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IMMORTALIZATION AND MORPHOLOGICAL AND ANCHORAGE-
INDEPENDENT TRANSFORMATION OF DIPLOID HUMAN FIBROBLASTS
by
Jiaxiong Weng
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Toxicology)
January, 1995
Copyright 1995 Jiaxiong Weng
UMI Number: 9621644
UMI Microform 9621644
Copyright 1996, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized
copying under Title 17, United States Code.
UMI
300 North Zeeb Road
Ann Arbor, MI 48103
UNIVERSITY OF SOUTHERN CALIFO RNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
Jiaxio n g Veng
under the direction of fti® . Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
C .. —
Dean o f Graduate Studies
Date
DISSERTATION COMMITTEE
ACKNOWLEDGMENTS
It is hard to believe that four and half years have passed since I
first arrived in the United States and started graduate studies at the
Institute for Toxicology in the fall of 1990. I owe a great deal to
many of the teachers and friends at the USC community.
I would like to thank my advisor, Dr. Joseph Landolph, who
introduced me to the field of chemical carcinogenesis. Throughout
my graduate training, he was the driving force of my intellectual
quest. I appreciate his guidance, support and the countless hours he
dedicated to reading and revising the draft of this dissertation and
other works. I would also like to thank the members of my advisory
committee, Drs. Timothy Chan, Alex Sevanian, Pradip Roy-Burman,
and Paul Hochstein for their invaluable suggestions, and support.
This dissertation could not have been written without their support
and constructive criticism.
In addition, I would like to thank Dr. Raji Pichika for helping me
with the experimental work presented in this dissertation and Drs.
Cecil Miller and Timothy Hutchin for their invaluable input,
comments, and advice in proofreading and shaping the presentation
of this dissertation. I also want to thank Dr. Anuradha Verma for her
help and encouragement and Hazel Peterson for her expertise and
technical support.
II
I would also like to give special thanks to my fellow graduate
students, Michael Dews, Laurent Ozbun, David Crawford, Evelyne
Gozal, and Laurie Mcleod for their friendship and scientific input.
Last but not the least, I want to thank the Institute for
Toxicology and Department of Molecular Pharmacology and
Toxicology for providing me the basis for my personal and
educational advancement for the past four years. I am grateful to the
many friends I made during the course of my studies, who lifted me
with support and friendship. I am deeply indebted to all of my
friends, without whom my dreams would not be realized.
Ill
TABLE OF CONTENTS
ACKNOWLEDGEMENTS 1 1
LIST OF TABLES V III
LIST OF FIGURES X
ABBREVIATIONS XV
ABSTRACT XVI
CHAPTER 1
INTRODUCTION 1
1.100 GENERAL INFORMATION 1
1.200 STRATEGIES 14
CHAPTER 2
MATERIALS AND METHODS 1 9
2.001
2.002
2.003
2.004
2.005
2.006
2.007
2.008
2.009
2.010
IV
Cells and Cell Culture 1 9
Plasmid Constructs 20
Antibodies Used in Our Studies 21
Transfection of Normal Human Fibroblasts and
Selection of Transfectants 22
Plating Efficiency Determination Assays 22
Calculation of the Number of Population
Doublings 23
Determination Of the Survival Curves of Normal
Human Fibroblasts in G418 24
Biological Characterization of Immortal Human
Fibroblast Cell Lines 24
1) Saturation Density Assays 25
2) Anchorage Independence Assays 25
3) Focus Reconstruction Assay 26
4) Assays to Determine Serum Requirements
for Cell Growth 27
Cytotoxicity Assays 28
Assays to Detect Chemically Induced
Morphological and Anchorage-Independent
Transformation 29
2.011 Immunostaining to Detect Specific Nuclear
Proteins (SV40 Large T Antigen) 30
2.012 Western Blotting Analysis 31
2.013 Measurement of Protein Concentrations 32
CHAPTER 3
RESULTS 34
3.100 Polyoma Large T Antigen 34
3.101 Introduction 34
3.102 Survival Curve Determination to Define
Concentrations of G418 to Be Used in
Transfection Assays 36
3.103 Transfection of Normal HFC and Selection of
Transfectants 37
3.104 Western Blotting Analysis of Polyoma Large T
Antigen 41
3.105 Subcultivation of Polyoma Large T Antigen
Transfected HFC Clones 41
3.106 Biological Characterization of Polyoma Large T
Antigen Transfected HFC Clones 45
3.107 Conclusions 49
3.200 Transfection of the Mutated p53 Gene into
Normal Diploid Human Fibroblasts 50
3.201 Introduction 50
3.202 Transfection of Normal HFC with a Mutated p53
Gene and Selection of Transfectants 51
3.203 Biological Characterization of Mutated p53 Gene
Transfected HFC Clone 57
3.204 Conclusions 57
3.300 SV40 Large T Antigen 61
3.301 Introduction 61
3.302 Tansfection of Normal HFC, Selection of
Transfectants, and Subcultivation of
Transfectant Human Fibroblast Clones 62
3.303 Detection of Immortal Human Fibroblast Clones 70
3.304 Assay for Immortality of Postcrisis Clones 71
3.305 Biological Characterization of Precrisis and
Postcrisis Human Fibroblasts 73
V
3.306
3.307
3.308
3.309
3.310
3.311
3.312
3.313
3.314
3.315
3.400
3.401
3.402
3.403
3.404
1) Morphological Changes
2) Saturation Density Assays
3) Reconstruction Assay for Focus Formation
4) Anchorage Independence Assays
