim e_t a_l., 1985) or fibroblasts (Sack, 1981) infected with SV40 virus, exhibit morphologic changes and altered growth characteristics i_n m but rarely give rise to infinte life span clones. Cultures of SV40 virus-transformed fibroblasts have a slightly prolonged but finite life span; rarely (< 1 in 107), an infinite life span variant will emerge at senescence (Huschtscha and Holliday, 1983). Similarly, proliferating clones of cells appear rarely in senescent cultures of SV40 virus-infected keratinocytes. Infection with SV40 virus is thought to prevent cells from entering a normal viable senescent state (Stein, 1985). How this change predisposes cells to immortalization is unknown, but clearly immortalization is not a direct effect of SV40 virus infection. The life span of human cells in culture can be prolonged by hydrocortisone (Grove and Cristofalo, 1976), but only very few investigators have reported the immortalization of human cells in culture by treatment with chemical carcinogens or radiation (Igel e_t a_l., 1975; Kakunaga, 1978; Namba e_t a_l., 1978 and 1981; Stampfer and Bartley, 1985; Namba, 1985). In only two instances were the parental cells preserved and the life span assay described. Namba e_t a_l. (1978 and 1981) reportedly generated three infinite life span fibroblast lines by prolonged treatment of human embryo fibroblasts with 4-NQO and gamma radiation. McCormick and Maher (1988) compared these cell lines to the parent cells by a variety of methods and concluded that the two of the lines are identical to each other, but that the two distinct lines obtained by these investigators are derived from the parent cells and represent 176 a EDIE fig example of immortalization. These infinite life span human cells exhibit other characteristics of transformed cells, such as morphologic changes, altered grth characteristics, and karyotypic abnormalities, but they are nontumorigenic in nude mice. Kakunaga (1978) reported isolating 15 infinite life span human fibroblast lines from foci of morphologically-altered cells appearing in cultures of dermal fibroblasts treated with 4-NQO and MNNG. The majority of these cell lines were later lost, but four were available for study (Leavitt and Kakunaga, 1980; Varma and Leavitt, 1988). However, these cells were later found not to be derived from the putative parent cells (McCormick and Maher, 1988). Instead, they are identical to the fibrosarcoma-derived cell line 8387, which itself already has an infinite life span (McCormick e_t a_l., 1988). McCormick and his colleagues generated an infinite life span fibroblast line, MSU- 1.1 (McCormick £11., 1989). This cell line arose within a clonal population of normal human foreskin fibroblasts transfected with a plasmid containing the pg gene and a v-m oncogene. Several G418-resistant cells were isolated and shown to express the V-M oncogene. The majority of the cultures went through crisis and a single cell line emerged. The cells grew slowly during the first several months in culture, but subsequently resumed a normal growth rate. These have been designated MSU-1.1 fibroblasts. MSU-1.1 fibroblasts have the same isozyme and HLA antigen pattern as the parent cells, are morphologically similar, and are nontumorigenic in nude mice. Their karyotype has been stable for over 200 population doublings. They have a near-diploid DNA content, but differ from the parent cells in having two marker chromosomes created by rearrangements between chromosomes 1 and 11, and 12 and 15. The relationship between the transfected my; oncogene and the cells’ infinite life span is under investigation. Since a diploid infinite life span cell line (MSU- 1.0) was derived from the same clone of cells transfected with the v-mjg oncogene (McCormick, unpublished studies), the chromosomal rearrangements observed in the MSU- 1.1 cell line are not causally involved in the infinte life span phenotype. INTRODUCTION Much evidence suggests that immortalization, or the acquisition of an infinite life span by cells, is fundamental to the process of carcinogenesis (for discussion, see Klein, 1979; Smets, 1980; O’Brien et al., 1986; Namba e_t a_l., 1988; McCormick and Maher, 1988). Indeed, the difficulty of transforming human cells to tumorigenic cells i_n m compared to mouse or rat cells appears directly related to the relative resistance of human cells to spontaneous or induced immortalization (DiPaolo, 1983). In contrast to mouse fibroblasts, infinite life span variants rarely appear in cultures of human fibroblasts and apparently are difficult to induce by treating