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dissertation entitled

Malignant Transformation Induced in Human Fibroblasts
by Expression of a Transfected T24 Hara§_0ncogene:
Requirement for an Infinite Lifespan

presented by

Peter J. Hurlin

has been accepted towards fulfillment
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Ph.D. Biochemistry

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MALIGNANT TRANSFORMATION INDUCED IN HUMAN FIBROBLASTS BY EXPRESSION OF
A TRANSFECTED T24 H-ng ONCOGENE: REQUIREMENT FOR AN
INFINITE LIFESPAN

by

Peter J. Hurlin

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1988

l

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ABSTRACT

MALIGNANT TRANSFORMATION INDUCED IN HUMAN FIBROBLASTS BY EXPRESSION
OF A TRANSFECTED T24 H-[gg ONCOGENE: REQUIREMENT OF AN
INFINITE LIFESPAN

by Peter J. Hurlin

This study was undertaken to identify the phenotypes induced in
human fibroblasts by expression of the T24 H-ras oncogene, one of several
members of the gas family of genes freqently detected in human tumors.
When the T24 H-ras oncogene, in a plasmid containing two sets of enhancer
sequences, was transfected into early passage, finite lifespan human
fibroblasts, colonies of morphologically transformed cells were generated.
Progeny of these cells expressed T24 H-ras p21, formed foci, and developed
colonies in soft agar. No such transformation was observed following
transfection with the normal H-ras gene. The majority of transformed
populations reverted to the normal phenotype, which correlated with loss
of expression of T24 H-ra; p21, even though the DNA persisted. The stably
transformed cells exhibited a finite lifespan and were not tumorigenic.
To determine whether the finite lifespan of these cells was a factor in
preventing tumor formation, the T24 H-ras oncogene was transfected into

an infinite lifespan human fibroblast strain, MSU-l.l, generated in this

laboratory following transfection with a v—myc oncogene. Foci of
morphologically transformed cells developed, and progeny of these cells
expressed the v-myc p110 and T24 H-L§§ p21, formed colonies in soft agar,
proliferated rapidly in serum-free medium containing 0.1 mM calcium, which
does not support growth of the parental cell line, and formed
progressively growing malignant fibrosarcomas in athymic mice. No such
transformation was observed with the control plasmid. Transfection of
additional infinite lifespan human fibroblast cell lines with the T24 H-
m oncogene also resulted in malignant transformation. The results
demonstrate the potent ability of the T24 H-gas oncogene to induce in
human fibroblasts characteristics of tumor cells and the fundamental and
complementary role that the infinite lifespan phenotype plays in T24 H-

;gs oncogene-induced malignant transformation.

ACKNOWLEDGEMENT

I would like to thank.my graduate advisor and research director, Dr.
J. Justin McCormick, for sharing with me his broad knowledge of cancer,
and his many ideas and perspectives that have shaped this thesis. I also
owe a special thanks to Dr. Veronica Maher for her direction and for her
critical review of the written manuscripts describing my work.

1 also would like to express my graditude to my parents. Shirley and
Kenneth Hurlin, whose friendship, guidance and love have been constant
throughout this endeavor. Finally, the love, support and smile of

Patricia Pillsbury, my'companion through the last two years, is gratefully

acknowledged.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES ................................................... vii
LIST OF FIGURES .................................................. viii
INTRODUCTION ..................................................... 1
CHAPTER I. LITERATURE REVIEW
A. Role of oncogenes in tumorigenesis ..................... 8
l. The genetic basis of cancer ......................... 8
2. Identification of oncogenes in the genomes of
acutely transforming retroviruses ................... 9
3. Identification of cellular oncogenes in human and
animal tumors ....................................... 11
a. Oncogenes identified in tumors by the NIH 3T3
transformation assay ............................. 11
b. Identification of oncogenes by genetic
rearrangement .................................... 13
i. Activation by proviral insertion ............ 13
ii. Activation by chromosomal translocation ..... 14
iii. Activation by amplification ................. 15
4. Function of oncogene encoded products in
tumorigenesis ...................................... 16
a. Growth factors and growth factor receptors ...... 16
b. Intracellular modulators of cell growth ......... 18
c. Nuclear proteins involved in contolling gene
expression ...................................... 18
B. Biochemical properties of La; ......................... 20
l. The gas genes ...................................... 20
a. Structural features of gas genes ................ 20
b. Mechanism of activation ......................... 22
2. The [as proteins ................................... 23
a. Structure of gas proteins .............................. 23
b. GTP binding and hydrolyzing activities .......... 25

c. Interaction with a putative effector protein.... 26
d. Involvement in the control of cell proliferation 27
3. Biological properties associated with expression of

gas oncogenes in mammalian cells ................... 28
a. Biological properties induced in rodent

fibroblasts ..................................... 28
b. Biological consequences of expression of [as

oncogenes in transgenic mice .................... 30
c. Use of human cells to study the biological

effects of gas oncgene expression ............... 32

References ............................................ 34

iv

CHAPTER II. MORPHOLOGICAL TRANSFORMATION, FOCUS FORMATION AND
ANCHORAGE INDEPENDENCE INDUCED IN DIPLOID HUMAN FIBROBLASTS BY
EXPRESSION OF A TRANSFECTED H-ggs ONCOGENE

Summary .................................................... 48
Introduction ............................................... 49
Materials and Methods
Cells and culture conditions ............................. 51
Plasmids ................................................. 51
DNA transfection ......................................... 51
Selection of G418 resistance ............................. 52
Assay for focus formation ................................ 52
Assay for anchorage independence ......................... 52
Southern blot analysis ................................... 53
RNA analysis ............................................. 53
Detection of p21 ......................................... 53
Results
Frequency of transfection to G418 resistance ............. 55
Transformation to altered morphology ..................... 55
Transformation to focus formation ........................ 58
Induction of anchorage independence ...................... 58
Analysis for the presence of transfected sequences ....... 61
Expression of the T24 H-ras oncogene in transformed cells 64
Tumorigenicity ........................................... 69
Discussion .................................................. 70
Acknowledgements ............................................ 73
References .................................................. 74

CHAPTER III. MALIGNANT TRANSFORMATION OF A HUMAN FIBROBLAST CELL
STRAIN, MSU-1.l, BY EXPRESSION OF A TRANSFECTED T24 H-Lgs ONCOGENE

Summary ..................................................... 80
Introduction ................................................ 81
Methods and Materials
Cells and culture conditions ............................. 84
Plasmids and DNA transfection ............................ 84
Assay for growth factor independence ..................... 85
Assay for anchorage independence ......................... 85
Tumorigenicity assays .................................... 85
DNA analysis ............................................. 86
Analysis of gas p21 and v-myg p110 ....................... 86
Karyotype analysis ....................................... 87
Results
Transformation to focus-formation by the T24 H-ras
oncogene ................................................. 88
Growth of MSU-l.l-T24 fibroblasts in serum-free medium... 88
Anchorage independence of MSU-1.l-T24 cell strains ....... 90
Tumorigenicity of MSU-1.l-T24 cell strains ............... 90
Presence of T24 H-ras and v-myg oncogenes ................ 94

Expression of T24 H-ras and v-myg oncogenes .............. 95

Karyotype analysis of MSU-1.l-T24 cell strains ........... 99
Tumorigenic transformation of infinite lifespan human
fibroblasts derived from other cell lines ................ 100
Discussion .................................................. 102
Acknowledgments ............................................. 106
References .................................................. 107
APPENDIX A ....................................................... 111
APPENDIX B ....................................................... 112
APPENDIX C ....................................................... 113

vi

LIST OF TABLES

Chapter II

Page
1. Frequency of transformation to altered cellular morphology
and focus formation .............................................. 56
Chapter III
1. Growth properties and tumorigenicity of MSU-1.1-T24 cell
strains .......................................................... 93

vii

LIST OF FIGURES

Chapter I

Page
1. Comparative amino acid sequence of [as proteins ............... 24
Chapter II
1. Comparison of normal and transformed morphologies ............ 57
2. The focus-forming ability of pHOGTl-transfected
fibroblasts ...................................................... 59

3. Southern blot analysis of cellular DNA from morphologically
transformed cells obtained following transfection with pH06T1, and

there morphologically normal progeny ............................. 62
4. Analysis of [as expression ................................... 65
5. H-Las RNA expression ......................................... 67

Chapter III
1. Morphology of LGI fibroblasts, MSU-1.l fibroblasts and

MSU-1.1-T24 fibroblasts .......................................... 89
2. Histology showing a poorly-differentiated human fibrosarcoma
formed in an athymic mouse by MSU-1.1-T24 strain 3 cells ......... 91
3. Southern blot analysis of plasmid pH06Tl-derived T24

H-Las sequences in MSU-1.l-T24 cell strains ...................... 95
4. Expression of T24 H-Las p21 in MSU—l.l-T24 cell strains ...... 96
5. Expression of v-myg encoded p110 in MSU-1.1-T24 cells

strains .......................................................... 98
APPENDIX A

Map of plasmid pH06T1 ............................................ 111
APPENDIX B

Athymic mouse with tumor formed by subcutaneous injection of
MSU-l.l-T24 strain 3 ............................................. 112

APPENDIX C

1. Comparison of gas p21 expression in focus-derived, anchorage
independent colony-derived and tumor-derived MSU-1.1-T24 cell

strains .......................................................... 114
2. Southern blot analysis of MC29 viral sequences in MSU-1.1-T24
cell strains ..................................................... 116
3. Analysis of 3;; p21 expression in KMST6-T24, GM0637-T24 and
SUSMl-T24 cell strains ........................................... 118

viii

Introduction

Malignant transformation of cultured mammalian cells is generally
recognized as a multistep process (Barrett and Ts’o, 1978; Thommasson and
DeMars, 1982; Perez-Rodrigez et al. 1982: Newbold et al., 1982; Land et
al., 1983; Ruley, 1983), as is in vivo carcinogenesis (Peto, 1977; Doll,
1978). Overwhelming evidence exists indicating that agents which damage
DNA are capable of mediating steps in the pathway leading to malignancies
in vivo and transformation in vitro. The first clues to the
identification of potential genetic targets for such agents came from
studies on RNA tumor viruses. Such studies showed that cellular genes,
transduced by RNA tumor viruses, were responsible for greatly enhancing
the ability of these viruses to cause tumors in host animals (Varmus,
1982). Studies on RNA tumor viruses converged with DNA transfection
'studies early in this decade resulting in the discovery that human tumors
contained mutated versions of cellular genes, which were homologous to
some of the cellular transforming genes of RNA tumor viruses (Bishop,
1983; Varmus, 1984). Cellular oncogenes were first detected in the DNA
of tumors by their ability to induce characteristics of tumor cells, for
example focus formation, when transfected into mouse NIH3T3 cells (Shih
et al., 1979). The oncogenes most frequently detected in human tumors
using the NIH3T3 transfection assay are homologous to the transforming gas
genes of Harvey and Kirsten murine sarcoma viruses, and members of a
family of cellular genes which have been designated H-gas, K-gas and N-

gas (Varmus, 1984). Sequence analysis of these oncogenes indicated that

2

point mutations occurring at or near codons 12 (Tabin et al., 1982; Reddy
et al., 1982; Taparowski et al., 1982) or 61 (Taparowski et al., 1983;
Yuasa et al., 1983) were responsible for their ability to transform NIH
3T3 cells. The detection of gas oncogenes in approximately 10-15%.of human
tumors (Barbacid, 1987), together with the known association between
carcinogen exposure and the generation of point mutations (Ames, 1979),
provides strong circumstantial evidence of a role for gas oncogenes in the
development of human malignancies.

Our understanding of the biological properties associated with expression
of gas_oncogenes has come primarily from transfection studies using rodent
fibroblasts. Early transfection experiments using finite lifespan diploid
rodent fibroblasts indicated that gas oncogenes could induce several
phenotypes characteristic of tumor cells, including morphological
transformation, focus formation and anchorage independence, but not an
infinite lifespan in culture or tumorigenicity (Land et al., 1983; Ruley,
1983; Newbold and Overell, 1983). However, co-transfection of such cells
with an H-gas oncogene and any one of a number of genes thought to cause
induction of an infinite replicative lifespan in culture resulted not only
in morphological transformation, focus formation and anchorage
independence, but also in an infinite lifespan in culture and
tumorigenicity (Land et al., 1983; Ruley, 1983). These results, along
with results showing that transfection of ms oncogenes into rodent
fibroblasts already exhibiting an infinite lifespan caused tumorigenic
transformation (Land at al., 1983; Overell and Newbold, 1983), led to the
hypothesis that oncogenes that induce the infinite lifespan phenotype act
in a complementary or cooperative fashion with gas oncogenes in a two step

model of in vitro carcinogenesis.

3
Although the use of rodent fibroblast transformation systems has been
instrumental in the identification and preliminary characterization of
oncogenes, it is not clear whether the results obtained using rodent
fibroblasts are directly applicable to cancer development in humans. This
is because exposure to various carcinogens (Barrett and Ts’o, 1978),
transfection of cloned oncogenes (Land at al., 1983; Ruley, 1983; Newbold
and Overell, 1983), or direct microinjection of oncogene-encoded products
(Feramisco et al., 1984; Stacy et al., 1984) can cause transformation of
rodent fibroblasts in culture, but attempts to reproduce such results
using human fibroblasts for the most part have failed (DiPaolo, 1983;
Sager et al., 1983; Spandidos et al., 1984; Feramisco et al., 1984; Stacy
et al., 1984). The failure of human fibroblasts to transform as readily
as rodent fibroblasts in culture may result from a fundamental biological
difference between the species, or may be related to the fact that, unlike
human fibroblast cell lines, many rodent fibroblast cell lines can
spontaneously transform into cells that exhibit characteristics of tumor
cells (Heidelberger, 1973). Because of this latter property of rodent
fibroblasts, one cannot dismiss the possibility that some transformed
characteristics attributed to the mutagenic effects of a carcinogen, or
expression of a transfected oncogene may actually be the result of, or

related to some spontaneous transformation event(s).

The goal of this thesis was to define the biological properties induced
in human fibroblasts following transfection of these cells with the T24
H-gas oncogene, an H-gas oncogene cloned from a human bladder carcinoma.
Results from earlier studies by others who tried to accomplish this same

goal suggested that human fibroblasts in culture are resistant to

4

transformation by gas oncogenes (Sager et al., 1983; Spandidos, 1985).
However, the considerable circumstantial evidence involving this family
of oncogenes in the development of human malignancies, as discussed above,
encouraged me to carry out a more comprehensive examination of the
problem. Transfection studies with the T24 H-gas oncogene cloned into
various vectors were carried out using both finite lifespan, and infinite
lifespan human fibroblasts in order to permit comparison of the results
to those of gas transfection studies that used comparable rodent

fibroblast cell lines as recipients.