5) Serum Dependence Assays
Western Blotting Analysis of SV40 Large T
Antigen in Immortal HFC Clones
Immunostaining of different SV40 Immortalized
Clones for SV40 Large T antigen
Correlation Analysis of the Relative Amounts
Intracellular SV40 Large T Antigen and specific
Transformed Phenotypes
Subcloning of Immortalized Clone with Less
Transformed Phenotypes
Conclusions
Chemical Transformation Assays in Immortalized
HFC Clones
1) Cytotoxicity Assays
2) Focus Formation Assays
3) Anchorage Independence and Saturation
Density Assays
Early Attempts to Induce Transformation in SV40
Large T Antigen Immortalized Cell Lines and
Expression Time for the Appearance of
Transformed Phenotype
In Vitro Chemical Transformation of HFC/SV9-1
Characterization of Cells Derived from Anchorage
Independent Colonies of MNNG treated HFC/SV9-1
Cells
Transfection of Immortal SV40 Large T Antigen
Clones with Oncogenes
Effects of the C-Myc Gene on Human Fibroblasts
Introduction
Transfection of Normal HFC and Selection of
Transfectants
Biological Characterization of c-myc Gene
Transfected HFC Clones
Conclusions
73
76
76
87
88
104
105
105
111
118
119
120
120
131
133
134
140
150
155
156
157
162
VI
CHAPTER4
DISCUSSION 163
4.100 Cellular Immortality Studies 163
4.200 In Vitro Chemical Transformation Studies 171
4.300 C-myc Oncogene 174
4.400 Summary and Prospectives 175
BIBLIOGRAPHY 180
V II
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
Table 10
Table 11
Table 12
LIST OF TABLES
Summary of Transfection Results with Polyoma
Large T Antigen 39
Summary of Biological Characterization of Polyoma
Large T Antigen Transfected Human Fibrobalst
Cells 4 6
Summary of Transfection Results with a Mutated
P53 Gene and a Control Vector 53
Summary of Biological Characterization of Mutated
p53 Gene Transfected Human Fibrobalst Cells 58
Transfection of SV40 Large T Antigen Encoding
Gene 64
Morphology of SV40 Large T Antigen Immortalized
Human Fibroblast Clones 7 4
Focus Reconstruction Assay of HFC/SV Cells
against Normal HFC 81
Assay of Precrisis Focus Forming Cells for
Im m ortality 82
The Ability of HT1080 to Form Foci against SV40
Large T Antigen Immortalized HFC Clones 84
i Growth of SV40 Large T Antigen Immortalized HFC
Clones in Medium without Serum 103
Summary of Phenotypes of SV40 Large T Antigen
Immortalized HFC Clones in in vitro
Transformation Characterization Assays 115
! Phenotypic Changes of Different HFC Immortal
Clones after being Treated with MNNG 131
VIII
Table 13 Induction of Anchorage Independence of HFC/SV9-1
by MNNG TreatmentP#8)
Table 14 Focus Formation Assay of HFC/SV1-1 after
Oncogene Transfection
Table 15 Focus Formation Assay of HFC/SV1-2 Cl 6 after
Oncogene Transfection
Table 16 Induction of Anchorage Independence in HFC/SV9-1
by Transfection of H-ras and myc Oncogenes
Table 17 Results of Transfection of c-myc and an activated
ras Oncogenes into Senescing Human Fibroblasts
Table 18 Results of Reconstruction Assays to Detect
Focus Formation of c-myc Immortalized Human
Fibroblast Clones against Normal HFC Cells
Table 19 Summary of the Effects of Various Genes on the
Phenotypes of Human Diploid Fibroblasts
139
151
152
153
158
159
176
IX
Figurel
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
LIST OF FIGURES
Survival Curve for Normal HFC Cells and a Neo-
SV40 Large T antigen Transfected Clone (HFC/SV1) 38
Western Blot Analysis of Polyoma Large T Antigen 42
Plating Efficiency of Polyoma Large T Antigen
Transfected HFC Clones 43
Population Doubling Curve of Polyoma Large T
Antigen Transfected HFC Clones 44
Saturation Density Curve of Polyoma Large T
Antigen Transfected HFC Clones 47
Focus Forming Efficiency of HT1080 against
Polyoma Large T Antigen and MLV Transfected HFC
Cells 48
Plating Efficiency of Mutated p53 and PCMV
Transfected HFC Clones 54
Population Doubling Curve of Mutated p53 and PCMV
Transfected HFC Clones 55
Focus Forming Efficiency of HT1080 against HFC
Cells Transfected with the Mutated p53 Gene or
PCMV 59
i Population Doubling Curve of SV40 Large T Antigen
Transfected HFC Clones 65
Plating Efficiency of Passaged SV40 Large T
Antigen Transfected HFC Clones 66
! Cumulative Population Doubling Curve for SV40
Large T Antigen Immortalized HFC Clones 72
X
Figure 13 Saturation Density Curve of SV40 Large T Antigen
Immortalized HFC Clones 77
Figure 14 Fold Increase in Saturation Density of SV40 Large T
Antigen Immortalized HFC Clones Compared to That
of Normal HFC 78
Figure 15 Plating Efficiency of SV40 Large T antigen
Immortalized HFC Clones 79
Figure 16 Focus Forming Efficiency of SV40 Large T Antigen
Immortalized HFC Clones 80
Figure 17 Anchorage Independent Growth of SV40 Large T
Antigen Immortalized HFC Clones 86
Figure 18a Growth of HT1080 Cells in Medium Containing
Different Concentrations of FBS 89
Figure 18b Growth Property of Normal HFC Cells in Medium
Containing Different Concentrations of FBS 90
Figure 18c Growth Property of HFC/SV1-1 Cells in Medium
Containing Different Concentrations of FBS 91
Figure 18d Growth Property of HFC/SV1-2 Cells in Medium
Containing Different Concentrations of FBS 92
Figure 18e Growth Property of SV1-2 Cl 6 Cells in Medium