cells with chemical carcinogens or radiation. On the other hand, available evidence on cell proliferation suggests that passage of human fibroblasts through the cell cycle is regulated at only a few key steps, called restriction points (Hull, 1981), and that passage through these restriction points is mediated by the products of a few key genes (Pardee, 1987; Pereira-Smith and Smith, 1988). Theoretically, mutation-like events that alter such growth regulating genes might induce or allow unregulated cell proliferation by abolishing the molecular controls that normally limit it. I hypothesized that human fibroblasts could be immortalized by a mutation-like event in a growth regulating gene; that is, by a. rare, permanent, heritable change in gene expression resulting from an alteration in the regulatory or coding sequences of the gene. In other words, I postulated that human fibroblasts could acquire an infinite life span (lose the ability to senesce or undergo terminal differentiation) in the same way they could gain or lose any other phenotypic characteristic. This hypothesis led me to two others: first, that infinite life span variants arise spontaneously in human fibroblast cultures at some low but detectable frequency, and second, that the frequency of immortalization could be increased by treating fibroblasts with agents that cause mutations or mutation-like changes in DNA. I expected 177 178 that testing these hypotheses would be no more difficult than conducting any other type of mutagenesis assay, provided two conditions could be met: infinite life span variants had to express that phenotype, and I had to be able to recognize them within fibroblast cultures. What I needed was an assay for infinite life span fibroblasts. At first glance, designing an assay for infinite life span cells seemed straightforward --if cells were continually passaged in culture, infinite life span variants should emerge as proliferating clones at senescence, when the rest of the cells in culture ceased proliferating. But the historical evidence from a quarter century’s experience with cultured human fibroblasts suggested that this approach would not work. Infinite life span variants have appeared very rarely, even among human fibroblasts treated with chemical mutagens and radiation. Investigators have taken this as evidence that human fibroblasts cannot be immortalized by mutation-like events. But I considered that previous results might indicate instead that infinite life span human fibroblasts could not be recognized under normal cell culture conditions. If infinite life span variants do occur, then why might they not appear as proliferating clones in senescent fibroblast cultures? One possibility is that infinite life span variants are inadvertently discarded during serial passages of cell cultures. Normal cell culture procedures involve discarding or freezing back a portion of the culture at intervals. This process might be repeated 10 times over the life span of a normal fibroblast culture that undergoes about 50 population doublings. The end result of such cell culture procedures is that the majority of the cells are discarded and relatively few are carried through their entire life span. In this situation, rare variants readily can be lost. If a variant has a slight competitive disadvantage, such as a slower growth rate, it is even more likely to be lost. Namba e_t a_l. (1988) suggested a second reason why infinite life span variants might not grow out of senescing fibroblast cultures. If expression of the infinite life span phenotype is inhibited by surrounding normal fibroblasts, such variants will remain latent and 179 undetected. There is evidence that this occurs. For instance, rodent fibroblasts can be immortalized by transfection with a myg oncogene (Mougneau e_t a_l., 1984). Bignami e_t a_l. (1988) found that proliferation of myfiransformed rodent fibroblasts is inhibited by contact with normal fibroblasts but not by conditioned medium from normal fibroblast cultures. Dotto e_t a_l. (1988) found that normal fibroblasts also can inhibit the i_n vim proliferation of mouse keratinocytes that are malignantly-transformed by Harvey murine sarcoma virus. Studies in our own laboratory with several infinite life span human fibroblast cell lines indicate that their proliferation can be inhibited by a soluble factor in the medium of normal fibroblast cultures and possibly by direct contact with normal fibroblasts as well (Sato, personal communication). Clearly, the problems of cell loss during passage and inhibition by