Chapter I of the thesis reviews the literature that provides the
conceptual framework of our understanding of how oncogenes can be
activated and how they may be involved in the induction of cancer and
reviews the literature directly concerning gas oncogenes. Chapter II is
primarily a manuscript by P.J. Hurlin, D.G. Fry, V.M. Maher and J.J.
McCormick that was published in the November, 1987 issue of Cancer
Research 51, 5752-5757, which describes my work showing that transfection
of the T24 H-ras oncogene into diploid human fibroblasts induces several
characteristics of tumor cells. The published manuscript has been
modified in Chapter II to include data showing that cells stably
transformed following transfection of the T24-H-gas oncogene are not
tumorigenic. These latter data were published in a review article by D.G.
Fry, P.J. Hurlin, V.M. Maher and J.J. McCormick which appeared in a
special issue of Mutation Research (vol. 199, 341-351, 1988) on the
transformation of human fibroblasts. Chapter III is a manuscript by P.J
Hurlin, V.M. Maher and J.J. McCormick submitted in May, 1988 for

consideration for publication in the Proceedings of the National Academy

5
of Sciences USA, which describes my work showing that the T24 H-ras
oncogene induces malignant transformation when it is transfected into
infinite lifespan human fibroblasts. Data relevant to this study that
were not shown in the submitted manuscript are shown in Appendices B and

C. Appendix A shows maps of plasmids important to these studies.

REFERENCES

Ames, B.N. (1979) Identiying environmental chemicals causing mutations
and cancer. Science 885, 587-593.

Barbacid, M. (1987) gas genes. Ann. Rev. Biochem. 88, 779-827.

Barrett, J.C. and Ts’o, P.0.P. (1978) Evidence for the progressive nature
of neoplastic transformation in vitro. Proc. Natl. Acad. Sci. USA 18,
3761-3765.

 

Bishop, J.M. (1983) Cellular oncogenes and retroviruses. Ann. Rev.
Biochem., 52, 301-354.

DiPaolo, J.A. (1984) Relative difficulties in transforming human and
animal cells lg vitro. J. Natl. Cancer Inst. 18, 3-8.

 

Doll, R. (1978) An epidemiological perspective of the biology of cancer.
Cancer Res. 88, 3573-3583.

Feramisco, J.R., Gross, M., Kamata, T., Rosenberg, M. and Sweet, R.N.
(1984) Microinjection of the oncogene form of the human H-gas (T-24)
protein results in rapid proliferation of quiescent cells. Cell 88, 109-
117.

Heidelberger, C. (1973) Chemical carcinogenesis in culture. Advances in
Cancer Research, Vol. 18, 318-365, Academic Press, Inc., New York and
London.

Land, H., Parada, L.F. and Weinberg, R.A. (1983) Tumorigenic conversion
of primary embryo fibroblasts requires at least two cooperating oncogenes.
Nature 885, 596-602.

Land, H., Parada, L.F. and Weinberg, R.A. (1983) Cellular oncogenes and
multistep carcinogenesis. Science 888, 771-778.

Newbold, R.F. and Overell, R.N. Fibroblast immortality is a prerequisite
for transformation by EJ Ha-gas oncogene. (1983) Nature 895, 648-651.

Perez-Rodriguez, R., Franchi, A., Deys, B.F. and Pouyssegur, J. (1982)
Evidence that hamster fibroblast tumors emerge in nude mice through a
process of two in vivo selections leading to growth factor "relaxation"
and to immune resistance. Int. J. Cancer 88, 309-314.

Peto, R. (1977) The origins of human cancer. Book C, Hiatt, H.H.,
Natson, J.D. and Hinsten, J.A. (Eds.), Cold Spring Harbor Laboratory, New
York, pp. 1403-1437.

Reddy, E.P., Reynolds, R.K., Santos, E. and Barbacid, M. (1982) A point
mutation is responsible for the acquisistion of transforming properties
by the T24 human bladder carcinoma oncogene. Nature, 888, 149-152.

7

Ruley, H.E. (1983) Adenovirus early region 1A enables viral and cellular
transforming agents to transform primary cells in culture. Nature 888,
602-606.

Sager, R., Tanaka, K., Lau, C.C., Ebina, Y. and Anisowicz, A. (1983)
Resistance of human cells to tumorigenesis induced by cloned transforming
genes. Proc. Natl. Acad. Sci. USA 88, 7601-7605.

Shih, C., Shilo, B.Z., Goldfarb, M.P., Dannenberg, A, and Weinber, R.A.
(1979) Passage of phenotypes of chemically transformed cells via
transfection of DNA and chromatin. Proc. Natl. Acad. Sci. USA 18, 5714-
5718.

Spandidos, D. (1985) Mechanism of carcinogenesis: The role of
oncogenes, transcriptional enhancers and growth factors. Anticancer Res.
8, 485-498.

Stacy, D.W. and Kung, H-. F. (1984) Transformation of NIH 3T3 cells by
microinjection of Ha-gas p21 protein. Nature 818, 508-510.

Tabin, C.J., Bradley, S.N., Bargman, C.I., Weinberg, R.A., Papageorge,
A.G., Scolnick, E.M., Dhar, R., Lowy, D.R. and Chang, E.H. (1982)
Mechanism of activation of a human oncogene. Nature 888, 143-149.

Taparowski, E., Shimizu, K., Goldfarb, M. and Wigler, M. (1983) Cell
88, 581-586.

Taparowski, E., Suard, Y., Fasano, 0., Shimizu, K., Goldfarb, M. and
Wigler, M. (1982) Activation of the T24 bladder carcinoma transforming
gene is linked to a single amino acid change. (1982) Nature 888, 762-
765.

Thommasen, D. G. and DeMars, R. (1982) Clonal analysis of the stepwise
appearance of anchorage independence and tumorigenicity in CAK, a
permanent line of mouse cells. Cancer res. 88, 4054-4063.

Varmus, H.E. (1982) Form and function of retroviral proviruses. Science
818, 812-820.

Varmus, H.E. (1984) The molecular genetics of cellular oncogenes. Ann.
Rev. Genet. 18, 553-512.

Yuasa, Y., Srivastava, S.K., Dunn, C.Y., Rhim, J.S., Reddy, E.P. and
Aaronson, S.A. (1983) Acquisition of transforming properties by

alternative point mutations within c-bas/has human proto-oncogene» Nature
888, 775-779.

CHAPTER I

LITERATURE REVIEH

A. Role of Oncogenes in Tumorigenesis
1. The Genetic Basis of Cancer

Cancer incidence data indicate that human populations living in
different geographical regions of the world tend to develop different
kinds of cancer (Waterhouse, 1976). For example, the inhabitants of third
world countries often have a high incidence of liver cancer, those of
western nations have a high incidence of cancer of the breast and colon,
and those living in Japan have a high incidence of stomach cancer.
Migrant populations tend to take on the pattern of cancer that is
characteristic of their new homes (Waterhouse, 1976), suggesting that such
variation in cancer incidence is due to diet, customs, or what can be
generally considered environmental factors.

It is now well established that the two most common cancers in the
western world, skin cancer and lung cancer, are caused primarily by known
environmental factors, i.e., sunlight exposure and cigarette smoking
respectivelyu Components of cigarette smoke and ultraviolet irradiations,
like most carcinogenes interact with DNA and are mutagenic (Haseltine,
1983). Because skin, lung and most other types of cancer are strongly age
dependent, it has been hypothesized that cancer induction must be a
multistep process, and that the accumulation of critical mutations,
resulting from carcinogen exposure or otherwise, over the life of an
individual is part of this process (Doll, 1978; Cairns, 1981; Moogavkar

and Knudson, 1981). Support for this hypothesis has come from a wide

9
range of studies including those on the induction of cancer in animals by
chemical carcinogens (Berenblum, 1940; Berenblum and Shubik, 1947),
studies on the relationship between carcinogen exposure and the induction
of mutation and cell transformation of cells in culture (Barrertt and
Ts’o, 1978; Spandidos and Siminovitch, 1978), and studies on the increased
mutagenicity of carcinogens in cells derived from individuals who have a
hereditary predisposition to cancer (Maher et al. 1976). There is new
experimental evidence to show that point mutations and deletions, gene
amplification and chromosomal translocation- -events that are known or
suspected to be induced by carcinogens- -can cause the "activation" of
specific cellular genes, referred to as "proto-oncogenes", into oncogenes

whose protein products contribute to the malignant phenotype.

2. Identification of Oncogenes in the Genomes of Acutely Transforming
Retroviruses

It was hypothesized by Temin in 1964 that as part of the lifecycle
of RNA tumor viruses, RNA acted as a template for the synthesis of a DNA
provirus which integrated into the host cells genome. The discovery of
the reverse transcriptase enzyme by Temin and Baltimore in 1970 explained
how the DNA-provirus mechanism could work. Experiments by Neiman (1972),
showing the presence of proviral DNA in the genome of infected cells,
provided more formal proof of the hypothesis. These discoveries led to
explanations for the profound transforming properties of some RNA tumor
viruses such as Rous sarcoma virus (Rous, 1911). It was proposed by Temin
(1976) that the genome of acutely transforming retroviruses evolved, in
part, from normal cellular sequences that had been previously mutated in

the host cell by exposure to carcinogens.

10

A similar hypothesis, the "viral oncogene hypothesis", proposed by
Huebner and Todaro in 1969, stated that the cells of all or most
vertebrate species contain genomes of potentially oncogenic RNA tumor
viruses that are transmitted vertically from parent to offspring. In a
modified version of this hypothesis (Todaro and Huebner, 1972) it was
proposed that the occurrence of cancer may be determined by the activation
of normally repressed viral genes by, perhaps, exposure to carcinogenic
agents or the normal processes of aging. Experiments by Neiman (1972)
and later Spector (1978) showing that sequences within the acutely
transforming Rous chicken sarcoma virus hybridized ‘with the DNA, of
uninfected cells appeared to support this hypothesis. However, it is now
known that the life cycle of RNA tumor viruses predisposes them to rare
genetic events that lead to the incorporation in their genome a group of
cellular genes that provide a selective advantage for proliferation to
cells that are infected by such transforming viruses. Ultimately, this
is manifested as malignant neoplasia.

Although it is not true, as originally hypothesised by Huebner and
Todaro (1969), that retroviral genomes are present in all of the cells of
any given host species, it is true that specific genes (proto-oncogenes)
which have the potential to contribute to the malignant phenotype do exist
in all cells. This concept, and the concept, put forth by both Huebner
and Todaro (1969) and Temin (1976), that carcinogen induced mutations can
be responsible for activating the transforming potential in such cellular
genes are now well established.

Since the first v-oncogene, v-sgs, was identified in 1976 (Stehelin
et al., 1976) there have been more than 20 other v-oncogenes identified

in the genomes of RNA tumor viruses isolated from tumors, mostly sarcomas,

11
formed in a number of different vertebrate species. Each of these
oncogenes has been found to be homologous to a cellular counterpart in the
genome of the viruses host species. Such cellular counterparts or proto-
oncogenes exist in evolutionarily distant eukaryotes including yeast and
drosophila and are sometimes regulated in a cell cycle or lineage-specific
manner, suggesting that they might play a central role in processes that
control the normal cellular growth and proliferation, processes that are

altered in cancer cells.

3. Identification of Cellular Oncogenes in Human and Animal Tumors
There are many possible mechanisms by which cellular proto-oncogenes
can be activated into oncogenes. These include overexpression,
inappropriate or ectopic expression (e.g., expression in cell type that
it is normally not expressed in), unscheduled expression, altered
biochemical properties caused by structural alterations within coding
sequences, or some combination of these mechanisms. In most cases,
putative cellular oncogenes have been detected in human and animal tumors
by the identification of’ genes that possess one or more of these
properties, as well as by exibiting homology to recognized retroviral

oncogenes.

a. Oncogenes Identified in Tumors by the NIH3T3 Transformation Assay
Experiments by Anderson et al. (1979) demonstrated that DNA
fragments of acutely transforming retroviruses, that carried putative v-
oncogenes, were capable of causing cellular transformation when
transfected into normal cells. These experiments suggested to Weinberg

and his colleagues that if cellular counterparts (c-oncogenes) existed in

12

chemically transformed cells, they too should be capable of exerting such
dominant transforming effects on normal cells. Experiments by Shih et al.
(1979), demonstrated that when DNA, isolated from chemically transformed
mouse cells, was transfected into non-transformed mouse NIH 3T3 cells
using the the calcium phosphate transfection technique developed by Graham
and Van der Eb (1973), foci of morphologically transformed cells were
generated. When DNA, isolated from normal mouse cells, was transfected
into the same recipient cells, it did not cause such transformation.
Although there was no formal proof, it was concluded that the induced
transformation was the result of the transfer of cellular alleles
(oncogenes) that had their transforming potential activated by carcinogen
induced mutations (Shih., 1979). The existence of such transforming
sequences has since been demonstrated in the DNA of a large number of
different human tumor cell lines and human tumor biopsy material.

The identification of tumor-derived sequences that caused fecus
formation and malignant transformation when transfected into mouse NIH 3T3
cells was first achieved by four separate groups (Parada et al., 1982;
Santos et al., 1982; Der et al., 1982; Goldfarb et al., 1982), using DNA
derived from human bladder carcinoma cell lines. By using molecular
probes developed from v-oncogenes, each of these groups discovered that
the sequences responsible for transformation in each case were homologous
to the v-oncogene, v-H-gas, of the Harvey strain of murine sarcoma virus.
Literature concerning gas genes and their products is reviewed in detail

in later sections.