Containing Different Concentrations of FBS 93
Figure 18f Growth Property of HFC/SV10-2 Cells in Medium
Containing Different Concentrations of FBS 94
Figure 18g Growth Property of HFC/SV10-3 Cells in Medium
Containing Different Concentrations of FBS 95
Figure 18h Growth Property of HFC/SV9-1 Cells in Medium
Containing Different Concentrations of FBS 96
XI
Figure 18i Growth Property of HFC/SV9-9 Cells in Medium
Containing different Concentrations of FBS 97
Figure 19a Growth Property of SV40 Large T Antigen
Immortalized HFC Clones in Medium Containing
Different Concentrations of Fetal Bovine Serum
Figure 19b Growth Property of SV40 Large T Antigen
Immortalized HFC Clones in Medium Containing
Different Concentrations of Fetal Bovine Serum
Figure 20 Western Blot Analysis of SV40 Large T Antigen
Figure 21 Relative Quantity of SV40 Large T Antigen per pg
Total Protein in Nuclear Extracts from Different
Immortal HFC Clones
Figure 22a Correlation Between Saturation Density and
Amount of Intracellular SV40 Large T Antigen in
SV40 Large T Antigen Immortalized Human
Fibroblast Clones
Figure 22b Correlation Between Focus-Forming Efficiency and
Amount of Intracellular SV40 Large T Antigen in
SV40 Large T Antigen Immortalized Human
Fibroblast Clones
Figure 22c Correlation Between Anchorage-Independent
Growth and Amount of Intracellular SV40 Large T
Antigen in SV40 Large T Antigen Immortalized
Human Fibroblast Clones
Figure 23a Saturation Density Curve of HFC/SV1-2 Subclones
Figure 23b Focus Forming Efficiency of HFC/SV1-2 Subclones
Figure 23c Anchorage Independent Growth of HFC/SV1-2
Subclones
98
99
106
107
108
109
110
112
113
114
XII
Figure 24a Survival Curve of HFC/SV1-1 Cells Treated with
M SN G
Figure 24b Survival Curve of HFC/SV1-2 Cells Treated with
M M M G
Figure 24c Survival Curve of HFC/SV1-2 Cl 6 Cells Treated
with MNNG
Figure 24d Survival Curve of HFC/SV10-2 Cells Treated with
M M S G
Figure 24e Survival Curve of HFC/SV10-3 Cells Treated with
M M M G
Figure 24f Survival Curve of HFC/SV10-4 Cells Treated with
I U N M G
Figure 24g Survival Curve of HFC/SV10-5 Cells Treated with
MVNG
Figure 24h Survival Curve of HFC/SV9-1 Cells Treated with
MN N G
Figure 24i Survival Curve of HFC/SV9-2 Cells Treated with
I W NG
Figure 24j Survival Curve of HFC/SV9-9 Cells Treated with
MN N G
Figure 25a Effects of Passage on the Efficiency of Anchorage
Independent Growth in MNNG Treated HFC/SV9-1
Cells
Figure 25b Effects of Passage on Saturation Density in MNNG
Treated HFC/SV9-1 Cells
Figure 25c Saturation Density Curve of HFC/SV9-1 after MNNG
Treatment (P8)
121
122
123
124
125
126
127
128
129
130
135
136
137
XIII
Figure 26a Growth Curve of HFC/SV9-1 (Control)
Figure 26b Growth Curve of HFC/SV9-1 (Acetone)
Figure 26c Growth Curve of HFC/SV9-1 (Al M-1)
Figure 26d Growth Curve of HFC/SV9-1 (Al M-2)
Figure 26e Growth Curve of HFC/SV9-1 (Al M-3)
Figure 27a Comparison of the Growth Properties of Anchorage
Independent Cells Derived from MNNG Treated
HFC/SV9-1 in Medium Containing 10% Fetal Bovine
Serum
Figure 27b Comparison of the Growth Properties of Anchorage
Independent Cells Derived from MNNG Treated
HFC/SV9-1 in Medium Containing 0% Fetal Bovine
Serum
Figure 28 Comparison of the Growth Properties of Anchorage
Independent Cells Derived from MNNG Treated
HFC/SV9-1 in Medium Containing Different
Concentrations of Fetal Bovine Serum
Figure 29 Anchorage Independent Assay of Al Cells Derived
from MNNG Treated HFC/SV9-1
Figure 30 Anchorage-Independent Growth of c-myc
Immortalized HFC Clones
Figure 31 Saturation Density Curve of c-myc Immortalized
HFC Clones
141
142
143
144
145
146
147
148
149
160
161
XIV
ABBREVIATIONS
Al: Anchorage Independence
DMSO: Dimethyl sulfoxide
EDTA: Ethylenediaminetetraacetic acid
FBS: Fetal Bovine Serum
HEPES: N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid
HFC: Human Fibroblast Cell
MEM: Modified Eagle’s Medium
MNNG: N-methyl-N'-nitro-N-nitrosoguanidine
NEAA: Nonessential Amino Acids
PBS: Phosphate Buffered Saline
PE: Plating Efficiency
SD: Standard Deviation
SDS: Sodium Dodecyl Sulfate
TPA: 12-0-tetra-decanoyl-phorbol-13-acetate
TRIS: Tris (hydroxymethyl) aminometane
Abstract
Carcinogenesis in humans is a complex process. To understand
chemical carcinogenesis, we developed a human model cell culture
system to study this process. We transfected four oncogenes into
human diploid fibroblast cells derived from circumcised, neonatal
foreskins, including the simian virus 40 (SV40) large T antigen
encoding gene, the polyoma virus large T antigen encoding gene, a
mutated human p53 gene, and the mouse c-myc gene. Transfection of
mutated p53 or polyoma large T antigen encoding genes into normal
human fibroblasts did not immortalize them, did not change their
morphology and did not induce other transformed phenotypes in the
cells. Mutated p53 gene extended the life span of human fibroblasts
by fourteen population doublings, and polyoma large T gene extended
the life span of normal human fibroblasts by four population
doublings. Constitutively expressed c-myc plasmid immortalized
human fibroblasts. Cells from c-myc immortalized human
fibroblasts were anchorage-independent but did not form distinctive
foci.