normal fibroblasts in co-cultures could be eliminated if latent infinite life span cells could be identified as they appeared, removed from bulk cultures, and grown as clonal populations. Infinite life span variants then might be free to express their unlimited proliferative potential. Studies in McCormick’s laboratory with the infinite life span human fibroblast line MSU-1.1 suggested a possible approach to identifying latent immortal fibroblasts, based on the calcium concentration of the culture medium. Calcium acts like a growth factor for human fibroblasts in culture. McCormick and his colleagues found that MSU-1.1 fibroblasts proliferated in low calcium medium but the parent normal fibroblasts did not (J. McCormick, personal communication). I reasoned that if the combination of infinite life span and reduced requirement for calcium was not coincidental, then ability to grow in medium with a reduced calcium content might serve as a phenotypic marker for infinite life span cells. I decided, therefore, to screen human fibroblast cultures for cells capable of proliferating in low calcium medium, clone any calcium-independent variants I found, and determine if those calcium- independent variants had infinite life spans. 180 Because I expected calcium-independent variants to be rare (Kraemer e_t a_l., 1986), Iwanted to screen large numbers of cells. I also wanted to screen cells under conditions that prevented cell—to-cell contact, in case such contact inhibited proliferation. Screening cells on plastic dishes at clonal density would prevent cell contact but would require hundreds of dishes per experiment. Instead, I decided to grow cells suspended in soft agar so that I could screen many cells with relatively few dishes. We had found previously in the laboratory that we could achieve high cloning efficiency in soft agar if we grew normal human fibroblasts in relatively defined medium containing adequate growth factors supplied by serum or as purified supplements (Ryan e_t al., 1987). For my purposes, growing cells in soft agar had two additional advantages over growing them on plastic besides enabling me to screen many cells per dish. First, I could pick proliferating clones cleanly from the agar, free of contamination by normal fibroblasts. Second, I could count proliferating clones using a computerized cell counter. In the studies reported here, I treated human fibroblasts with EN U or 5-AzaC. ENU is an alkylating agent which causes base substitutions and occasional small deletions that inactivate genes (Aust e_t a, 1984; Drinkwater and Kleindinst, 1986; Skopek, 1986; DuBridge e_t al., 1987). 5-AzaC interferes with methylation of newly-synthesized DNA; decreased methylation is generally thought to activate genes (Jones gt a_l., 1982; Bird, 1984). I passaged both treated and untreated cells as bulk cultures to, see if infinite life span variants emerged when the cultures senesced. In addition, I assayed cells at intervals by growing them in soft agar under low calcium conditions, to identify and quantify calcium-independent variants. Clones of fibroblasts that proliferated in low calcium conditions were isolated and tested to see if the calcium-independent phenotype was stable and if the cells had infinite life spans. I found that normal fibroblast cultures did indeed contain a few cells that were calcium- independent and that I could induce such variants with ENU, but that the calcium- independent phenotype was not stable and that such cells had normal life spans. MATERIALS AND METHODS Cells and Cell Culture SL88 and LGl are normal human fibroblast lines derived from neonatal foreskins. MSU-1.1 fibroblasts are an infinite life span human fibroblast line derived from a population of LGl fibroblasts transfected with a v-m oncogene (McCormick _e_t a_l., 1989). HT1080 cells are derived from a human fibrosarcoma (Rasheed gt a_l., 1974). Cells were grown in 75 mm tissue culture flasks at 37°C in a 5% CO2 atmosphere and fed with FM medium supplemented with 5-10% FCS, 100 units/ml penicillin, 100 ug/ml streptomycin, and 10 ug/ml hydrocortisone. Unless otherwise noted, cultures were fed weekly and passaged when nearly confluent. Cultures were passaged by loosening cells with trypsin, harvesting them in FM medium, counting a 0.5 ml aliquot in an automated cell counter (Coulter Electronics, Inc., Hialeah, FL) to keep track of the age of the cells in population doublings, and replating cells at 500,000 cells / flask. At each passage, cells not put back into culture were stored at -120°C. Treatment with ENU and S-AzaC Cells were plated at 800,000/ 150 mm-diameter petri dish. ENU solution was prepared by dissolving ENU in DMSO to a final concentration of 400 mM. Cells were exposed to serum—free FM medium containing 100 uls of ENU solution (final concentration = 2 mM) for 1 hour. Control cell cultures were exposed to 100 uls of DMSO in serum-free FM medium for 1 hour. Following treatment, cells were harvested, counted, and replated into 75 mm tissue culture flasks to allow an expression period. Cells were also plated into 100 mm-diameter petri dishes to determine % survival. 