Several other putative oncogenes have been detected in human tumor

cell DNA by the NIH3T3 transformation assay (Padhy et al., 1982; Cooper

13
et al., 1984; Padua et al., 1984; Shimizu et al., 1985; Eva and Aaronson,
1985; Martin—Zanca et al., 1986; Fusco et al., 1987). For at least two
of these oncogenes the putative mechanism of activation has been
identified. The gag oncogene, isolated from a rat neuroblastoma, codes
for a transmembrane, receptor-like protein and shows a 50% sequence
identity with the human epidermal growth factor receptor gene (Schechter
et al., 1984) appears to be activated by point mutations (Bargman et_al.,
1986). The gag oncogene, isolated from a human colon carcinoma, is a
fusion gene consisting of sequences derived from the tropomyosin gene and
a gene that codes for a protein tyrosine kinase (Martin-Zanca et al.,
1986). This fusion gene appears to have been generated by a mechanism

involving chromosomal translocation (Martin-Zanca et al., 1986).

b. Identification of Oncogenes by Genetic Rearrangements
i. Activation by Proviral Insertion

Retroviruses that do not have oncogenes may nevertheless be involved
in tumor induction. This occurs by means of insertional mutagenesis as
was first shown in the study of chicken lymphomas (Hayward et al., 1981).
In these tumors, the cellular myc gene has been activated by insertions
of retroviral DNA upstream, or within the gene (Hayward,et al., 1981;
Payne et al., 1981; Neel et al., 1981). Altered transcriptional
regulation of c-myc is generally thought to be one of several steps in
tumorigenesis (Thompson and Neiman, 1987). Such altered regulation and/or
overexpression of a previously regulated or silent gene results because
the integrated viral DNA provides a strong promoter from which
transcription of the cellular gene is initiated and/or a transcriptional

enhancer that stimulates increased transcription from the cellular gene’s

14
own promoter (Bishop, 1987). Because proviral integration is thought to
occur randomly throughout the host cells genome (Varmus, 1983), commonly
interrupted cellular domains identified in tumor cells are likely to
represent sites that are functionally significant to tumor induction. In
addition to c-mys, c-mas (Gattoni-Celli et al. 1983; Cohen et al., 1983;)
and c-ag1)_8 (Fung et al., 1983), which has been identified as the
epidermal growth factor receptor gene (Downward et al. 1984), are
frequently activated in animal tumors by proviral insertional mutagenesis.
Further support for a role in tumor induction by these genes is based on
the fact that each is homologous to v-oncogenes (Varmus, 1984). Other
genetic loci that do not show homology to v-oncogenes have also been
identified as putative oncogenes based on the observance of repeated

adjacent retroviral insertion (Varmus, 1984).

ii. Activation by Chromosomal Translocations

Although most human tumors fail to show specific chromosomal
translocations, there are highly regular translocations associated with
some tumor types, most notably chronic myelogenous leukemia (CML), acute
lymphoblastic leukemia (ALL), and several B-cell malignancies (Sandberg
et al. 1980). In approximately 95% of CML patients and 20% of ALL
patients (Sandberg, 1980), there exists a cytogenetic marker, the
Philadelphia chromosome, that is generated by a reciprocal translocation
between chromosomes 22 and 9 (Nowell and Hungerford, 1960). The presence
of the Philadelphia chromosome in such patients has important prognostic
and diagnostic value (Sandberg, 1980). The c-a81 gene, a homologue of the
v-a81 oncogene that codes for a protein tyrosine kinase (Davis et al.

1985), has been localized to the portion of chromosome 9 that contributes

15

to the Philadelphia chromosome (de Klein et al., 1982). The c-a81 gene
is translocated into a region of chromosme 22, designated the breakpoint
cluster region (gag), and the biochemical consequence of this molecular
rearrangement is the production of an abnormal fusion protein, gag-aal
p210, with enhanced protein kinase activity (Konopka and Witte, 1985; Chan
et al. 1987; Kurzrock et al. 1987).

Whereas the translocations that characterize the formation of the
Philadelphia chromosome in CML and ALL apparently activate the a81 gene
by altering the biochemical properties of its product, translocations
characteristic of a number of B-cell malignancies appear to activate the
c-mya gene by altering its normal transcriptioal regulation (Leder et al.,
1983, Marcu et al., 1983).

The specific translocations existing in a large number of B-cell tumor
lines join the c-mys gene on chromosome 8 with the an immunoglobulin locus
on one of three different chromosomes (2, 14 and 22) (Croce et al., 1979;
Erickson et al., 1982; Malcolm et al., 1982). Altered transcriptional
regulation of the c-mys gene (overexpression and/or unscheduled
expression), caused by the influence of adjacent transcriptional
regulatory elements at the immunoglobulin loci, appears to be the
mechanism of activation of the c-mgs gene in such B-cell malignancies

(Leder, 1983).

iii. Activation by Amplification

Several cellular genes, that are homologous to v-oncogenes, have
been identified in some tumors and tumor-derived cell lines in multiple
copies. For example, the mys proto-oncogene is amplified 30 to 50-fold

in the human promyelocytic leukemia cell line HL-60 (Dalla-Favera 1982),

16

and in small cell carcinoma-derived cell lines (Little et al., 1983). The
k-gas proto-oncogene is amplified 3- to 5-fold in a human colon carcinoma
cell line (McCoy et al., 1983), and a member of the mys family of genes
designated N-mys is amplified in many human neuroblastomas (Schwab et al.,
1983). Such amplification has been found to correlate with advanced
disease stage (Brodeur et al., 1984). In these cases, the increased gene

copy number correlates with an increased level of transcription.

4. Function of Oncogene Encoded Products in Tumorigenesis

For the most part, the physiological function of the products of
oncogenes is unknown. However, the preliminary characterization of
oncogene encoded products has revealed that they all appear to share
properties with various gene products that are known to play a role in
cellular mechanisms that control growth. The following descriptions of
oncogene products are not meant to be complete, but to relate the above
sections on mechanisms of oncogene activation to their role, or speculated

role, in the transformation process.

a. Growth Factors and Growth Factor Receptors

The only retroviral oncogenes identified as genes that code for a
peptide growth factor is the v-si_s oncogene of simian sacoma virus
(SSV)(Devare et al., 1983) and the transforming gene of the Parodi-Irgenes
feline sarcoma virus (Besmer et al., 1983), which both code for the B-
chain of platelet-derived growth factor (PDGF). In vitro studies of SSV-
transformed cells indicate that the mature v-sis gene product enters the
cellular secretion pathway and interacts with the PDGF receptor to cause

unrestricted growth in an autocrine fashion (Heldin et al., 1987).

17
Expression of the c-sis gene is not detectable in most normal cell lines

or in most tissues (Eva et al., 1982), but has been found expressed in a
number of human tumor cell lines (Heldin et al., 1987). In such cells
expressing PDGF receptors, activation of c-sis gene expression may affect
cells in a manner similar to SSV transformation, i.e. induce autocrine
growth stimulation.

A connection between oncogene products and growth factor receptors
was established with the discovery that the v-aga oncogene of avian
erythroblastosis virus, and the v-fms oncogene of the McDonough strain of
feline sarcoma virus code for’ products homologous to the epidermal growth
factor receptor (Ullrich et al., 1984; Xu et al., 1984: Lin et al., 1984)
and colony stimulating factor-1 receptor (Rettenmeier' et al., 1985)
respectively. In addition, the product of the gag oncogene, mentioned
above, is thought to be the rat homolog of the human EGF receptor (Shector
et al., 1984). The features common to the EGF and CSF-l growth factor
receptors, as well as to the growth factor receptors for PDGF and insulin,
is a glycosylated extracellular domain that binds ligand, a transmembrane
domain consisting primarily of hydrophobic amino acid residues, and an
intracelluar domain that exhibits intrinsic protein kinase activity. The
tyrosine kinase activity of these receptors is dependent on the binding
of the appropriate growth factor (Heldin et al., 1987), and appears to be
an integral part of their function in stimulating cellular proliferation.
However, there are many substrates and it has been difficult to find a
molecular scheme to explain which tyrosine phosphorylations are important
for such stimulation. In the case of the v-ag8 B oncogene, the region
coding for the extracellular, ligand binding domain is deleted (Downward

et al., 1984) and, as a consequence, its tyrosine kinase activity is

18

activated (Gilmore et al., 1985; Kris et al., 1985). Thus, alterations
in the proportions of phoshorylated derivatives of EGF receptor-targeted

intracellular proteins may act to cause cellular transformation by v-ag8

B.

b. Intracellular Modulators of Cell Growth

In addition to the growth factor receptor products of oncogenes,
several other oncogene products that reside at the plasma membrane or in
the cytoplasm code for protein tyrosine kinases, as well as
serine/threonine kinases. Although the functions of these kinases are
not known, it is thought that they control cell proliferation by
modulating the activity or function of their various cytoplasmic and
plasma membrane substrates. The oncogenic versions induce transformation
through inappropriate or excessive phosphorylation of such substrates
(Hunter, 1987). Therefore, it is not suprising that the gene that codes
for protein kinase C, whose protein product is known to induce profound
effects on cell growth and proliferation by substrate phosphorylation, can
induce transformation of NIH3T3 cells if it is linked to strong viral
transcriptional elements (Housey et al., 1988).

The protein products of gas oncogenes, which are localized to the
plasma membrane, appear to exert their profound effects on cell
proliferation through a pathway involving protein kinase C and possibly

other protein kinases. This will be discussed in a latter section.

c. Nuclear Proteins Involved in Controlling Gene Expression
The viral and cellular my; genes code for proteins that are located

in the nucleus and have DNA-binding properties (Abrams et al. 1982). The

19

rapid increase of c-mys gene expression following mitogenic stimulation
of resting cells (Leder et al. 1983) suggests that the c-mys protein plays
a role in the cell cycle, possibly regulating entry into S-phase. As
discussed above, the transforming properties of my; oncogenes are
activated by unscheduled expression and/or overexpression. Thus, it is
thought that such activation leads to the continuous supply of signals for
entry into S-phase, and an inability of cells to cease dividing.

Another nuclear oncogene product, that of the 188 oncogene,
functions as a trans-acting transcriptional regulator (Angel et al.,
1988). It was recently discovered that the cellular jag product forms a
complex with the cellular product of another known nuclear oncogene, gas,
and that this complex can interact with the regulatory sequences of many
different genes (Rauscher et al, 1988). Like the cellular myc product,
the cellular figs gene product is rapidly induced in various cell types in
responce to mitogens (Mitchel et al., 1985; Muller et al., 1985; Treisman,
1985). Thus, interactions between the jag and figs gene products appear
to be a part of a network of mechanisms that regulate the expression of
genes involved in normal, and potentially abnormal, cellular proliferation

in mammalian cells.

20

B. Biochemical properties of gas

Using the NIH 3T3 transformation assay, gas oncogenes have been
detected in approximately 10-15% of human tumors (Barbacid, 1987). The
recent development and employment of more sensitive analytical
methodologies for detection of gas genes indicate that a higher percentage
of human tumors may contain gas oncogenes (Winter et al., 1985; 305 et
al., 1986; 805 et al., 1987; Forrester et al., 1987). However, it is not
possible to demonstrate a causative role for ,gas_ oncogenes in the
development of human tumors simply by being able to detect their presence
there. The use of carcinogen-induced animal tumor model systems provides
one means of determining if there is a causal relationship between tumor
induction and somatic mutation of gas genes. Several studies have shown
that tumors which arise in animals following administration of chemical
or other carcinogens contain activated gas genes, and that such activation
is reproducible and carcinogen-specific (Balmain and Pragnell, 1983;
Sukumar et al., 1983; Balmain et al., 1984; Zarble et al., 1985;
Quintanilla et al., 1986; Roop et al., 1986). These in vivo studies
provide strong evidence that gas genes play a role in mammalian

tumorigenesis.

1. The gas Genes
a. Structural Features of gas Genes

Three functional gas genes have been identified in the human genome.
These are designated H-gas, K-gas and N-gas (Shimizu et al., 1983). The

H-gas gene, and the K-m gene were found to be homologous to the

21
transforming genes of Harvey murine sarcoma virus (Der et al., 1982,
Parada et al., 1982; Santos et al., 1982) and Kirsten sarcoma virus (McCoy
et al., 1983) respectively, hence their names. The N-gas gene was
originally identified as a transforming gene in a human neuroblastoma cell
line by the NIH 3T3 transformation assay (Hall et al., 1983). The three
mammalian gas genes code for proteins that have a molecular weight of
approximately 21 kilodaltons with 187 or 188 amino acid residues (Capon
et al., 1983; Fasano et al., 1983; Reddy, 1983; Hall and Brown, 1984).
The p21 coding sequences of each of the human gas genes is contained in
4 exons (K-gas also has an alternative 4th exon) with the first three
exons being nearly identical, and each of the exon splice junctions for
all three gas genes being identical (Capon et al., 1983; Fasano et al.,
1983; Reddy, 1983; McGrath et al., 1983; Shimizu et al., 1983). Such
sequence conservation suggests a common origin of r_as genes from one
ancestral gene. Because variation in the size of the intervening
sequences exists between H-, K- and N-gas genes, the genetic locus of
these genes ranges in size from 4.5 kilobase pairs for H-gas to 50
kilobase pairs for K-gas (Fasano et al., 1983; McGrath et al., 1983;
Shimizu et al.,1983; Capon et al., 1983). The promotor regions of each of
the three functional mammalian gas genes lack a characteristic TATA or
CAAT box (Breathnach and Chambon, 1981), but contain multiple
transcriptioal start sites and copies of the hexanucleotide sequence
CCGCCC or GGGCGG (GC box) (Hall and Brown, 1985; Ishii et al. 1985;
McGrath et al., 1983). The GC box consensus sequence was originally found
in the simian virus 40 early promoter (Byrne et al., 1983) and is
characteristic of a number of cellular housekeeping genes (Ishii et al.,

1986),

22

c. Mechanism of Activation of gas Genes

Mammalian gas genes acquire the ability to transform cultured
mammalian cells by single missense point mutations within their coding
sequences (Tabin et al., 1982; Reddy et al., 1982; Taparowski et al.,
1982). Sequence analysis of gas oncogenes derived from carcinogen induced
tumors in animals, carcinogen transformed rodent cells, and from naturally
occurring human tumors indicate that the mutations responsible for
conferring transforming properties are located at codons 12 (Tabin et al.,
1982; Reddy, et al., 1982; Taparowski et al., 1982), 13, (Bos et al.,
1985), 59, (Dhar et al., 1982), and 61 (Yuasa et al., 1983). Missense
mutations at several other positions, introduced by in vitro mutagenesis,
also can confer transfoming properties to gas genes (Fasano et al., 1984;
Walter et al., 1986; Sigal et al., 1986).
Activation of gas genes by alterations affecting position 12 have been
studied most thoroughly. Analysis of the transforming potential (focus
formation in transfected Rat 1 cells) of 20 different H-gas genes, each
encoding a different amino acid at this position, revealed that
substitution of the normal amino acid (glycine) with any one of the other
20 amino acids, except proline, resulted in an activated p21 (Seeburg et

al., 1984).

There is some evidence that overexpression of normal gas genes, can
play a role in cell transformation in vitro and tumor development in vivo.
For example, it has been shown that normal gas genes can cause malignant
transformation of NIH 3T3 cells when linked to a strong promotor (Chang

et al., 1982), or when multiple copies are incorporated into the genome

23
of these cells (Pulciani et al, 1985). Transformation in each of these
cases correlated with a 30-100-fold increase in the level of g_s
expression over that observed in non-transfected NIH 3T3 cells (Chang et
al., 1982; Pulciani et al., 1985). Amplification and overexpression of
normal gas genes, usually K-gas, has been observed in a number of human
tumors (Kahn et al., 1987), but the levels of expression were much below
that which was capable of causing transformation of NIH 3T3 cells.
Furthermore, there has been no consistent results from studies examining

gas expression in tumor tissues that would implicate a role for

overexpression of normal gas alleles in tumorigenesis (Barbacid, 1987).