Transfection of the SV40 large T antigen encoding gene into
normal human diploid fibroblasts resulted in fifteen immortal human
fibroblast cell clones. Fourteen clones formed foci in reconstruction
experiments, grew to higher saturation densities, grew in soft agar,
and acquired serum-independent growth, indicating they acquired
transformed phenotypes. The transformed phenotype of individual
XVI
clones correlated with the quantity of SV40 large T antigen
expressed inside cells of that clone. One clone expressed a low level
of SV40 large T antigen, did not grow in soft agar, had highly serum-
dependent growth properties, and had only a slightly higher (1.3-
fold) saturation density than normal diploid fibroblasts. Treating
this immortal clone with the carcinogen N-methyl-N'-nitro-N-
nitrosoguanidine (MNNG), caused it to acquire a more transformed
cell morphology and anchorage-independent growth. Cells derived
from anchorage-independent colonies remained stably anchorage-
independent and showed serum-independent growth.
We believe this model cell culture system will provide insight
into mechanisms of carcinogen-induced neoplastic
transformation and information on potential genes that can be
targeted for intervention in the prevention, detection, and
treatment of chemically-induced human cancer.
XVII
CHAPTER 1
INTRODUCTION
1.100 GENERAL INFORMATION
Cancer is a costly public health problem worldwide, both
economically and in terms of human suffering. It has been the second
leading cause of death in the United States for decades, second only
to cardiovascular diseases (Daly, 1993). It is reported that 32% of
the U.S. population contracts cancer and 16% of the U.S. population
dies from it (Daly, 1993). As the number of deaths caused by
cardiovascular diseases continues to decline due to better diets,
smoking cessation, cholesterol lowering and exercise, it is
projected that that cancer will be the number one killer by the turn
of the century (Tomatis, 1990). Since the present cure rate for the
major cancers of the population is approximately 50% overall, and
only 5% for lung cancer, understanding the etiology and the
mechanisms of carcinogenesis has become very important (Vainio,
1986).
Traditionally, two approaches have been employed to detect the
possible causes of human cancer; one is epidemiological studies of
human populations, and the other is laboratory experiments using
various animal models or in vitro cell culture systems. The major
strength of epidemiological studies is their focus on human
populations. They are the most direct way of investigating the
1
possible causes of human cancer, thereby avoiding the need to
extrapolate data from animals to humans. However, the
interpretation of epidemiological studies is complicated by the
heterogeneity of the human population, and difficulties in accurately
measuring both the exposure and the outcome variables. It is also
important to note that epidemiological studies are at best
correlations compounded by many uncontrollable variables. Animal
models and cell culture studies are basically an effort to overcome
the limitations of the direct studies of cancer in humans and can be
excellent tools to confirm or disprove the results of epidemiological
studies. Both animal and cell culture studies provide a simplified
and controlled environment, with the animal subjects and cultured
cells regarded as uniform population. Laboratory experiments are
therefore more precise than epidemiological investigations.
Laboratory studies are often used to confirm or disprove the
suggested correlations of epidemiological studies. Epidemiological
studies are used to derive suggested etiologic information, while
laboratory studies are employed to confirm the epidemiological
findings and discover mechanisms of carcinogenesis (Alderson,
1986).
In vitro cell culture techniques have been used extensively to
study the process of carcinogenesis and neoplastic cell
transformation caused by viral, physical, or chemical agents in the
past five decades. The term "transformation", when used in cell
culture, refers to a process whereby a cell line of either finite or
infinite life span in culture has assumed one or more characteristics
of tumor-derived cells, such as focus formation, anchorage-
independence, serum-independent growth, or the ability to form
progressively growing tumors when injected into animal hosts
(reviewed in Landolph, 1985, 1989, 1994).
Using carcinogen treatment to transform normal cells, or cells
with an infinite life span in culture, into cells which have acquired
various characteristics of tumor cells provides a useful tool to
study the mechanism of carcinogenesis, and a means of evaluating
potentially carcinogenic agents (Landolph, 1994; McCormick, 1994).
Cell culture transformation studies utilizing human cells have a
special relevance since, ethically, one cannot knowingly expose
humans to carcinogenic agents. Therefore, studies of human
carcinogenesis in vivo have been limited to individuals accidentally
exposed to such agents in an occupational setting or to individuals
who have chosen to expose themselves to carcinogenic agents, e.g.,
cigarette smokers. Studies of such exposure may indicate that an
agent is carcinogenic, but they can yield only a limited
understanding of mechanisms of carcinogenesis. The strength of cell
culture transformation assays is precisely their ability to yield
mechanistic information (reviewed in Landolph, 1985, 1989, 1994).
The history of studying the process of transformation with in
vitro cell culture systems began in the early 1950's. Cell
transformation assays measuring focus formation were first used as
3
a quantitative assay for titrating viruses (Temin and Rubin, 1958).