181 182 For azacytidine treatment, 5-AzaC was added to cells in regular cell culture medium to a final concentration of 10 uM. .‘I J Assav for (‘alch-m ' ‘ Variants I assayed cells in 100 mm petri dishes as follows. Low calcium agar solutions were prepared by dissolving Seakem agar in McM medium prepared as described by Ryan L211. (1987), except that the calcium concentration was 0.1 mM instead of 1.0 mM. Medium contained 0.5% FCS, 1.0 mg/ ml calcium-free BSA, 100 units/ ml penicillin, 100 ug/ ml streptomycin, 900 ng/ml dithiothreitol, 180 ng/ml glutathione, 1.9 ug/ml phosphoenolpyruvate, 8.1 ng/ml prostaglandin E1, 1 ug/ml insulin, 1.4 ug/ml FeSO4, 13.5 ng/ ml cholesterol, 11 ng/ ml soybean lecithin, 4.5 ng/ ml sphingomyelin, 4.7 ng/ ml vitamin E, and 10 ug/ml hydrocortisone. I first poured a bottom layer of 2% agar and allowed it to harden. On top of this I poured a top'layer of 0.3% agar containing 150,000 cells per dish. Cells were fed 24 hours after plating and weekly thereafter with the same medium used to prepare the original agar. Cells were also plated into plastic petri dishes and into agar made with 4% serum to compare cloning efficiencies. Cells on plastic and in agar made with 4% serum were fed weekly with medium containing 10% and 4% serum, respectively. Agar dishes were examined twice weekly under a microscope for evidence of proliferation. Proliferating clones greater than 40 u in diameter were counted using a computerized agar counting system. Once counted, proliferating clones were removed in an agar plug, teased gently free of the agar, and inoculated into 60 mm culture dishes. RESULTS I initially tested the low calcium agar assay to see if it discriminated among cells with different calcium requirements by comparing the grth of SL88 fibroblasts, MSU-1.1 fibroblasts, and HT1080 cells. The ability of these cells to form proliferating colonies in low calcium agar (Table 2) paralleled their growth curves on plastic under low calcium conditions. Table 2. Growth of Untreated Fibroblasts in Low Calcium Agar Cloning Efficiency (clones per 10‘5 cells) Cell Line Plastic (10% serum) Low Cal Agar (0.5% serum) SL88a 190,000 564 MSU-1.1 680,000 7,289 HT1080 290,000 43,195 SL88-2b < 1,000 < 10 a. SL88 fibroblasts at age 18 population doublings b. SL88-2 fibroblasts at age 40 population doublings HT1080 cells grew readily in medium with reduced calcium concentration, MSU- 1.1 fibroblasts grew less well, and normal fibroblasts grew very poorly. However, there were a few variants within young SL88 fibroblasts (about 6 / 10,000 cells) that could proliferate in low calcium conditions. I picked these clones and attempted to expand them into populations large enough to retest in low calcium agar and assay for infinite life 183 184 span. Only 1 of 21 clones, SL88-2, yielded enough cells to retest in low calcium agar; the rest senesced within 20 population doublings of being isolated as clones in agar. When SL88-2 cells were plated into agar once more, no proliferating clones were detected (Table 2), indicating that, at least in this one case, calcium-independence was not a stable phenotype. I next treated SL88 and LGl fibroblasts with EN U or 5-AzaC at population doublings 18 and 30, respectively. ENU treatment resulted in 18% survival of SL88 fibroblasts and 10% survival of LGl fibroblasts. 