2. The gas Proteins
3. Structure of gas Proteins

Several different functional domains of gas p21 proteins have been
identified (Figure 1). These domains were identified using primarily
three approaches: comparing the deduced amino acid sequence of p215 that
exhibit transforming properties and those that do not (Taparowski et al.
1982; Dhar et al., 1982; Seeburg, et al., 1984; Fasano et al., 84; Walter
et al., 1984; Sigal et al., 1986); comparing the biochemical properties
of mutant proteins expressed in as 2911 with their biological properties
in cells microinjected with the mutant protein or in cells transfected
with the mutated gene (Willumsen et al., 1985; Willumsen et al., 1986;
Sigal et al., 1986; Der et al., 1986); and determining the biochemical and
biological properties of gas p21 following reaction with antibodies with
known p21 epitopes (Clark et al., 1985). The binding domain of the Y13-
259 monclonal antibody (Furth et al., 1981), which has been utilized

extensively for characterization of gas proteins, is located in a highly

24

no
I I I I I I
“am" ' to
muons

 

 

 

 

ODflGfl' "”““““'M”P

Efloetor

 

vnpzn
umuuyuumn 5:3

Membrane
“WI

MW nglon

Figure 1. Schematic representation of the structural and functional
domains within mammalian p21 gas proteins.

Printed with permission from
Ann. Rev. Biochem. Inc.

25

conserved, but functionally expendible region (Willumsen et al., 1986;
Sigal et al., 1986; Lacal et al., 1986). The only region of gas proteins
that shows sequence diversion (hypervariable region) is near the carboxy
terminus, suggesting that some as yet unknown feature of this region will
provide an explanation as to why three so functionally similar gas genes

are expressed in mammalian cells.

b. GTP Binding and Hydrolysing Activities

The known biochemical activities associated with gas p21 proteins
include GDP and GTP binding (Scolnick et al, 1979; Shih et al. 1980) and
GTPase activities (McGrath et al., 1984; Gibbs et al., 1984; Sweet et al.,
1984). In addition, gas proteins possesing threonine at amino acid
position 59 (v-H-gas p21) exhibit autophosphorylation at that residue
(Shih et al., 1982). In all g_s p21’s, palmitic acid is post-
translationally attached to a cysteine residue near the carboxyl terminus
which facilitates their association with the inner surface of the plasma
membrane (Willingham et al., 1980; Shih et al., 1982; Sefton et al., 1982;
Willumsen et al., 1984). A biochemical marker that seems to correlate
well with mutations that activate the transforming properties of gas

oncogenes is decreased GTPase activity (McGrath et al., 1984; Gibbs et

al., 1984: Sweet et al., 1984).

The biochemical properties of ES p21 proteins closely resemble
those of the G proteins involved in transmembrane signaling systems
(Gilman, 1984). Such G proteins cycle between active GTP-bound forms,

and inactive GDP-bound forms, and are involved in the stimulation or

26

inhibition of adenyl cyclase (Gilman, 1984). The observation that many
oncogenic mutants of p21 are reduced in GTPase activity led to the
proposal that transformation by p21 is the result of abnormally high
levels of p21 in the active GTP-bound state (Gibbs et al., 1984; McGrath
et al.,1984; Sweet et al., 1984). However, analysis of GTPase activities
associated with a large number of p21 mutants failed to reveal a clear
quantitative relationship between GTPase activity and transforming
potential (Trahey et al. 1987; Colby et al., 1986; Der et al., 1986).
Furthermore, no such association between mammalian gas p21 proteins and

adenylate cyclase has been observed (Beckner et al., 1985).

c. Interaction with a Putative Effector Protein

The putative gas p21 effector domain, the region or regions that
interact with molecules either upstream or downstream in the gas pathway,
was ascribed to a region of the NHZ-terminal end of gas proteins (Figure
1) by analyzing deletion mutants (Sigal et al., 1986). Mutations in the
region that includes amino acids 35-40 reduce the biological effects of
gas proteins in NIH 3T3 cells, but do not disrupt the in vitro biochemical

properties of 8. coli expressed gas proteins (Sigal et al., 1986).

Recently, it was discovered that a protein, designated GAP (GTPase
activating protein) interacts with the gas p21 (Trahey et al., 1987), and
that such interaction occurs at the putative effector domain (Cales et
al., 1988). The GAP protein is a cytosolic protein that stimulates the
GTPase of normal gas p21 by loo-fold with no apparent effect on the GTPase
of oncogenic gas p21 (Trahey et al., 1987). The studies by Cales et al.
(1988) suggest that association of GAP with the normal gas p21-GTP bound

27
complex leads to a conformational change in the protein that increases its

intrinsic GTPase activity, and that due to the mutations in the oncogenic
forms of ras p21, such conformational changes are prevented from
occurring, resulting in a reduced intrinsic GTPase activity. Formal proof
of a role for GAP as the target or a regulator of gas p21 has yet to be

obtained.

d. Involvement in the Control of Cell Proliferation

The normal physiological role of the gas proteins remains unknown.
Studies on the expression of gas genes in mammalian cells indicate that
they are expressed at low levels, in most, if not all cell lineages (Chesa
et al., 1987; Furth et al., 1987). Such data imply that the physiological
role of ,gas, proteins is shared by most cell types. Because the
biochemical properties of {as genes are similar to those of proteins
involved in signal transduction, and because expression of ras oncogenes
in various rodent cells lead to the abnormal growth characteristics
exhibited by transformed cells, gas genes are thought to participate in

biochemical pathways essential to normal cellular proliferation.

It has been known for a long time that various growth factors cause
changes in inositol lipid metabolism (Durell et al., 1969). More
recently, it has been shown that biochemical activities correlated with
the action of GTP binding/hydrolyzing proteins are involved in pathways
linked to phosphotidylinositol turnover (Brown et al., 1984).
Intermediates in this pathway, particularly diacylglycerol (DAG) and
inositol 1,4,5-triphospate (1P3), can act as second messengers to mediate

cellular responses initiated by the binding of growth factors, as well as

28
neurotransmitters and hormones to their cell surface receptors (Berridge,

1984). In as transformed NIH3T3 cells the steady state levels of
phoshotidylinositol 4,5-diphosphate (PIPZ) and DAG are elevated compared
to their untransformed counterparts (Fleischman et al., 1986), and it has
been suggested that gas p21 may act to affect such levels by activation
of a specific enzyme, phospholipase C (Fleischman et al., 1986), which
catalyses the breakdown of PIP2 to IP3 and DAG. IP3 itself stimulates
release of calcium into the cytosol from intracellular stores (Berridge,
1984) and DAG activates a calcium- and phospholipid-dependent kinase
(protein kinase C) (Nishizuka, 1986) and acts as a substrate for
arachidonic acid release during prostaglandin synthesis. Both the DAG-
activated protein kinase C and the IP3-stimulated Ca“ release cause a
plasma membrane NaI-H’ exchanger, which elevates cytoplasmic Na+ and raises
the pH (Streb et al., 1983). Recent evidence suggests that generation of
these signals through stimulation of phosphinositol turnover is a critical
event in growth factor stimulation of cell proliferation (Berridge et al.,
1984). Although there is little direct data supporting a role for gas
genes in this pathway, it has been speculated that the loss of GTPase
activity in oncogenic gas proteins causes these proteins to activate
phospholipase C and the concomitant production of 1P3 and DAG in an
uncontrolled way, independent of signals supplied by growth factor binding

(Berridge, 1984; Fleischman et al., 1986).

3. Biological Properties Associated with Expression or gas Oncogenes in

Mammalian Cells

a. Biological Properties Induced in Rodent Fibroblasts

29

Studies by several groups (Land et al., 1983; Ruley et al., 1983;
Newbold and Overell, 1983; Thomassen et al., 1985) have indicated that
transfection of gas oncogenes into early passage rodent fibroblasts can
cause such cells to exhibit some properties of tumor cells, such as a
transformed morphology and anchorage independence, but not the infinite
lifespan phenotype or tumorigenicity. Malignant transformation of finite
lifespan rodent fibroblasts can be attained if a ,gas oncogene is
transfected along with any one of a number of different genes, such as v-
myc, c-myc or polyoma large T (Land et al., 1983), adenovirus ElA (Ruley,
1983), N-myc (Schwab et al., 1985), or p53 (Parada et al., 1985), that are
thought to be capable of causing the infinite lifespan phenotype in rodent
cells in culture. Malignant transformation can also result following
transfection of gas oncogenes into rodent fibroblasts that already exhibit
an infinite lifespan in culture (Land at al., 1983; Newbold and Overell,
1983). Together, these results suggest that the infinite lifespan
phenotype is a prerequisite for’ malignant transformation of rodent
fibroblasts induced by expression of a transfected ras oncogene. However,
it has been reported that
transfection of a ga_s oncogene alone is sufficient for the malignant
transformation of finite lifespan mouse, rat, or Chinese hamster
fibroblasts (Pozatti et al., 1986; Spandidos and Wilkie, 1984; Land et
al., 1986; Spandidos, 1985), and it appears that a high level of gas
oncogene expression was necessary for such transformation. Such malignant
transformation has not been achieved following transfection of gas
oncogenes into Syrian hamster fibroblasts (Thomassen et al., 1985, Newbold
and Overell, 1983), which have a more stable karyotype and a lower

incidence of spontaneous transformation into cells having an infinite

30
lifespan than do mouse, rat or Chinese hamster fibroblasts (Newbold and
Overell, 1983; Thomassen et al., 1985). This latter result has led to
speculation that the malignant transformation reported to occur following
transfection of a gas oncogene into finite lifespan mouse, rat and Chinese
hampster fibroblasts was caused by the increased propensity of such
fibroblasts to aquire an infinite lifespan (Thomassen et al., 1985).
Therefore, the hypothesis that the acquisition of an infinite lifespan in
culture is a prerequisite for the malignant transformation of rodent

fibroblasts is still widely accepted.

b. Biological Consequences of Expression of gas Oncogenes in Transgenic
Mice

An important limitation of experiments involving the use of cultured
cells for evaluating the action of oncogenes, is that they do not
accurately reflect transformation as it occurs in vivo. Such experiments
cannot assess the influence of various factors, such as blood supply,
humoral and cell-mediated Igrowth controls, and physical barriers to
growth, that are thought to be crucial to the development of tumors in
vivo (Stewart et al., 1984). In contrast, the use of transgenic mice,
i.e., mice containing exogenous sequences introduced into their germ
lines, permits the evaluation of the action of oncogenes in the context

of the living organism.

Three studies utilized transgenic mice to investigate the effects
of gas oncogenes (Quaife et al., 1987; Andres et al., 1987; Sinn et al.,
1987). In each of these studies an H-gas oncogene, linked to

transcriptional regulatory elements that direct expression in a tissue-

31
specific manner, was introduced into the germ line of mice (Quaife et al.,
1987; Andres et al., 1987; Sinn et al., 1987). Expression of an H-gas
oncogene under the control of the rat elastase regulatory elements which
direct transcription only in the pancreas (MacDonald et al., 1987),
resulted in the neoplastic proliferation of all, or most of the pancreatic
acinar cells (Quaife et al., 1987). However, such neoplastic
proliferation does not necessarily represent a true malignant tumor, but
more likely represents a benign, but lethal neoplasm. In contrast to
these results, expression of an H-gas oncogene under the control of either
a mouse mammary virus tumor (MMTV) promoter (Sinn et al., 1987), or under
the control of the mouse whey acidic protein promotor (Andres et al.,
1987), each of which directs transcription primarily in mammary
epithelium, resulted primarily in the development of mammary tumors
(adenocarcinomas) that were apparently of clonal origin and occurred only
after a relatively long latent period (usually 6-12 months) (Sinn et al.,
1987; Andres et al., 1987). Such results suggest that the cells of
certain tissues are capable of responding directy to the transforming
effects of gas oncogenes by exhibiting benign neoplastic growth and that
other cells require additional changes at the cellular level before

neoplastic proliferation or malignant tumor development occurs.

In experiments by Sinn et al. (1987), mice matings were performed
between transgenic mice that harbored an H-gas oncogene linked to the MMTV
promoter, and mice that harbored a cellular mys gene linked to the same
promoter (Stewart et al., 1984), to examine the combined effects of these
genes in target cells of progeny animals. Coexpression of these genes

resulted in'a change in the spectrum of cell types that developed tumors

32

and a decrease in the time of tumor onset compared to that observed in
animals expressing the gas or my; transgenes separately (Sinn et al.,
1987). Nevertheless, the tumors formed appeared to be of clonal origin,
and tumor incidence in these animals increased in an age dependent manner
(Sinn et al., 1987), suggesting that events additional to co-expression
of the gas and mys transgenes were required for tumor development by the

targeted cells.

c. Use of Human Cells to Study the Biological Effects of gas Oncogene
expression

For obvious reasons humans cannot be experimentally manipulated in the
same manner as mice or other animals. Therefore, in the case of the human
species, cell culture transformation systems take on special relevance
for the study of cancer. However, no reproducible method has been
developed to bring about the malignant transformation of human cells in
culture using carcinogenic agents or transfection oncogenes (DiPaolo,
1984; McCormick and Maher, 1988). The failure of human fibroblasts to
transform as readily as the rodent cell lines to date prevents a direct
comparison of the tranformation process in human and rodent cells, and
raises the question of whether the results achieved using rodent cells can
be applied to cancer development in humans. Such dissimilarities between
rodent and human cell transformation systems may reflect a fundamental
biological difference between the two species, or may merely relect a
significant difference in the frequency of acquiring some required

phenotype, such as infinite lifespan (McCormick and Maher, 1988).

There have been several studies that have attempted to identify the

33

biological consequences of expression of transfected gas oncogenes in
human fibroblasts. These studies are discussed in Chapters 11 and III of

the thesis.

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44

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CHAPTER II

Morphological Transformation. Focus Formation
and Anchorage Independence
Induced in Diploid Human Fibroblasts by

Expression of a Transfected H-ras OncogeneI

Peter J. Hurlin, Dennis 6. Fry, Veronica M. Maher

and J. Justin McCormick

Carcinogenesis Laboratory, Fee Hall
Department of Microbiology and Department of Biochemistry
Michigan State University, East Lansing, MI 48824-1316

46

47
Footnotes
1This research was supported in part by the Department of Energy
Grant DE-FGOZ-87-ER60524 and by the Department of Health and Human
Services Grant CA21289 from the National Cancer Institute. DGF is

a recipient of a Leukemia Society of America Special Fellowship Award.

2The abbreviations used are:

DMSO, dimethylsulfoxide; EMEM, Eagle's minimal essential medium;
FBS, fetal bovine serum; Staph A buffer, 0.2 M phosphate buffer,
pH 7.4, l% triton X-lOO, 0.l% SDS, 0.5% sodium deoxycholate, 0.1%
NaN3. 0.1 M NaCl.

3Peter J. Hurlin, Dennis G. Fry, Veronica M. Maher and J. Justin

McCormick, unpublished data.