With this assay, cells infected with viruses can form foci. Foci are
dense formations due to the piling-up of morphologically
transformed cells growing on the top of a confluent layer of
nontransformed cell. The first chemical carcinogen-induced
mammalian cell transformation assay was reported by Berwald and
Sachs in 1963, in which they treated early passage golden hamster
embryo cells in culture with benzo(a)pyrene. Colonies arose in
which cells exhibited a random growth pattern. This transformed
phenotype was recognized because the altered morphology and
growth pattern of those chemically transformed colonies appeared
similar to those previously obtained when cells were infected with
polyoma virus. These transformed cells were shown to form tumors
after extended subculture (Berwald and Sachs, 1965).
Since that time, several rodent fibroblast cell lines have been
developed that could be induced by carcinogen treatment to form
distinctive foci of morphologically transformed cells. Among those
are the BALB/c 3T3 Clone A31 cell line developed by Kakunaga and
Kamahora (1970), and the C3H/10T1/2 clone 8 mouse embryo
fibroblast cell line developed by Reznikoff and Heidelberger in 1973.
Following carcinogen treatment, both these cell lines can form
distinct foci on the top of a confluent monolayer in a dose-dependent
manner after carcinogen treatment (reviewed in Kakunaga, 1985 and
Landolph, 1985, 1989, 1994). This focus-based transformation assay
has gained broad acceptance and is one of the most widely used
4
assays for assaying agents for their potential carcinogenicity by
measuring cell transforming ability and for studying the molecular
mechanisms of neoplastic transformation caused by these agents.
Cloned cell lines derived from these foci show the phenotypes of
tumor-derived cells, such as anchorage-independent growth and a
reduced serum requirement for growth, and many transformed cell
lines yield tumor after extended subculturing (35-70 population
doublings). This assay is a good predictor of carcinogenicity, and
investigators therefore use this assay to test for carcinogens,
obviating the need for the long-term rodent carcinogenicity
bioassays (reviewed in Landolph, 1985, 1989, 1994). The assay for
morphological transformation in 10T1/2 cells shows focus
formation after the cells are treated with a wide variety of
polycyclic aromatic hydrocarbons (Reznikoff et al., 1973; Landolph
and Heidelberger, 1979); carcinogenic nickel compounds (Miura et al.,
1980); carcinogenic arsenic compounds (Landolph, manuscript in
preparation) and carcinogenic chromium salts (Patierno et al., 1989)
and anti-neoplastic and anti-parasitic agents (Nianjun et al., 1994).
Since the first report of in vitro transformation of hamster cells
with chemical carcinogens (Berwald,et al, 1963), extensive studies
on the mechanisms of carcinogenesis have been conducted with in
vitro mammalian cell transformation assay systems. The recent
identification of proto-oncogenes and tumor suppressor genes in the
mammalian genome, which has led to a new understanding of how
cancer develops, has further accelerated the acquisition of our
5
knowledge concerning molecular mechanisms of carcinogenesis from
in vitro transformation studies (Spandidos et al, 1987; Weinberg,
1992). These studies, however, were conducted mainly with primary
cultures or permanent cultures of cells derived from mice, hamsters,
rats, and chickens.
Consequently, a question arose as to whether or not these data on
animal cells could be extrapolated to the development of human
cancers. For this reason, in vitro chemical transformation studies
have recently been undertaken with primary cultures of normal
human cells during the last two decades, but no reproducible
transformation of human cells has as yet been reported. For reasons
that remain unclear, primary cultured human cells are extremely
refractory to agents that readily induce the neoplastic conversion of
rodent cells. To date, the transformation of primary cultured normal
human cells with chemical carcinogens is still a rare event
(McCormick, 1994; Landolph, 1994; Biedermann and Landolph, 1987,
1990; reviewed in Landolph, 1985, 1989, 1994). No one has
successfully produced malignantly transformed cells capable of
making progressively growing invasive tumors in athymic mice by
carcinogen treatment alone of normal primary cultured human cells.
Most of the data on human cell transformation is obtained from the
studies of clinically obtained tumor samples. Since these cells are
already transformed, they are unable to illustrate a dynamic cause-
effect relationship between chemical carcinogen treatment and
neoplastc transformation of a normal human cell in vivo. For this
6
reason, establishing a human cell culture model system like
C3H101/2 Clone 8 or Balb/c 3T3 Clone A31 cells which can be used
for in vitro transformation assays has become one of the most active
areas in the fields of cancer research, and this is also one of the
research goals in our laboratory.
To establish a human cell transformation assay analogous to the
rodent C3H101/2 Clone 8 or Balb/c 3T3 Clone A31 cell culture
system, two questions need to be addressed. First, what makes
primarily cultured human cell so refractory to in vitro m a lig n a n t
transformation by chemical carcinogens? Second, can immortality
be separated from other transformed phenotypes, such as focus
form ation, anchorage-independent growth, reduced serum
requirements, or most importantly, tumorigenicity, in human cells?
Understanding these two questions will give us a hint as to where to
begin our project and how to achieve our goal in the most efficient
way.
To answer the first question, we needed to compare some of the
phenotypic differences between normal human cells and human
tumor-derived cells. Most, if not all human tumor-derived cells grow
continuously under in vitro culture conditions and therefore are
considered immortal, while normal primary human diploid
fibroblasts only have a life span of 50-80 population doublings (Shay
et al, 1989), after which they go into senescence. Second, various
data suggest that tumor cells have acquired multiple independent
7
genetic changes, in addition to immortality (Shay et al, 1993a). For
example, one of the fibrosarcoma-derived fibroblastic cell lines
(HT1080) synthesizes platelet-derived growth factor (Pantazis et
al., 1985) and an epidermal growth factor precursor (Burbeck et al.,
1984). Normal fibroblasts do not synthesize these proteins.