5-AzaC was nontoxic to cells. Treated and control fibroblasts were passaged to senescence as bulk cultures. At intervals, cells were harvested and tested for growth on plastic and in agar under normal and low- calcium conditions (Tables 3 and 4). Calcium-independent variants were not detected in control LGl cultures, nor were they induced by ENU or 5-AzaC. Calcium-independent variants did appear with increased frequency in ENU-treated SL88 cultures at population doubling 28, but 3 population doublings later these variants were no longer detectable, suggesting that the altered phenotype had been transient. I picked proliferating clones from low calcium agar and attempted to expand them into populations large enough to retest in low calcium conditions. Nine LGl clones failed to proliferate more than 1 or 2 population doublings. Of 12 SL88 clones, only 1 proliferated more than a few population doublings and it failed to yield enough cells to retest before senescing. None of the calcium-independent clones had an extended life span. W/I/fl’" 185 Table 3. Growth of ENU- and 5-AzaC-treated LG1 Fibroblasts in Low Calcium Agar Cloning Efficiency (clones per 10‘5 cells) Plastic Agar Low Cal Agar Treatment Agea (L)% serum) (4% serum) (0.5% serum) Untreated 33 3,750 399 < 10 ENU 35 < 1,000 263 <10 5-AzaC 33 20,000 722 < 10 a. Age is expressed in population doublings Table 4. Growth of ENU- and 5-AzaC-treated SL88 Fibroblasts in Low Calcium Agar Cloning Efficiency (clones per 106 cells) Plastic Agar Low Cal Agar Treatment Agea (L)% serum) (4% serum) (0.5% serum) Untreated 26 60,625 2,933 < 10 28 14,000 13,047 < 10 30 . 37,500 ‘ 3,331 < 10 ENU 28 60,625 5,298 125 31 45,000 36,179 <10 5-AzaC 26 20,000 2,429 < 10 29 10,000 5,083 <10 a. Age is expressed in population doublings 186 I also picked proliferating clones from regular agar fed with 4% serum to see if they would prove immortal. Of 9 LG1 clones and 63 SL88 clones, none had an extended life span; only 3 SL88 clones underwent more than a few population doublings, and these ultimately senesced. No infinite life span variants emerged from any of the cloned populations at senescence. When the bulk cultures of treated and control fibroblasts senesced at about population doubling 40, I refed them weekly and maintained the cultures for several months. During that time, foci of proliferating cells occasionally appeared. I picked these foci, transferred them to 60 mm culture dishes, and fed them weekly with medium containing 10% serum. The 38 such foci I isolated underwent 5-15 more population doublings but all ultimately senesced. DISCUSSION The agar assay designed for these studies did discriminate among fibroblasts on the basis of calcium requirements. Unlike normal fibroblasts, infinite life span MSU-1.1 fibroblasts proliferated in 0.1 mM calcium, but not as efficiently as fibrosarcoma-derived cells. Young cultures of normal fibroblasts contained a few cells with reduced calcium requirements, but these variants disappeared or were lost as the culture aged and was passaged. Treatment with the mutagen ENU could enhance the number of calcium- independent variants, but only transiently. These observations do not support the idea that reduced requirement for calcium results from the activation or inactivation of a specific gene. Instead, they suggest that the calcium-independent variants identified in this assay represent one end of a spectrum of biological variation. My failure to derive infinite life span fibroblast cell lines from chemically-treated cultures is similar to the experience of others. Hypomethylating agents reportedly reduce rather than prolong the life span of human fibroblasts in bulk culture (Holliday, 1986; Fairweather e_t a_l., 1987), although no attempt was made in previous studies to look for rare infinite life span variants. With mutagens, infinite life span cells are produced only by prolonged or repeated treatment, and they typically have other transformed characteristics such as altered morphology and gross karyotypic abnormalities (Milo and DiPaolo, 1978; Borek, 1980; Namba e_t al., 1978 and 1981; Namba, 1985: Stampfer and Bartley, 1985). This may mean that human cells acquire an infinite life span infrequently because multiple or gross genetic changes are required (Cairns, 1981). But it may mean instead that human cells only appear to immortalize infrequently because infinite life span variants cannot be recognized under normal cell culture conditions unless they also acquire additional characteristics like altered morphology, or unless most of the normal 187 188 cells in culture with them are destroyed (DiPaolo, 1983). If that is true, then immortalization may require only a single genetic change, not many. Human fibroblasts exposed to a single treatment with propane sultone, MNNG, or 4-NQO can be transformed to growth factor independence and anchorage independence (Silinskas e_t al., 1981; Miyaki e_t al., 1982), suggesting that altered growth characteristics could result from a single mutational event. Stampfer and Bartley (1985) regularly extended the life span of human mammary epithelial cells be 2 treatments with BPDE, and even generated two infinite life span cell lines that way. In these experiments I tried to find