48

In an attempt to determine how normal human fibroblasts respond
to high expression of the T24 H-ras oncogene, we transfected such
cells with the plasmid vector pH06Tl (Spandidos and Wilkie, Nature
3l0:469-475, 1984), containing the T24 H-ras oncogene with 5' and
3' enhancer sequences, and the aminoglycoside phosphotransferase
gene which confers resistance to the drug G418. Approximately 1.5%
of the G418 resistant colonies obtained after transfection and
selection consisted of cells exhibiting obvious morphological
transformation, i.e., they were highly refractile, and more rounded
than normal fibroblasts. DNA hybridization analysis showed that
the morphologically transformed cells contained the transfected T24
H-ras oncogene. and radioimmunoprecipitation analysis showed that
they were expressing the T24 H-ras protein product, p21.
Morphologically transformed cells formed colonies in soft agar at
a frequency at least 60 times higher than that of cells that had
been transfected with the control plasmid containing the normal
cellular H-ras gene. Cells transfected with plasmid pH06Tl could
also be identified by their ability to form distinct foci when grown
to confluence in non-selective medium following transfection. This
study demonstrates that normal diploid human fibroblasts in culture
can be transformed by transfection with a H-ras oncogene. and that

such transformation correlates with expression of the mutant p21.

49
IITRINNICTIN

A significant pr0portion of human tumors from various sites
in the body have been shown to contain activated oncogenes from the
ras family (1-3). This finding, which suggests that the ras oncogenes
are involved in causing such tumors, has prompted investigations
into the role ras oncogenes play in bringing about transformation
of cells in culture. Transfection studies using primary or early
passage rodent fibroblasts as recipients of ras oncogenes indicate
that these genes act in a dominant manner to cause transformation
(for review see ref. 4). The transformed phenotypes observed in
ras oncogene-transfected rodent fibroblasts include morphological
alteration (5.6), focus formation (6), anchorage independence (5,8,9),
and, in some instances. tumorigenicity (6-9). Several of these studies
indicate that the level of expression of the ras oncogene can be
an important factor governing the degree of transformation by ras
oncogenes (7-9) and that high expression of the transfected ras
oncogene is required for the induction of the tumorigenic phenotype
in such fibroblasts (6-9).

In contrast to the results achieved using rodent fibroblasts,
the results of most DNA transfection studies in which early passage
diploid human fibroblasts were used as the recipients suggested that
these cells are more resistant to transformation by 31s oncogenes.
For example, several groups have reported that following ras oncogene
transfection, human fibroblasts failed to exhibit morphological
transformation (9,10), focus formation (10), anchorage independence

(9), or tumorigenicity (9-ll). Sutherland g_tal. (ll) reported that

50

transfection of human fibroblasts with the T24 H-ras oncogene in
a plasmid devoid of transcriptional enhancer sequences caused the
target population to exhibit a very high frequency of cells able
to form colonies in soft agar. Sager at a_l_. (10,12) reported that
following transfection of normal human fibroblasts with the same
oncogene, but in a plasmid which contains SV40 transcriptional enhancer
sequences. expression of the H-ras p2l could be detected. However.
this did not result in morphological transformation or focus formation,
or tumorigenicity. These investigators did not assay the transfectants
for anchorage independence.

An explanation for the negative results with human fibroblasts.
suggested by the transfection studies with animal fibroblasts, is
that the transfected ras oncogene was not expressed or that the level
of expression of the ras oncogene was not high enough to cause
measurable effects. In an attempt to achieve high expression of
the T24 H-ras oncogene in early passage, diploid human fibroblasts
we used a plasmid, pH06Tl, obtained from Spandidos and Hilkie (8),
in which the oncogene is inserted between two sets of transcriptional
enhancer sequences. We here report that the transfectants exhibited
transformation to morphological alteration and focus formation, that
progeny of mmrphologically altered colonies exhibited anchorage
independence, and that such transformation was correlated with
expression of the T24 H-ras oncogene protein product p21 in the human

fibroblasts.

5 1
MTERIALS AND m5

Cells and Culture Conditions. Diploid human fibroblasts were
initiated from foreskins of clinically normal newborn infants as
described previously (13). The human bladder carcinoma cell line
T24 was purchased from the American Type Culture Collection (Rockville.
MD). EMEMZ, supplemented with 0.2 mM serine, 0.2 mM aspartate, 1.0
mM pyruvate, 10% FBS (Grand Island Biological Co., Grand Island,
NY), penicillin (100 units/ml), and streptomycin (lOOug/ml) (complete
EMEM) was used for routine culturing of cells. Cells were grown
at 37°C, in 5% C02, water saturated incubators.

Plasmids. Homer 6 plasmids (8) containing the T24 H-ras oncogene
(pH06Tl), the cellular H-ras gene (pH06Nl), or no additional sequences
(pH06) were kindly provided by Dr. Neil Wilkie. These plasmids contain
the selectable marker gene, aminoglycoside phOSphotransferase, which
confers resistance to the drug G418.

DIA Transfection. The Polybrene/DMSO method, adapted for
transfection of diploid human cells by Morgan _e_t_a_l. (14) was used.
Briefly, 1 x 105 cells (passage 7-10) were plated into loo-mm diameter
dishes in complete EMEM and incubated at 37°C. After l6-24 h, the
medium was removed and replaced with transfection medium, i.e., 2.5
mls of complete EMEM containing 7.5 ug Polybrene per m1 (Sigma Chemical
Co., St. Louis, MO) and 1.2-2.0 ug plasmid per ml, and the dishes
were returned to 37°C. The transfection medium was distributed over
the cells by shaking once every hour. After 6 h the transfection
medium was removed and the cells were treated for 4 min at room

temperature with 5 mls of complete EMEM containing 30%

52
freshly-distilled DMSO. Following DMSO “shock“ the cells were rinsed

2 times with EMEM, refed with complete EMEM, and incubated at 37°C.

Selection of 6418 Resistance. If the transfected population
were to be selected for drug resistance, 24 h after DMSO shock the
medium was exchanged for complete EMEM containing 200 ug of active
G418 (GIBCO) per ml. Colonies were allowed to develop for 14 days.
with one refeeding with this selective medium.

Assay for Focus Formation. If the transfected cells were to
be assayed for the ability to form macroscOpic foci piled up on tap
of a monolayer of contact-inhibited cells, they were not selected
for G418 resistance, but instead were allowed to grow to confluence
in complete EMEM following DMSO "shock'. The cells were fed once
per week for three weeks and then stained with methylene blue and
the foci counted.

Assay for Anchorage Independence. Cells were trypsinzed and
resuspended in a modified version of Ham's MCDBllO (15) formulated
in this laboratory for use with serum replacements in studies requiring
absence of serum (16). For the present assay, 1% FBS was supplied.
The cells were counted electronically (Coulter Corp., Hialeah, FL)
and plated into top agar at a density of 4,000 cells per ml. The
tap agar consisted of the modified MCDBllO medium supplemented with
1% FBS and 0.33% SeaPlaque agar (FMC Corp., Rockland, ME). The bottom
agar was prepared with the same medium, but supplemented with 2%
agar. One ml of top agar was plated per 35-m diameter dish on tap
of 2 ml solidified bottom agar. The following day and weekly
thereafter 1 m1 of modified MCDBllO medium with 1% FBS was added

to the tap agar to compensate for evaporation. After 3 weeks the

53

number of colonies with a diameter larger than 60 um was determined
mi crosc0pi cal 1y.

Southern Blot Analysis. Genomic DNA was isolated as previously
described (17). DNA was digested with restriction enzymes under
conditions reconmIended by the supplier (New England Biolabs, Beverly,
MA). Ten ug of digested genomic DNA or 5.6 ug plasmid DNA was
electrOphoresed in 0.8% agarose gels and transferred to nylon filters
(Gene Screen Plus, New England Nuclear, Boston, MA) using standard
techniques (18). Nylon filter prehybridization, hybridization and
high strigency washing conditions were carried out as recomended
by the supplier. The 2.9 kb _Sas 1 fragment of the T24 H-ras oncogene,
nick translated with 3"’P-labeled dNTPs (New England Nuclear, Boston,
MA, 600Ci/IImIol) to a specific activity of 1-2 x 108 cpm/ug, was used
for hybridization. Filters were exposed to Kodak XAR X-ray film
(Kodak, Rochester, NY) with the aid of intensifier screens (Cronex
Lightning Plus, Dupont, Inc., Wilmington, DE) for 72 h at -70°C.

RNA Analysis. Cyt0p1asmic RNA dot blots were performed as
described by White and Bancroft (19).

Detection of p21. Protein labeling and iIIInunOprecipitation
were carried out essentially as described previously (20,21) with
the following modifications. Cells growing exponentially in the
modified version of MCDBllO medium supplemented with 10% FBS were
plated into Ioo-m diameter dishes at 106 cells/dish. After 24 h
the medium was exchanged for 3 ml of this medium lacking methionine
and F85, but supplemented with serum replacements (16). After 45
min, 1.0 mCi of 35S-methionine (1148 Ci/Innol, New England Nuclear,

Boston, MA) was added and the cells were incubated for 24 h. Cells

54

were lysed with 1.0 m1 Staph A buffer2 containing 2.0 mM
phenylmethanosulfonyl fluoride (Sigma Chemical Co., St. Louis, MO)
and 135 kallikrein inactivator units/m1 aprotinin (Sigma Chemical
Co., St. Louis, MO). Lysed cells were centrifuged at 35,000 rpm
in a Beckman 50Ti rotor for 35 min. The supernatant, containing
108 cpm of trichloroacetic acid precipitable counts, was
imunOprecipitated for 2 h at 4°C with the monoclonal antibody Y13-259
(22), kindly supplied by Dr. M. Anderson. Protein A-sepharose
(Pharmacia Chemical Co., Piscataway, NJ) coated with goat-anti-rat
196 (Cooper Biomedical, Malvern, PA) was added and the mixture was
incubated for 30 min at 4°C. The mixture was centrifuged, the
supernatant was removed and the sepharose fraction was washed twice
with Staph A buffer, once with a solution of 50 mM Tris-Cl (pH 7.5),
1 M MgClz and one more time with Staph A buffer. Electrophoresis
sample buffer was added and the samples were heated at 90°C for 5
min. The samples were centrifuged and aliquots of the supernatant,
along with pre-stained protein molecular weight markers (Bethesda
Research Laboratories, Bethesda, M0), were electrOphoresed (23) through
12.5% SDS-polyacrylamide gels for 4 h at 200 Y. The gels were fixed,
treated with autoradiographic enhancer (Enlightning, New England
Nuclear, Boston, MA), dried, and exposed to X-ray film at -70°C for
24-72 h.

55

RESULTS

Frequency of Transfection to G418 Resistance. Plasmid pHOGTl,
which contains the T24 H-ras oncogene, was chosen for transfection
of diploid human fibroblasts because of its demonstrated ability
to transform early passage rodent fibroblasts into malignant cells
(8) and because in our preliminary experiments, other plasmids
containing the T24 H-ras oncogene proved to be ineffective in
transforming human fibroblasts3. Plasmid pH06N1, which contains
the normal, endogenous H-ras gene, and plasmid pHOG» which has no
ras gene were used as controls in the transfection experiments.
Recombinant plasmids were introduced into early passage (passage
7-10) diploid human fibroblasts using a modified Polybrene/DMSO
transfection technique. This transfection technique was chosen because
the frequency of G418 resistant clones obtained was shown to be 10
to 40 times that acheived using calcium phosphate (1). To increase
the probability that colonies selected for resistance to G418
represented unique transfection events, transfection and selection
of cells were performed in the same culture dish. The frequency
of G418 resistant colonies obtained after transfection of human
fibroblasts with the various Homer 6 plasmids is shown in Table 1.
Each plasmid construction yielded approximately the same number of
G418 resistant colonies per ug of transfected plasmid, i.e., 200
per 106 target cells.

Transformation to Altered Morphology. G418 resistant colonies
were microsc0pica11y examined for evidence of altered cellular

morphology two wks following transfection and selection in G418 medium.

56

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57

 

Figure 1. Comparison of normal and transformed morphologies. (a)
normal human fibroblasts; (b) fibroblasts transfected with pH06Tl.
Confluent cultures were fixed with methanol and stained with methylene

blue. (Magnification, 100x.)

58

Only pHOGTl induced morphological transformation, as indicated in
Table 1. A representative colony of morphologically transformed
cells is shown in Fig. l. The morphologically transformed cells
divided rapidly, were highly refractile and grew in irregular patterns,
rather than the highly-oriented pattern of normal human fibroblasts.

Transformation to Focus Formation. Cells transformed following
transfection with pH06Tl could be also identified by their ability
to grow into a 3-dimensional array of cells, a focus, on top of a
monolayer of contact-inhibited cells. This type of aberrant growth
behavior is a cannon characteristic of tumor cells, including those
derived from human fibrosarcomas3. Macroscopic foci, and an individual
focus of pH06Tl transfected fibroblasts surrounded by adjacent
non-transformed fibroblasts are shown in Fig. 2. The number of foci
observed per lOO-Inn diameter dish following three weeks of growth
under non-selective conditions was approximately lO-fold greater
than the number of morphologically transformed colonies observed
after two weeks of G418 selection (see Table 1). No foci were
generated following transfection of the control plasmids pH06N1 and
pH06 which lack the oncogene. MicroscOpic inspection of the morphology
of the cells making up the fbcus revealed that not all cells capable
of forming foci exhibited the transformed morphology. Thus, the
number of foci is a more sensitive determination of the frequency
of transformed cells following transfection with pHOGTl than is the
number of morphologically transformed colonies.

Induction of Anchorage Independence. Morphologically transformed,
G418 resistant colonies obtained following transfection with pH06Tl

and G418 resistant colonies obtained following transfection with

59

Figure 2. The focus forming ability of pH06Tl transfected fibroblasts.
Following transfection with pHOGTl which contains the T24 H-ras
oncogene, or pH06N1 which contains the normal cellular H-ras gene,
cells were allowed to grow for 3 weeks under non-selective conditions.
Confluent cells were trypsinized and replated at a 1 to 3 dilution
and then allowed to grow for an additional 3 weeks before being stained
with methylene blue. (a) Foci generated in a dish containing pH06Tl
transfectants; (b) confluent monolayer in a dish containing pH06N1
transfectants; (c) 40x magnification of a focus from the dish shown

in (a).

6O

 

61

the control plasmid, pHOGNl, were compared for their ability to form
colonies in soft agar. The cell p0pulations assayed were derived
from pooled colonies that had been isolated 24 h earlier. No colonies
were observed in the control dishes, representing a total of 8,000
cells assayed (frequency, (1.25 x 10'4). In contrast, 122 colonies
with a diameter equal to or larger than 60pm were observed with the
oncogene transformed cells, out of a total of 16,000 cells assayed
(frequency, 76 x 10'4).

A concomitant comparison of the ability of the two pepulations
to form colonies on plastic showed that the cloning efficiency of
the control cells was twice that of the transformed cells (data not
shown). Therefore, when measured under the stringent conditions
(1% FBS) employed in this assay, cells exhibiting the transformed
morphology formed colonies in soft agar at a frequency at least 60
times that of the control cells.