Furthermore, HT1080 cells have an activated N -ras gene, which
causes disturbance in the G-protein associated signal transduction
pathway, which prevents cell from entering the Go state of the cell
cycle (Hall et al., 1983). Another human fibrosarcoma cell line,
SHAC, which also has an activated N -ras gene, in addition it has
overexpression of the c-myc gene, and is immortal (Suarez et al.,
1987). Obviously, immortal or extended life span is a prerequisite
for normal human cells to acquire those further genetic changes
necessary for the transformation of that cell into a tumor cell
(Newbold and Overell, 1983; Landolph, 1985). However, unlike rodent
fibroblasts in culture, which readily give rise spontaneously to
karyotypically abnormal immortal cell population, human fibroblasts
in culture exhibit extremely stable diploid karyotypes (Thompson
and Holliday, 1975) and very rarely give rise to variants possessing
an infinite life span. Therefore, the difficulty in inducing
transformation of human cells in culture may be due in part to the
stringent genetic control of the life span of the normal human cell
(senescence), which limits its proliferative ability.
The second question we should ask is can immortality be
separated from other other transformation phenotypes in human
8
cells under in vitro culture conditions? Is it possible to generate a
cell line with infinite life span but otherwise normal phenotype?
Available information suggests that immortality is not necessarily
linked to the phenotype of malignant transformation, because
malignant but mortal human tumor cells may exist (Stamps et al,
1992). McCormick et al. (1980) reported that MNNG-treated normal
diploid human fibroblastic cells formed foci, and that the progeny
cells from such foci formed small nodules after those cells were
injected into athymic mice, but each of these nodules eventually
regressed. Borek reported in 1980 that tretament of normal human
fibroblasts with ionizing radiation induced focus-formation and the
focus forming cells produced "non-invasive fibrosarcomas" in nude
mice. However, those fibrosarcom a-derived cells eventually
senesced in culture. Milo et al. (1978), used MNNG and Biedermann
and Landolph used MNNG and carcinogenic metal salts (1987) to show
that chemical carcinogen treatment could induce anchorage-
independent growth of normal human fibroblasts, which is one of the
phenotypes of tumor-derived cells. Milo et al. (1978) also showed
that cells isolated from those anchorage-independent colonies
produced nodules when they were injected into nude mice, but, those
nodules could not grow progressively, and cells isolated from those
anchorage-independent colonies or the nodules senesced after being
subcultured in tissue culture dishes. Price et al. (1994) also show
that by controlling the amount of SV40 large T antigen expressed
inside the human fibroblast cells, immortal human fibroblast cells
9
could be obtained which did not possess transformed phenotypes. All
these experimental data suggested that immortalization might be an
event independent of other malignant phenotypes in human cells.
Immortality is known to be independent of these transformed
phenotypes in rodent cells. Immortal but non-tumorigenic human cell
lines have also been established from human bronchial epithelial
cells, human keratinocytes and human fibroblasts (Yang, D et al.,
1992; Klein-Szanto et al., 1992; Yang, J. et al., 1992). These human
cell lines have been used in in vitro chemical transformation assays.
So far, most of the available human cell lines are already
transformed, and exhibit transformed phenotypes when studied with
in vitro assays to detect various transformed phenotypes (Namba et
al., 1988). Currently, the three most commonly used human cell lines
for in vitro chemical transformation assays are the SV40 large T
antigen immortalized human bronchial epithelial cell line, BEAS-2B,
established by Dr. Harris at NCI (Klein-Szanto et al., 1992), the SV40
large T antigen immortalized keratinocyte cell line, RHEK-1,
established by Rhim at NCI (Yang, J. et al., 1992), and the v-myc
oncogene immortalized human fibroblast cell line, MSU-1.1
established by McCormick at Michigan State University (Yang, D et
al., 1992). The BEAS2B cell line, from our own experience and the
published literature, has already gained transformed phenotypes
prior to carcinogen treatment. Most of the chemical transformation
assays with this cell line had to be carried out in animal subjects,
i.e. cells were treated in vitro with chemical carcinogens, and then
10
injected into athymic mice for assaying tumorigenicity. This
tumorigenicity assay is extremely expensive, and more difficult to
manipulate than simple cell cultures. Obviously, it is not the ideal
cell line for in vitro chemical transformation assays. The data on
the v-myc oncogene immortalized human fibroblast cell line, MSU-
1.1, is limited. It was reported that this cell line had a normal
fibroblast morphology, did not grow in soft agar, but it forms foci
and acquires anchorage independent growth after chemical
carcinogen treatment (McCormick, 1994). However, we were unable
to obtain this cell line as the investigator will not send it to other
research groups.
Our goal was therefore to establish a human cell line of our own
which can be used to detect and study the in vitro malignant
transformation of human cells following chemical carcinogen
treatment. Prior work from our laboratory showed that MNNG and
carcinogenic arsenic, nickel, and chronium salts induced anchorage-
independence in diploid human foreskin fibroblasts, but no foci, and
cells strains derived from these anchorage-independent cells
eventually senesced (Biedermann and Landolph, 1987, 1990). We
therefore decided to immortalize the diploid human fibroblasts, and
then to attempt to transform them with carcinogen. We therefore
would like to derive an immortal cell line which still retained an
otherwise normal cellular phenotype. Our view is that immortality
and the other phenotypes possessed by transformed cells are derived
from independent genetic changes. It should, therefore, be possible
11
to establish a human cell line with normal cell phenotypes.
Historically, most transformation assay with mammalian cells,
including human cells, have utilized fibroblasts. Fibroblasts were
among the first cells grown successfully in culture. In retrospect,
this was an accidental finding because fibroblasts show a strong
response to mitogenic proteins found in serum, such as platelet-
derived growth factor, are not as fastidious as epithelial cells in
their cell culture requirements, and are not as sensitive to calcium-
induced terminal differentiation as epithelial cells (Bettger et al,
1981, reviewed in Landolph, 1985). Therefore, when serum was
added to an adequate nutrient medium, fibroblasts were found to
proliferate. It is now clear that many other mammalian cells of
different types can also be induced to grow in culture if specific
mitogenic proteins are added.