potentially-immortal cells and remove them from co-culture with normal fibroblasts. My inability to do so does not mean they did not exist, but only that I did not hit upon the right way to look for them. Possibly I failed because there is no connection between reduced calcium requirement and infinite life span. Admittedly, my results suggest that such a connection does not exist, since all the calcium-independent variants I isolated senesced. But in no case was the calcium- independent phenotype stable. These studies therefore do not rule out that a stable, permanent reduction in calcium requirement is related to infinite life span. On the contrary, McCormick and his colleagues have found that MSU-1.1 fibroblasts share the stable phenotypes of infinite life span and calcium-independence, and Mougneau e_t a_l. (1984) found that myg-transfected rat fibroblasts acquire both reduced growth factor requirements and an infinite life span. The polyoma virus large T antigen produces the same effect as 111% when introduced into rat fibroblasts (Rassoulzadegan e_t a_l., 1982). When Griffin and Karran (1984) transfected monkey kidney cells with a fragment of Epstein-Barr virus, the cells became able to grow in very low serum and acquired an infinite life span. Immortalization of human fibroblasts by SV40 virus also frequently reduces their requirement for growth factors (Sack, 1981). Thus, although 189 immortalization is not always linked to reduction in growth factor requirements (Newbold and Overell, 1983), the connection between the two warrants further exploration. My inability to find infinite life span fibroblasts also may mean that expression of their infinite life span was inhibited by mortal fibroblasts in the same culture, even though cell-to-cell contact was prevented by growing cells suspended in soft agar. Experiments are currently underway to investigate this possibility and to find other ways to search for infinite life span variants. Several possibilities suggest themselves, such as using phorbol esters to block gap junction-mediated cell-to-cell communication. Blocking inhibition caused by a soluble factor released from normal fibroblasts would be more difficult, but if the factor could be partially purified then antibodies might be generated that could be used to neutralize it. SUMMARY In these studies I attempted to address the question of whether human fibroblast cultures contain spontaneously-arising infinite life span variants that go undetected by current cell culture procedures, and whether such variants could be induced by activating or inactivating genes with mutagens or hyopmethylating agents. I attempted to find infinite life span variants by looking for cells able to grow in low calcium conditions. By growing cells in soft agar to prevent cell—to—cell contact I was able to isolate calcium- independent variants, but they were not phenotypically stable and did not have extended life spans. Unfortunately, by the nature of my original question, negative results did not indicate that infinite life span variants were absent from fibroblast cultures, but only that I was unable to identify them. I undertook the search for infinte life span variants with some hope for success, partly because an infinite life span fibroblast line had arisen in McCormick’s laboratory from the same LG1 fibroblasts that I used in my experiments. I found myself in much the same position as Professor J .L.B. Smith in 1938. In that year he identified a fish pulled from the waters off Madagascar as a coelocanth, a prehistoric species thought extinct for 60 million years. Despite intense efforts he did not find another one until 1952. Since then, dozens of specimens of this prehistoric fish have been captured and studied (Fricke, 1988). I suspect the search for infinite life span fibroblasts will ultimately meet with the same success. All evidence points to infinite life span as a prerequisite for malignant transformation (McCormick and Maher, 1988). If that is true, then human cells must acquire an infinite life span as readily as rodent cells i_n m, since tumors arise frequently in both humans and rodents. That human cells don’t appear to do so as readily as rodent 190 191 cells i3 vitro probably has more to do with cell culture techniques than with intrinsic species differences. LIST OF REFERENCES Aust, A.E., Drinkwater, N.R., Debien, K., Maher, V.M., McCormick, J .J . Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts. Mut Res 125: 95-104 (1984) Ballard, F .J ., Read, L.C. 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