Analysis for the Presence of Transfected Sequences. G418-resistant
colonies that exhibited the transformed morphologically were isolated,
pooled in groups of several clones, and expanded for several population
doublings. High molecular weight DNA was extracted and digested
with restriction endonucleases Xba I and Mind 111 (double digestion)
to liberate specific size fragments (2.5kb and 4.5kb) that are
diagnostic for an intact region of plasmid pH06Tl containing the
T24 H-ras oncogene, its endogenous promoter and exogenous 5' and
3' enhancer sequences (8). The DNA was analyzed by the method of
Southern (18). As shown in Fig. 3, cells exhibiting the transformed
morphology contain the 4.5 kb and 2.5 kb fragments. The intensity

of these two fragments was approximately half that of the endogenous

62

Figure 3. Southern blot analysis of cellular DNA from morphologically
transformed cells obtained following transfection with pH06Tl.
Following transfection of pH06Tl, and G418 selection, colonies of
morphologically transformed cells were isolated and pooled so that
each pool represented 3-4- separate clonal papulations. The clonal
populations were expanded and high molecular weight DNA was extracted
from them and from the parental strain of normal diploid human
fibroblasts used for transfection. lOug of DNA from each sample
was digested with Xba I and Hind 111 (double digest), electrOphoresed
through a 0.8% agarose gel, and transferred to nylon filters. The
filters were hybridized with the 2.9 kb Sac I fragment of the T24
H-ras oncogene radioactively labeled by nick translation. (a) Lane
1, non-transfected normal diploid human fibroblasts; lane 2-5, pooled
populations of morphologically transformed cells. Hybridizing
fragments of 4.5 and 2.5 kb are diagnostic for the presence of
transfected T24 H-ras oncogene sequences. (b) Southern blot analysis
of high molecular weight DNA extracted from progeny cells of those
analyzed in Fig. 4a, but which had reverted to the normal fibroblast
morphology. DNAs were analyzed as above. Lanes 2'-5' are the progeny

cells from the respective lanes in Fig. 4a, kb, kilobases.

63

 

64

ras sequences, indicating that the cells contain only' one capy' of
the transfected ras gene.

We found that if the morphologically transformed G418-resistant
cells were passaged extensively, even in the presence of selective
medium, the majority of them eventually reverted to a normal
morphology. Of the 38 morphologically transformed colonies analyzed,
36 reverted to a normal morphology within 25 pepulation doublings
following transfection. These reverted cells ‘were assayed for 'the
ability to form foci, to form colonies in soft agar, and for the
presence of the 'T24- H—ras oncogene and its protein product. They
did not form foci or colonies in agar. However, Southern blot analysis
showed that the revertants still possessed the transfected oncogene
(Fig. 4b).

Expression of the T24 H-ras Oncogene in Transformed Cells.
Substitution of valine for glycine at the 12th amino acid position
of the T24 H-ras-encoded p21 alters its electrOphoretic mobility,
making it distinguishable from normal, endogenous p21 (24). Seven
independent G418 resistant, morphologically transformed clonal
p0pulations obtained after pH06Tl transfection and 5 clonal populations
that reverted to the normal fibroblast morphology were analyzed for
the presence of the mutated, form of p21 by radioimunOprecipitation
analysis using monoclonal antibody Y13-259 (22). Fig. 4 shows a
comparison of p21 expression in pH06Tl transfectants exhibiting a
transformed morphology, and in cells which reverted to a normal
morphology. In cells transfected with the control vector (lanes
1 and 7) and in pH06Tl transfectants that reverted (lanes 3-5), the

characteristic p21 doublet (20) is expressed (see dashes). The pH06Tl

65

Figure 4w Analysis of ras gene expression. [35S]-methionine labeled
cellular extracts were immunoprecipitated with monoclonal antibody
Y13-259, the immunOprecipitates electrophoresed on 12.5% polyacrylamide
gels, and the gels analyzed by fluorography. Lanes: 1 and 7, cells
transfected with the control vector, pH06; 2 and 8, the human bladder
carcinomoa cell line T24; 3-5 independent clonal papulations that
reverted from a transformed morphology to a normal morphology following
transfection with pH06Tl; 6 and 9, pH06Tl transfected, morphologically
transformed cells. Lanes 7-9 represent a second analysis of samples
1, 2, and 6, respectively. Dashes: endogenous p21 doublet. Arrows:

mutant (T24) H-ras p21.

 

1 23456

66

 

789

67

Figure 5. H-ras RNA expression. Cytoplasmic samples were extracted
from 8 x 105 cells, denatured, diluted to the equivalent cell number
indicated, and spotted onto nitrocellulose filters. (a) Hybridization
with the Sac 1 3kb T24 H-ras fragment, nick translated with [32P]-NTPs.
Lanes: 1, cells transfected with the control vector, pH06; 2 and
3, cells transfected with pH06Tl and exhibiting a transformed
morphology. (b) Hybridization of the same samples as in (a) with
a /3-2 microglobulin probe to control for variability in the amount

of cyt0plasmic extract loaded.

68

 

A
. 1 2 3
'91 . 2:1105
4 O . 1x105
1“. s 2.5x104
6.25x103
3
1 _2__ 3 g
0 '0' a. 2x105

69

transfectants exhibiting a transformed morphology expressed an
additional p21 species that co-migrated with a p21 species expressed
in the human bladder carcinoma cell line T24. The mutant species
migrated immediately below the slower' migrating, normally present
p21 species (see arrow). A similar pattern of expression has been
observed in all seven of the morphologically transformed populations
analyzed. The additional p21 species was not observed in any' of
the reverted papulations, or in control, vector (pH06) transfected
p0pulations tested.

RNA dot blot analysis of cytoplasmic RNA samples indicated that
transfectants maintaining the transformed morphology expressed higher
than normal levels of H-ras specific RNA. Fig. 5 shows that samples
from such cells (lanes 2 and 3) expressed approximatley four times
the amount of H-ras RNA expressed in control cells (vector transfected;
lane 1). This higher level of expression of H-ras RNA correlates
with expression of the T24-H-ras p21 species in pH06Tl transfected,
morphologically transformed cells.

TI-origenicity. Two G418-resistant, morphologically transformed
clonal populations obtained following transfection with pH06Tl that
maintained their altered morphology when expanded into large
papulations were assayed for tumorigenicity in athymic mice as
described previously (25). Three mice were used per test and 107
cells were injected subcutaneously in each animal. No tumors or
indication of growth at the site of injection were observed over
a period of greater than one year. When the cells were continuously
passaged in culture as part of a lifespan assay, they senesced at
a population doubling level comparable to that of normal fibroblast

populations.

7O
DISCUSSIN

Results of the present study show that normal diploid human
fibroblasts can be transformed into morphologically altered, focus
forming, and anchorage independent cells following transfection of
the T24 H-ras oncogene. Our results, showing that the transformed
cells exhibited anchorage independence, support the results obtained
by Sutherland e_t_ _a_1_ . (11) and extend that study by showing that
the transformed cells expressed the T24 H-ras protein. However,
unlike Sutherland fl; al. (11) we did not use anchorage independence
to identify transformed papulations of cells directly following
transfection. Therefore, it is not possible to directly compare
our frequency of anchorage independent cells with theirs.

If the anchorage independence exhibited by cells transfected
with the 124 H-ras oncogene in the enhancer-free plasmid used by
Sutherland _e_t_ g. (11) is an indication that the transfectants were
expressing the mutant p21 protein, then it is difficult to understand
why Sager _e_t_ al, (10,12), did not observe any evidence of
transformation. They used the same oncogene, but in a plasmid which
contains a set of transcriptional enhancer sequences, and they found
expression of mutant p21. An obvious explanation for the difference
between the results of Sager _e_t_ _a_l. (10,13) and those we present
here is a difference in the level of expression of p21 resulting
from the use of different plasmid constructions containing the 124
H-ras oncogene. When we began transfection studies using normal
diploid human fibroblasts as recipients, like Sager gal. (10,12)

we constructed and used a pSV2-derived plasmid containing the T24

71

H-ras oncogene and found, as they did, no evidence of morphological
transformation or focus formation3. What is more, there was no
induction of anchorage independence3. The positive results reported
in the present study were generated using a plasmid (pHOGTl) that
was specifically designed by Spandidos and Wilkie (8) to elicit high
expression of the T24 H-ras oncogene in mammalian cells by inserting
the oncogene between two sets of transcriptional enhancer sequences.
Therefore, we interpret our results to indicate that a sufficiently
high level of expression of the T24 H-ras oncogene was achieved to
cause the transformation of normal human fibroblasts, and that the
pSV2-T24 plasmid of Sager _et _a_l_. (12) was not capable of generating
such levels. This interpretation does not explain the negative results
of Spandidos (9), who recently reported that transfection of normal
human fibroblasts with pH06Tl did not cause anchorage independence
or any obvious morphological transformation. However, Spandidos
used a different method of transfection (calcium phosphate) then
we did (DMSO/Polybrene), and examined a relatively small number of
independent G418 resistant colonies (ni200) compared to the total
number we examined (>2461).

The results of numerous ras oncogene transfection studies carried
out with rodent fibroblasts suggest that high expression of the T24
H-ras oncogene can be attained, and that with such expression the
tumorigenic phenotype is induced (6-9). Our studies with human
fibroblasts indicate that the transfected oncogene is expressed at
low levels, compared to what has been observed in rodent fibroblasts,
and that such expression is lost in most cells as they are passaged.

Only two papulations of pH0611 transfectants out of 38 analyzed could

72

be isolated that maintained a transformed morphology when extensively
passaged, and both of these proved to have a finite lifespan and
were not tumorigenic. However, there is evidence that ras oncogenes
are involved in ne0p1astic transformation of human fibroblasts.
For example, transfection of diploid fibroblasts derived from Bloom's
syndrome patients with DNA from NIH 3T3 cells that had been transformed
by Harvey murine sarcoma virus (Ha-MSV) DNA resulted in cells able
to form nodules in athymic mice (26). These nodules subsequently
regressed (26). Also, Harvey (27) or Kirsten murine sarcoma virus
(28) infection of human fibroblasts that had acquired an indefinite
lifespan in culture following SV40 virus transformation or repeated
exposure to ganlna radiation resulted in cells able to form tumors.
One group (27) indicated that the tumors regressed, the other (28)
found they did not. Three groups (29-31) have succeeded in causing
ne0plastic transformation of human epithelial cells by ras oncogene
transfection. Further studies to determine if diploid human
fibroblasts that continue to express the T24 H-ras oncogene are
tumorigenic and whether this gene cooperates with other* oncogenes
(e.g., myc) in causing malignant transformation of human fibroblasts

are in progress.

73
ACKIOHLEDGEMENTS

We thank Dr. Neil Wilkie for kindly supplying us with the pH06
series of plasmids and Dr. Marshall Anderson for the Yl3-259 antibody

to H-ras p21, and Carol Howland for typing the manuscript.

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Eva, A., Tronick, S. R., Gol, R. A., Pierce, J. H., and Aaronson,
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Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A.,
Della Porta, G., and Barbacid, M. J. Malignant activation of
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Balmain, A. Transforming ras oncogenes and multistage
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Land, H., Parada, L., and Weinberg, R. A. Tumorigenic conversion
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Pozzatti, R., Muschel, R., William, J., Padmanabhan, R., Howard,
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Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F., and
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Spandidos, D. A., and Wilkie, N. M. Malignant transformation

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Spandidos, D. A. Mechanism of carcinogenesis: The role of
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Sager, R., Tanaka, K., Lau, C. C., Ebina, Y., and Anisowicz,
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Sutherland, B., Bennet, P. V., Freeman, A. G., Moore, S. P.,
and Strickland, P. T. Transformation of human cells by DNA
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Sager, R. Resistance of human cells to transformation. 13;:
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Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1984.
McCormick, J. J., and Maher, V. M. Measurement of colony-forming
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Morgan, T. L., Maher, V. M., and McCormick, J. J. A procedure
of high efficiency DNA-mediated gene transfer in normal human
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Bettger, W. J., Boyce, S. T., Walthall, B. J., and Ham, R. C.

Rapid clonal growth and serial passage of human diploid
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Ryan, P. A., McCormick, J. J., and Maher, V. M. Modification
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Heflich, R. H., Dorney, D. J., Maher, V. M., and McCormick,
J. J. Reactive derivatives of benzo[a]pyrene and
7,12-dimethylbenz[a]anthracene cause S] nucleases sensitive
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Southern, E. M. Detection of specific sequences among DNA
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White, B. A., and F. C. Bancroft. Cytoplasmic dot blot
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Der, C. J., and C00per, G. M. Altered gene products are
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Reynolds, S. H., Stowers, S. J., Maronpot, R. R., IAnderson,
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Furth, M. E., Davis, L. J., Fleurdelys, and Scolnick, E. M.
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family. J. Virol. 13: 294-304, 1982.
Laemmli, V. K. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature (London),

2_27: 680-685. 1970.

Tabin, C. J., Bradley, S. M., Berman, C. 1., Weinberg, R. A.,
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and Chang, E. H. Mechanism of activation of a human oncogene.
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Fry, 0. G., Milam, L. D., Maher, V. M., and McCormick, J. J.
Transformation of diploid human fibroblasts by DNA transfection
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Namba, M., Nishitani, K., Fukushima, F., Kimoto, T., and Nose,
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O'Brien, W., Stenman, G., and Sager, R. Suppression of tumor
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Yoakum, G. H., Lechner, J. F., Gabrielson, E., Korba, B. E.,
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Rhim, J. S., Jay, G., Arnstein, P., Price, F. M., Sanford, K.
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Boukamp, P., Stanbridge, E. J., Cerutti, P. A., and Fusenig,
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lines by Harvey-gas oncogene. J. Inves. Der., g: 131, 1986.

CHAPTER III

Malignant Transformation of a Human Fibroblast Cell Strain,

MSU-1.l, By Expression of a Transfected T24 H-gas Oncogene

Peter J. Hurlin, Veronica M. Maher and J. Justin McCormick

Carcinogenesis Laboratory, Fee Hall
Department of Biochemistry and Department of Microbiology
Michigan State University, East Lansing MI, 48824-1316

79

80
SUMMARY

We showed previously that normal diploid human fibroblasts that
express a transfected 124 H-gas oncogene exhibit several characteristics
of transformed cells, but are not tumorigenic. To extend these studies of
the transforming ability of the 124 H-gas_oncogene in human cells, we have
utilized an infinite lifespan, but otherwise phenotypically normal human
fibroblast cell strain, MSU-1.1, which was developed in this laboratory
following transfection of diploid fibroblasts with a plasmid carrying a
v-mys oncogene. Transfection of MSU-1.1 cells with the 124 H-gas oncogene
flanked by 2 transcriptional enhancer elements (pH0611) caused foci of
morphologically transformed cells to develop. No such transformation
occurred if the plasmid containing the 124 H-gas oncogene had only one
enhancer or none at all, or if the normal H-ras gene was transfected in
the pH06 vector. Cells derived from such foci expressed high levels of
124 H-ras p21, formed colonies in soft agar at high frequency,
proliferated rapidly in serum-free medium1containing 0.1 mM calcium, which
does not support growth of the parental cell line, and formed
progressively growing, invasive fibrosarcomas in athymic mice. No
karyotypic changes accompanied the transformation of MSU-l.l fibroblasts,
suggesting that chromosome instability' did not play a role in the
conversion of the cells to malignancy. Transfection of two other infinite
lifespan fibroblast cell lines with the T24 H-gas oncogene in pH06Tl also
resulted in malignant transformation, suggesting that the infinite
lifespan phenotype of MSU-l.l cells, and not necessarily expression of the
v-myc oncogene, was the factor that complemented 124 H-ras expression to

cause malignant transformation.