Since fibroblasts remained the most common cell type used for
experiment studies because of the ease in culturing them, we chose
to utilize human fibroblasts. Another advantage of fibroblast cells
is that they are found in all organs of the body, although they are not
the dominant cells of any organ. Fibroblasts can become malignant,
producing malignant fibroblastic tumors, termed fibrosarcomas
(Pories et al., 1983). For this reason, we used human fibroblast cells
as the source cell to establish an immortal cell line that can be used
for the general purpose of studying chemical carcinogenesis in vitro.
Such a cell line, if successfully established, could help us identify
12
agents that might cause human cancer, and could help us to consider
preventive procedures against human cancer development. Such a
model cell culture system would also help us to avoid the
extrapolation of data from animal cell experiments to human
carcinogenesis. We could utilize human cell and extrapolate from
human cells to humans for cancer risk assessment. In addition, such
a human model cell system could help us to elucidate the molecular
and cellular mechanisms of human tumor development.
13
1.200 STRATEGIES
It has been reported that many DNA tumor viruses and activated
oncogenes from RNA tumor viruses are able to immortalize
mammalian cells in culture. The immortalizing ability of those DNA
tumor viruses has been localized to specific DNA fragments. These
immortalizing genes include the v-m yc (Land et al., 1983),
adenovirus E1A (Shay et al, 1993b), mutated p53 (Rovinski et al.,
1988), polyoma large T antigen (Cherington, 1986), SV40 large T
antigen (Shay et al., 1989), and papillomavirus E6 genes, among
others (Shay, et al., 1993b). All of these genes code for proteins
that appear to function in the regulation of cell cycle. All the above
genes can immortalize rodent cells, and SV40 large T antigen and v-
myc also immortalize human fibroblasts (Shay, et al., 1989; Yang, D.
et al., 1992).
There are many in vitro assays or morphological examinations
that distinguish tumor cells from their normal counterparts. These
assays are mostly based on the difference in the morphology and
growth properties between tumor and normal cells (reviewed in
Landolph, 1985), and are listed and discussed below.
(1) Morphological Aberrations: The morphology of tumor cells is
often different from that of normal cells. Tumor cells often have an
increased nuclear size, increased nuclear-cytoplasmic ratios,
irregular chromatin distributions, and prominent nucleoli. When
observed through phase-contrast microscopy, the tumor cell is often
14
seen to be rounded, refracts more light, and produces a bright
highlight at the rounded edge. In contrast, the edges of the flattened,
normal cells refract little light. This is particularly true of
transformed rodent and human fibroblast cells (Milo et al., 1988).
(2) Focus Formation and Focus Reconstruction Assays: Cells
explanted from normal tissues, when cultured in vitro, have an
ordered growth pattern, characterized by regular and predictable
relationships with neighboring cells, and form a monolayer of cells.
These cells will then stop dividing upon contact with each other
(Abercrombie and Heaysman, 1954). Within such a confluent cell
monolayer, each cell maintains contact with other cells, and the cell
population as a whole stops dividing; this is the phenomenon of
contact inhibition. Tumor cells have escaped the controls that
normally regulate orderly tissue growth. When grown in culture,
tumor cells display a disordered growth pattern. They grow until
contact is made with neighboring cells, then continue to divide,
forming disordered, multiple layers of transformed cells. Tumor
cells in culture dishes can grow to a higher saturation density than
normal cells. These piles of transformed cells are termed foci. This
loss of contact inhibition forms the basis for chemically induced in
vitro focus formation and focus formation in reconstruction assays
of transformed and normal cells. This change of behavior in vitro
usually serves as a good predictor of in vivo behavior. Cells isolated
from foci in culture usually exhibit the attributes of naturally
arising tumor cells, such as the ability to form invasive malignant
15
tumors (reviewed in Landolph, 1985).
(3) Anchorage Independence: With the exception of cells from
hematopoietic lineages, normal cells grown in culture must have a
solid substrate upon which to grow. If normal cells are prevented
from adhering to a substrate by being suspended in a viscous medium
or a semi-solid medium containing agar, they stop dividing and are
said to exhibit anchorage-dependent growth. Transformed cells in
culture frequently grow when they are suspended in a semisolid
medium, like agar (Mcpherson and Montagnier, 1973). This property
is designated anchorage-independence.
(4) Serum-Oependence of Cell Growth: Normal human cells in
culture typically require fetal bovine serum (FBS) for growth,
because it contains essential growth factors such platelet-derived
growth factor, and attachment factors, such as fibrobronectin
(Morgan et al., 1991). The growth of human cancer cells is usually
much less dependent upon serum factors. Tumor cells frequently can
be grown in reduced concentrations of fetal bovine or even in bovine
serum. When normal cells are placed in medium containing reduced
levels (< 1%) of serum, they become arrested in the Go phase of the
cell growth cycle and stop dividing. Tumor cells often will continue
to grow even under conditions of significantly reduced serum levels
(Morgan et al., 1991).
Our strategy for deriving immortal, non-transformed human
fibroblastic cell lines was to transfect immortalizing genes like the
16
SV40 large T antigen, the polyoma large T antigen, a mutated p53
gene, and a normal c-myc gene under SV40 promoter control into
normal human fibroblasts, we then planned to isolate cell clones
harboring the immortalizing gene by neomycin selection, and to
subculture the transfectants until they passed their senescence
point or underwent sufficient population doublings to become
immortal, and then to select immortal clones with otherwise normal
phenotypes.