81
INTRODUCTION

Oncogenes of the gas family have been detected in numerous and
diverse human malignancies. A growing body of evidence from both in vivo
and in vitro studies suggests that the p21 products of gas oncogenes are
involved in the genesis of such malignancies. For example, transgenic
mice harboring the 124 H-gas (1), or v-H-gas oncogenes (2) linked to a
murine mammary protein gene promoter developed mammary tumors following
a long latency. 'These tumors were clonal in origin, suggesting that
additional changes at the cellular level were required to cause tumor
formation. Clues to the nature of the steps or events that may complement
mutational activation of gas genes in tumorigenesis have come primarily
from transfection experiments utilizing rodent fibroblasts in culture (for
review see ref. 3). When primary or early passage rodent fibroblasts are
transfected with a combination of a ras oncogene and any one of a number
of genes thought to be involved in causing cellular imortalization,
malignant transformation results (4-6). Transfection of gas oncogenes
into infinite lifespan rodent fibroblast cell lines usually results in
malignant transformation (4,7,8), but not in all cases (9). However,
several studies have reported that high levels of gas oncogene expression
can convert finite lifespan, early passage rodent fibroblasts into
infinite lifespan malignant cells without the need for transfection of
another oncogene (IO-12).

To test models proposed for r_as transformation generated using rodent
fibroblasts for their relevance to human cell transformation, we have been
using human fibroblasts as recipients of transfected ,gas oncogenes

(13,14). Several groups reported that early passage diploid human

82

fibroblasts are resistant to transformation by transfected gas oncogenes
(15,16), or microinjection of gas oncogene encoded p21 (17,18). But,
Hurlin et. al. (13) showed that if such cells are transfected with the
pH0611 plasmid which contains the 124 H-gas oncogene flanked by Moloney
murine sarcoma virus LTR and SV40 virus transcriptional enhancer
sequences, they become morphologically transformed, develop distinct foci,
and form colonies in soft agar. In a related study, Tubo and Rheinwald
(19) showed that diploid human fibroblasts transfected with the 124 H-gas
oncogene in a plasmid which contains an SV40 virus enhancer become
morphologically transformed and exhibit reduced growth factor
requirements. However, the transformed cells in these two studies did not
acquire an infinite lifespan and were not tumorigenic (14,19).

For the most part, oncogenes have been detected and defined by their
transforming effects on rodent fibroblasts which have an infinite lifespan
in culture, but are otherwise normal. No comparable human cell line has
been available for use in testing the applicability of the results to
human fibroblasts. This is because human fibroblasts in culture are
extremely difficult to transform into cells with an infinite lifespan
(20,21), even by SV40-virus infection (22) and the few infinite lifespan,
non-tumorigenic fibroblast cell lines that have been generated (22-24)
consist of cells that are morphologically transformed and exhibit other
transformation characteristics. Recently, a human fibroblast cell strain
with an infinite lifespan in culture, designated MSU-l.1, was generated
in this laboratory in experiments examining the properties induced in
finite lifespan, diploid human fibroblasts by transfection of a plasmid
carrying a v-mgs oncogene. MSU-l.1 cells have a normal morphology and a

stable, near diploid karyotype. They do not form foci, or produce colonies

83
in soft agar, and are non-tumorigenic. Details on the generation of this
cell strain, and its properties will be described eleswhere. In the
present study we show that transfection of MSU-1.l cells with the 124 H-
ras oncogene in pH06Tl caused morphological transformation and focus
formation and that cells derived from such foci exhibited reduced growth
factor requirements, formed colonies in soft agar at high frequency and

gave rise to rapidly growing, malignant fibrosarcomas in athymic mice.

84
METHODS AND MATERIALS

Cells and Culture Conditions. Cell line LG1, from which the MSU-1.l cell
strain arose was initiated from foreskin material of a clinically normal
newborn male as described (25). The infinite lifespan cell strain
MSU-1.1, described above, is resistant to G418 (Grand Island Biological
Co., Grand Island, NY) because the plasmid used to transfect the original
LGl population carried the aminoglycoside phosphotransferase gene. The
infinite lifespan human fibroblast cell lines KMST-6 and SUSM-I, which were
generated following repeated carcinogen treatment (23,24), were obtained
from M. Namba. The SV40 virus transformed, infinite lifespan human
fibroblast cell line GM0637B was purchased from the NIGMS Human Genetic
Mutant Cell Repository, Camden, NJ. The human bladder carcinoma cell line
124 was purchased from the American Type Culture Collection (Rockville,
MD), and the chicken hepatoma, MC29 virus-producer cell line LSCC-DU72 was
provided by K. Nazerian. The cells were routinely cultured in Eagle’s
minimum essential medium, supplemented with 0.2 mM serine, 0.2 mM
aspartate, 1.0 mM pyruvate, 10% fetal bovine serum (FBS) (Gibco), 100
units/ml penicillin, and 100 ug/ml streptomycin at 37°C, in 5% C02, in
water saturated incubators. For studies of growth factor independence, a
serum-free medium, McM, a version of MCDBllO (26) formulated for use with

serum-replacements (27), was employed.

Plasmids and DNA Transfection. Homer 6 plasmids (10) containing the 124
H-gas oncogene (pH0611), the normal human H-yas gene (pH06N1) or no
additional sequences (pH06), were kindly provided by N. Wilkie. Plasmid
p124 was purchased from the American Type Culture Collection, and plasmid

85
pSV2-124 was constructed in this laboratory by inserting the T24 H-gas
oncogene into the BamHI site of the plasmid pSV2neo (28).'arget cells (2
x 105) were transfected with plasmid DNA at 1.3 ug per ml in transfection
medium, using the Polybrene/dimethylsulfoxide method (29) as previously
described (13).

Assay for Growth Factor Independence. Cells (5 x 10‘) were plated in McM
medium supplemented with 1% FBS. After 24 h, the number of cells was
determined by electronic counting and the medium was exchanged for McM
medium containing 0.1 mM calcium and supplemented with the serum
replacements of Ryan et al. (27), but lacking epidermal growth factor.
The medium was renewed 3 days later, and the number of cells was

determined after another 3 days.

Assay for Anchorage Independence. Cells were assayed for the ability to
form colonies in soft agar as described (13) with the following
modifications. Cells (5000 per dish) were plated in top agar consisting
of McM medium supplemented with 2% FBS and 0.33% SeaPlaque agar (FMC
Corp., Rockland ME.) McM medium containing 2% FBS was provided to
cultures weekly. Colonies having a diameter larger than 80 um*were counted
and sized electronically (Olympus Cue-2 Image Analyser, Olympus Corp. Lk.

Success, NY) after 3 wk.

Tumorigenicity Assays. The ability of cells to form tumors was assayed
by injecting 1 x 107 exponentially growing cells subcutaneously into 4 wk
to 8 wk old BALB/c athymic mice which had been irradiated with 300 rads

from a °°Co source 24 h previously. To test for the ability to metastasize,

86
5 x 10‘ cells were injected into the tail vein of athymic mice, and after

8 mo the mice were sacrificed and complete necropsies were performed.

DNA Analysis. DNA was isolated as previously described (30) and analysed
by the method of Southern (31) for transfected sequences.

Analysis of gas p21 and v-mys p110. Cells to be analysed for expression
of r_as p21 were labeled with [3SS]-Met (New England Nuclear, Boston, MA)
at 250uCi per ml for 18 h in McM medium lacking methionine, but
supplemented with 1% FBS. Cell lysis, immunoprecipitation with the gas
p21 specific antibody Y13-259 (32), NaDodSOg-PAGE and fluorography were
performed as previously described (13). For analysis of v-mys p110, cells
were labeled as above, but for only 1.5 h. Cells were washed once in ice-
cold phosphate-buffered saline, lysed in lysis buffer (10 mM Tris
hydrochloride (pH 8.2), 150 mM NaCl, 1% sodium deoxycholate, 1% TRITON X-
100, 0.1% NaDodSO‘, 5 mM EDTA, and 2 mM phenylmethanosulfonyl fluoride),
and clarified by centrifugation at 10,000 x g for 20 min. Supernatants
containing 7.5 x 107 cpm were immunoprecipitated with a sheep antibody
(Cambridge Research Biochemicals, Valley Stream, NY, antibody OA-11-801)
that reacts broadly with the proteins of the guys family. Protein A-
Sepharose (Pharmacia Chemical Co., Piscataway, NJ) coated with goat anti-
sheep 196 (Cooper Biomedical, Malvern PA) was added, and the mixture was
shaken for 30 min at 49C. The Sepharose fraction was washed twice with
lysis buffer and once with 10 mM Tris hydrochloride (pH 7.5), 1 M MgClZ,
and one more time with the lysis buffer. NaDodSO‘-PAGE sample buffer
containing 8 M urea was added, and the samples were heated for 5 min at

95°C. The samples were centrifuged, and aliquots of the supernatant, along

 

 

87

with prestained molecular weight markers (Bethesda Research Laboratories,
Bethesda, M0) were electrophoresed (33) in 10% NaDodSO4-polyacrylamide
gels. The gels were fixed, treated with autoradiographic enhancer

(ENHANCE, NEN, Boston, MA), dried, and exposed to X-ray film at -80°C.

Karyotype1Analysis. G-banded chromosomes were prepared as described (34).
For each cell line, at least 25 G-banded karyotypes were prepared to
examine for chromosomal rearrangements, and 100 conventially stained

metaphases were counted to determine the modal chromosome number.

88
RESULTS

Transformation to Focus-Formation by the 124 W-gas Oncogene. Following
transfection of MSU-1.1 fibroblasts with the various plasmids, the cells
were cultured for 3 wk in Eagle’s medium containing 10% FBS, and screened
for colonies of cells that overgrew the monolayer (focus formation). With
cells that had been transfected with pH06Tl, distinct foci of
morphologically altered cells were found at a frequency of approximately
10 per 10° cells transfected. No foci, or indications of transformation
were observed following transfection with control plasmids containing
either the normal human H-gas gene (pH06N1), or lacking a gas gene (pH06).
Transfection of 5 x 10° MSU-1.1 cells with plasmids containing the 124 H-
gas oncogene, but no enhancer sequences (p124), or with only one SV40
transcriptional enhancer (pSV2-124) did not induce focus-formation. Six
populations of morphologically transformed fibroblasts (designated
MSU-1.1-124 strains 1-6) were isolated from foci in separate dishes, and
the populations were expanded for subsequent characterization. An example
of the morphology of MSU-1.1-124 cells, which are smaller, rounder and
more refractile than their parental cells, is shown in Fig. 1. These cells
have maintained their transformed morphology for more than 40 population
doublings thus far. This stability contrasts with what Hurlin et a1. (13)
found with finite lifespan cells transformed following transfection of the

124 H-gas oncogene in pH06Tl.

Growth of MSU-1.1-T24 Fibroblasts in Serum-Free Medium. Schilz et. a1.
(35) showed that human fibrosarcoma-derived cell lines, but not normal

diploid human fibroblasts, were capable of proliferating in McM medium

89

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90

containing 0.1 mM calcium, instead of the usual 1 mM, and supplemented
with the serum replacements of Ryan et. al. (26), but lacking epidermal
growth factor. The six MSU-1.1-124 cell strains, the parental MSU-1.1
cells and the LGl cell line from which the latter arose were compared for
their ability to proliferate in such medium. MSU-1.1 cell strains
proliferated to numbers 6 to 7 fold higher than the parental MSU-1.l cells
did, and as expected, LG] cells could not proliferate under these
conditions (Table 1). The number of cells attained by the MSU-l.1-124 cell
strains indicated that, under these conditions, they double at a rate

approximately equal to that achieved in medium containing 10% FBS, i.e.,

once every 24 h.

Anchorage Independence of MSU-1.1 124 Cell Strains. The six MSU-1.l-124
cell strains were tested for their ability to form colonies in 0.33% agar
medium. LG] cells, and the parental MSU-1.1 cell strain formed colonies
with an efficiency of less than 0.007%, i.e., no colonies were detected
out of a total of 15000 cells plated. The six MSU-1.1-124 cell strains
formed colonies in agar at efficiencies ranging from 3.5% to 23% (Table
1). There was variation in the size range of colonies detected. For
example, although MSU-1.1-124 strain 6 exhibited a cloning efficiency of
only 3.5%, it gave rise to the highest proportion of very large colonies

(i.e., having diameters of >300um).

Tumorigenicity of MSU-1.1-124 Cell Strains. Four of the six MSU-I.1-124
cell strains tested for the ability to form tumors in athymic mice formed
progressively growing tumors (Table 1). Four to 5 wk were required for the

tumors to reach 1 cm in diameter. They were diagnosed as

91

Figure 2. Histology showing a poorly-differentiated human fibrosarcoma
formed in an athymic mouse by MSU-1.1-124 strain 3 cells. A 2.5 cm
diameter tumor was removed 8 wk following subcutaneous injection of 107
cells, and was sectioned, and stained with hematoxylin-eosin. Mouse
skeletal muscle is indicated by the arrows. Magnification; (A) 40x, (8)

200x.

 

92

 

93

Table 1. Growth properties and tumorigenicity of MSU-1.1-124 cell strains

 

 

Cell no.
Cloning attained in Days for
efficiency serum-free tumor to
Cell in agar 0.1 mM Ca++ Tumor reach
strain (%) medium (x10'5)a incidenceb 1 cm diam
1 15 16 2/2 36
2 23 NTc 6/6 30
3 10 NT 6/6 30
4 18 17 0/5 --
5 8.5 19 0/3 --
6 3.1 17 4/4 34
MSU-1.1 (0.01 2.7 0/9 --
LG1 cells <0.01 0.4d 0/9 --

 

aDuplicate cultures were counted 7 d after plating 5 x 104 cells/dish.
bAnimals with tumors per animals injected subcutaneous. Animals were examined
for tumor formation for at least 8 mo following injection.

cNT, not tested.

dThis is the same no. of cells as was determined 1 d after plating.

94

poorly-differentiated, invasive human fibrosarcomas with a high mitotic
index and a moderate degree of anaplasia. A low proportion of giant
multinucleated cells were observed in several of’ the tumors. A
histological section of a representative tumor is shown in Fig. 2. Two
criteria indicated that the tumors were formed by the injected MSU-1.1-
124 fibroblasts. First, the tumor-derived cells that were returned to
culture were capable of proliferating in medium containing G418, and
second, they contained the marker chromosomes characteristic of MSU-1.1
cells (see below). One of the tumorigenic MSU-1.l-TZ4 strains was tested
for metastatic properties by being injected into the tail vein of mice.
No tumors were found from necropsies performed on 5 such mice 8 months
later.

Presence of 124 H- as and v-mys_0ncogenes. Following digestion of genomic
DNA with 18a 1 and glad III, fragments of 4.5 and 2.5 kb that hybridize
with the 8as I, 2.9 kb fragment of the 124 H-gas oncogene are diagnostic
for plasmid pH0611 sequences which include the 124 H-gas oncogene and
flanking 5’ and 3’ transcriptional enhancer sequences (10). As shown in
Fig. 3, genomic DNA from each of the MSU-1.1-T24 cell strains contained
H-gas_hybridizing fragments close to these predicted sizes. Although the
data are not shown, v-mys hybidizing DNA fragments were detected in the
six MSU-1.1-124 cell strains and they'were of identical size and intensity
as those detected in the parental MSU-1.1 cell strain, indicating that no
rearrangements or amplification of this oncogene had occurred subsequent

to introduction of plasmid pH06Tl.

Expression of 124-H-gas and v-mys Oncogenes. Radioimunoprecipitation

analysis of the MSU-1.1-124 cell strains (Fig. 4) using the r_as p21

95

1.0.1
. MSU—l.’l

MSU-1.1-T24 Cell Strains
2

 

Figure 3. Southern blot analysis of plasmid pH0611-derived 124 H-gas
oncogene sequences in MSU-1.1-124 cell strains. LGl is the diploid human
fibroblast cell line from which the MSU-1.1 cell strain was derived. DNA
was digested with 818d III and 18a I and hybridized with the 2.9 kb 8as
I fragment of the T24 H-gas oncogene. The locations of the expected 4.5

kb and 2.5 kb plasmid pH0611 specific fragments are indicated.

96

Figure 4. Expression of 124 H-gas p21 in MSU-1.1-T24 cell strains. The gas
encoded p21 proteins were immunoprecipitated with antibody Y13-259 (25)
from f”$]methionine-labeled cell lysates and analysed by electrophoresis
in 12.5% SDS-NaDod504 polyacrylamide gels and flourography. The location
of the endogenous gas encoded p21 protein doublet is indicated by the
bars, and the 124 H-gas encoded p21 location is indicated by the arrow.
124 is the human bladder carcinoma cell line. 3a is a clonal derivitive
of MSU-1.1-124 strain 3. The location of molecular weight (kilodaltons)

markers on the gel is indicated.

97

 

'T‘
a a :1. MSU- 2.1 1—1‘24 cell strains
.4 E E- 1 4 3 3a
42 - - ‘1 3 '5
H
. a. 1 " ‘ J‘ .

25-

98

Ms_u-1.1-Tz4 Cell Strains
1 2 3 4 5 6

LSCC-DU72

MSU-l.l

H
‘5
_l

      

    

                    

206 -

100 -

42 -

Figure 5. Expression of v-myg encoded p110 in MSU-1.1-T24 cell strains.

v-myg_p110 was immunoprecipitated fronl[35$]methionine-labeled cell lysates

with an antibody that reacts with proteins of the my; family and analysed
by electrophoresis in 10% polyacrylamide gels and fluorography. The
location of the v-myg encoded p110 is indicated by the arrow. LSCC-DU72
is a chicken hepatoma, M629 virus-producer cell line. The location of

molecular weight (kilodalton) markers on the gel is indicated.

99
specific monoclonal antibody Yl3-259 (28), indicated that each of them
overexpressed a 21 kd protein (see arrow) that migrated to the same
position as the overexpressed T24 H-[as oncogene encoded p21 of the T24
bladder carcinoma cell line. To determine the relative levels of_ra§ p21
expression, the radiolabeled p21 bands were cut out of duplicate gels and
quantitated by scintillation counting. The level of total [as p21
expression in the MSU-l.l-T24 cell strains was 3 to 5 times that expressed
in the parental MSU-l.l cell strain, or in the LG] cell line from which
the latter was derived, and was only slightly elevated over that observed
in T24 bladder carcinoma-derived cells. Radioimmunoprecipitaion analysis
of [as p21 was also carried out on MSU-l.l—T24 fibroblast cell strains
derived from tumors generated in athymic mice. T24 H-Las p21 levels in
these cells were comparable to levels determined in their parental
focus-derived cells prior to injection into athymic mice, indicating that
no in vivo selection of cells expressing higher or lower levels of the

transfected oncogene had occurred (data not shown).

Radioimmunoprecipitation analysis was also used to compare the level of
v-myg p110 expression in the MSU-l.l-TZ4 cell strains, with that in the
parental, MSU-l.l cell strain, using as a positive control a chicken
hepatoma cell line LSCC-DU72 that expresses a v-myg p110. Such analysis
indicated that in the MSU-l.l-T24 cell strains, v-myg p110 was expressed
at levels comparable to that found in the parental MSU-l.l cells (Fig. 5,

see arrow). As expected, no v-myg p110 was detected in the LEI cells.

Karyotype Analysis of USU-1.1-T24 Cell Strains. Analyses of G-banded,

and conventionally stained chromosomes from MSU-1.l fibroblasts and

100
MSU-l.l-T24 fibroblasts were carried out to determine karyotype and
chromosome stability respectively. A detailed description of the banding
patterns for MSU—l.l fibroblast chromosomes will be reported elsewhere.
The MSU-1.l-T24 cell strains analysed (strains 2 and 3), as well as cells
taken from the tumors derived from them, showed the same karyotype and
modal chromosome number, i.e., 45, as the parental MSU-l.l cell strain,

as well as the two marker chromosomes characteristic of this cell line.

Tumorigenic Transformation of Infinite Lifespan Human Fibroblasts Derived
from Other Cell Lines. To determine whether expression of T24 H-ras in
infinite lifespan, non-tumorigenic human fibroblast cell lines other than
MSU-l.l also caused tumorigenic transformation, we transfected pH06Tl, or
the control plasmid, pH06N1, into the SUSM-l (23) and KMST-G (24) cell
lines, as well as into a cell line (GM0637B) generated following SV40
virus infection. The transfectants were selected for 6418 resistance,
rather than focus formation, because unlike MSU-l.l cells, these target
cell lines already exhibit a transformed morphology and aberrant growth
behavior. G418-resistant colonies were pooled, and their progeny cells
tested for tumorigenicity in athymic mice. Within 4.5 mo, four out of five
athymic mice injected with pH06Tl-transfected KMST-6 cells (KMST-G-T24) and
three out of six injected of pH06Tl-transfected GM0637B cells (GM0637B-
T24) developed progressively growing malignant fibrosarcomas within that
reached a 1 cm diameter. No tumors were formed in 5 mice injected with
pH06Tl—transfected, SUSM-l cells (SUSM-l-T24) generated in two separate
experiments, or' with any of the pHOGNl transfected, G418-resistant

controls. Because of the long time required for tumor development by these

101

cell strains relative to MSU-1.1—T24 tumorigenic cell strains, cells were
cultured from tumors formed by these cell strains and reinjected in
athymic mice to determine if an in vivo selection for tumorigenic cells
had occurred. Two out of 2 mice injected with tumor-derived KMST-G-T24
cells developed 1 cm diameter tumors within 25 days, (a period similar to
that of MSU-l-T24 tumorigenic strains), but no decrease in time for tumor
development with tumor-derived GM0637B-T24 cells. Both KMST-6-T24 cells
and GM0637B-T24 cells expressed T24 H-Las oncogene, but the SUSM-l-T24
cells did not (data not shown), which correlates with their
tumorigenicity.

102

DISCUSSION

In an earlier study (13) we demonstrated that expression of a transfected
T24 H-ras oncogene in normal diploid human fibroblasts resulted in their
transformation to morphologically altered cells that form foci and exhibit
anchorage-independent growth. Analysis of the relationship between
expression of the transfected [as oncogene and tumorigenicity was not
possible because expression of the transfected La; oncogene and the
transformed phenotype were lost concomitantly during expansion of
transformant cell populations. Later, we succesfully isolated several T24
H-ras transformed diploid human fibroblast populations that were stable,
i.e., did not revert during expansion in culture subsequent to
transfection, and showed that these cells had a finite lifespan in culture
and were not tumorigenic (14). In the present study we have extended this
research by demonstrating that expression of an exogenous T24 H—m
oncogene in a human fibroblast strain that has an infinite lifespan in
culture resulted not only in morphological transformation, focus formation
and anchorage independence, but also in tumorigenicity. ‘The tumors formed
by MSU-l.1-T24 cell strains grew progressively in athymic ice and were
diagnosed as invasive fibrosarcomas. It must be noted that cell strains
4 and 5 did not give rise to tumors (Table 1) even though they expressed
the T24 H-ras oncogene and exhibited the other transformed
characteristics. These 2 strains were retested in a separate experiment
and again proved nontumorigenic. The reasons for the lack of tumor

formation by these strains are not yet understood.

Three other studies have investigated the transforming potential of gas

103

oncogenes in infinite lifespan human fibroblast cell lines. O’Brien et.
al. (36) utilized an SV40-virus transformed human fibroblast line, VaZ,
and Namba et. al. (37,38) utilized the KMST—6 cell line' .Athough neither
of these target cell lines are tumorigenic, they exhibit many
characteristics of ras-transformed rodent fibroblasts, i.e., they are
morphologically transformed, exhibit aberrant growth behaviour, and are
highly aneuploid. Introduction of the r_as oncogene by either virus
infection protocols (36,37), or by transfection (38), resulted in cells
able to form tumors in athymic mice, although the tumors produced with
the VaZ cells did not grow progressively (36). Our results, using cells
of the KMST-6 cell line as recipients for transfection of plasmid pH06Tl,
confirm the results of Namba et. al. (37,38), and our results using the
SV40-transformed infinite lifespan human fibroblast cell line, GM0637B,
are similar to those of O’Brien et. al. (36), although our cells formed

progressively growing tumors.

Because the near-diploid MSU-l.l cell strain exhibits no
transformation-related phenotypes, other than its infinite lifespan in
culture, it permitted a more complete evaluation of the spectrum of
transformation characteristics induced by .:a§_ oncogenes in infinite
lifespan human fibroblasts, that are predicted from studies with rodent
fibroblasts. Indeed, we feund that expression of the transfected T24
H-Lii oncogene in MSU-1.l fibroblasts induced several
transformation-related phenotypes including malignancy. In addition,
because of the chromosome stability of MSU-1.1 fibroblasts, we conclude
that the transformed phenotypes that resulted following transfection of

the T24 H-rgs oncogene was not the result of the selection of tumorigenic

104

variants arising from a karyotypically unstable population of

transfectants.

Each of the focus-derived MSU-1.1-T24 cell strains generated by
transfection of plasmid pH06Tl expressed a high level of T24 H-Las
oncogene encoded p21. Transfection of the normal H-ras gene in the same
plasmid construct, or the T24 H-ras oncogene in plasmids containing only
one or no transcriptional enhancer elements did not result in focus
formation. These data suggest that a sufficiently high level of T24 H—ras
oncogene expression was necessary to transform MSU-1.1 fibroblasts. Thus,
the potent transforming activity of 'the ‘T24 H-[a§_ oncogene appears
dependent not only on mutational activation, but also on its enhanced

expression.

Although the MSU-1.1 cell strain arose following transfection of finite
lifespan diploid human fibroblasts with a plasmid containing a v-m
oncogene, it is not clear that expression of this oncogene was responsible
for induction of the infinite lifespan phenotype since we have only one
such cell strain. Likewise, it is not known whether, as proposed for rat
embryo fibroblasts (4), the expressed v-myg protein acts in a cooperative
fashion with the T24 H-gas oncogene to cause transformation. However, our
results showing that ras oncogene expression induces tumorigenic
transformation of non-v-myc-transfected infinite lifespan human
fibroblasts suggest that the infinite lifespan phenotype, rather than v-
myg expression, is the characteristic of MSU-l.1 cells that complemented
ras oncogene expression to cause tumorigenicity. Taken together with

results showing that finite lifespan human fibroblasts transfected with

105

La; oncogenes exhibit a transformed phenotype, but do not have an infinite
lifespan in culture and are not tumorigenic (13,14,19), the results
described here indicate both the fundamental and practical importance of
the infinite lifespan phenotype for in vitro transformation systems that

use tumorigenicity as an endpoint (21).

106

ACKNOWLEDGEMENTS

We thank Dr. J. Dillberger for histological examination, Dr. D. Yang for
chromosome analysis, Dr. N. Hilkie for kindly supplying us with the pH06
series of plasmids, Dr. M. Anderson for the Yl3-259 antibody to ras p215,
Dr. M. Namba for the KHST-6 and SUSM-l human fibroblast cell lines. This
research was supported by Department of Energy Grant DEF602-87-ER60524,
National Cancer Institute Grant CA21289 and National Institute for
Environmental Health Sciences Contract NOI-ES-65152.

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110

APPENDICIES

111

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APPENDIX A

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112
APPENDIX B

Athymic mouse with tumor formed by subcutaneous injection of MSU-1.1-T24
strain 3 cells.

 

113

APPENDIX C

Data 'not shown“ in Chapter III

114

Figure 1. Comparison of nae p21 expression in focus-derived, anchorage
independent colony-derived and tumor-derived MSU-1.1-T24 cell strains.
Analysis was performed as described in the Methods and Materials section
of Chapter 11. T24 is the T24 bladder carcinoma cell line. 2T, 3T1 and
3T2 are were cultured from independent tumors formed by MSU-l.l-T24 cell
strains 2 and 3. 381 and 3B2 are cell strains derived from anchorage
independent colonies formed by MSU-1.1-T24 cell strain 3. The bars
indicate the location of the normal p21 doublet and the arrow indicates

the approximate position of T24 H-[aa p21.

115

 

116

Figure 2. Southern blot analysis of MC29 sequences in MSU-l.1-T24 cell
strains. Genomic DNAs were digested with XhQ I, and Cam, HI, and
hybridized with the Sag I fragments of the MC29 virus (Reddy et al. (1983)
Proc. Natl. Acad. Sci. USA CC, 2500-2504). Hybridizing sequences specific

to MC29 viral sequences are indicated.

117

 

H
7'
a a MSU—LI-T24 Strains
*3 E 1 2 6
23-
9.4-
6.6-
4.4-
-> v- s“ —

 

118

Figure 3. Analysis of pa; p21 expression in SUSM-l-T24, KMST-6-T24 and
GM0637-T24 cell strains and in their parent cell lines. Analysis was
performed as described in Chapter II Maaterials and Methods with the
exception that immunoprecipitates were solubilized in SDS-PAGE buffer
containing 8 M urea. This modification caused a shift in the mobility of
the mutant p21 so that it migrated to a position above the characteristic
p21 doublet (see arrow). T24 is the human bladder carcinoma cell line.
The bars indicate the location of the normal p21 doublet and the arrow

indicates the location of the T24 H-Laa encoded p21.

‘_—r—*‘_

119

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