We used two assays to demonstrate the immortality of the
transfected cell clones. One is the plating efficiency assay which
measures the cell's ability to attach and form a colony on the dishes
(Landolph et al., 1979, 1985). The plating efficiency of primary
cells normally decreases as the cell clones are being subcultured.
During the senescence stage, the plating efficiency declines to zero
(Biedermann and Landolph, 1987, 1990) . If immortal cells appear,
the plating efficiency should then increase again. Therefore, if we
were able to obtain a significant plating efficiency with the
transfected cells before and significantly after they passed their
normal life span of 50-80 population doublings, it is likely that we
would have derived immortal cells. We would confirm this by
growing the presumptive immortal cells for one year and
periodically determining their plating efficiency.
The other assay used is the population doubling calculation (Shay
et al, 1989). Normal human fibroblasts have a life span of 50-80
17
population doublings. If an isolated clone has undergone significantly
more than 50-80 population doublings, and the cells were still
growing, there is a very high probability that those cells had become
immortalized or at least have extended life span.
After we obtained the immortal cell lines, we planned to
characterize them by using in vitro transformation characterization
assays to determine whether they had acquired transformed
phenotypes. The clone which retained the normal phenotype would be
chosen for further in vitro chemical transformation assays.
18
CHAPTER 2
MATERIALS AND METHODS
2.001 Cells and Cell Culture
Normal human diploid fibroblasts were obtained from circumcised
foreskins from infants at the Huntington Hospital, Pasadena,
California, courtesy of William Callouette, M.D. After we derived
primary cultures in our laboratory (Biedermann and Landolph, 1987,
1990), we stored the cells frozen in liquid nitrogen until use. Cells
were then thawed and cultured in Eagle's minimum essential medium
(MEM) (No. 410-1500, Grand Island Biological Co., Grand Island, NY),
supplemented with 0.1 pM sodium pyruvate, 1 x nonessential amino
acids, 0.2% sodium bicarbonate, 2.5 mM 4-(2-hydrxyethyl)-1-
piperazineethanesulfonic acid buffer, and 10% fetal bovine serum.
This medium was used for cell culturing and all assays. All human
fibroblast cultures used in these studies were routinely checked for
mycoplasma contamination by Hoescht staining and fluorescent
microscopy by the methods of Chen (1977) and were found to be
negative. The MEM medium was supplemented with either 50pg/ml
gentamicin (GIBCO) or 500 units/ml penicillin G and 500pg/ml
streptomycin (Irvine Scientific, CA) only for the initial derivation of
the cell cultures. Noble agar, insulin, and tryptose phosphate broth
were purchased from DIFCO (Detroit, Ml). Hydrocortisone was
purchased from Sigma (St Louis, MO). The medium was supplemented
with 10% fetal bovine serum for growing the cells. The cells were
19
cultured in Corning 60-mm tissue culture dishes or T-75 flasks and
incubated at 37°C in humidified incubators under a constant flow of
5% C02/air. Tissue culture plastic dishes and flasks were purchased
from the Corning Glass Company, Corning, NY. Trypsin was purchased
from GIBCO. Fetal bovine serum was prescreened for the ability to
support acceptable plating efficiency and not to induce
transformation and was purchased from Gemini Bio-Products
(Calabasas, CA).
2.002 Plasmid Constructs
The plasmid constructs used in our study included the following:
(1) PSV3neo, a pBR322 construct which contains the SV40 virus
early region driven by the SV40 promoter and expresses the SV40
large T antigen and a selectable marker coding for neomycin
phosphotransferase which confers the resistance to antibiotic G418,
was purchased from ATCC (Rockville, MD, Southern, et al., 1982); (2)
Zip, a pBR322 vector carrying the G418 resistant gene under SV40
promoter control, was used as a control for pSV3neo, was obtained
from the laboratory of Dr. Dan Broek of USC (Cepko et al, 1984); (3)
P53-143, a plasmid expressing a mutated p53 gene which carries a
single point mutation and causes substitution of alanine for valine
at p53 codon 143 (Baker et al., 1990); (4) pCMV-Neo-Bam, the vector
for P53-143; both plasmids (#3 and #4) were driven by the
cytomegalovirus (CMV) promoter and were obtained from Dr. Bert
Vogelstein of Johns Hopkins University (Baker et al., 1990); (5)
20
pNEOPLTMLV, a plasmid containing the polyoma large T antigen
driven by the murine leukemia virus promoter, and (6) pNEOMLV,
vector pNEOPLTMLV, derived from MLV virus (Kaplan etal., 1987),
were two kind gifts from Dr. Suzanne Simon at the Salk Institute, La
Jolla, California; (7) myc, a construct which encodes exons 2 and 3
of the mouse c-m yc gene was cloned from the Balb/c mouse
myeloma MOPC 315 cell line (Land et al., 1983), and was purchased
from ATCC (Rockville, MD). All of the above plasmids, except myc,
contain the neo-resistant gene, coding for resistance to G418. All of
the above plasmids were transfected into the competent E. Coli
strain HB101 by the method of Sambrook et al (1989a), and the
plasmids were purified by the CsCl2 gradient method. The purity of
the plasmids was determined by electrophoresing the plasmids on
agarose gels and observing the plasmid bands under UV light. The
restriction pattern of each plasmid was also examined to ensure
that the correct plasmid was purified (Sambrook et al, 1989b).
2.003 Antibodies Used in Our Studies
PLT AB-4, a mouse lgG1 monoclonal antibody directed against the
C-terminal of polyoma large T antigen, was purchased from
Oncogene Science (Cambridge, MA). Pab 101, an lgG2 monoclonal
antibody directed against the C-terminal of SV40 large T antigen,
and Pab 122, an lgG2 monoclonal antibody against the C-terminal
residue of the P53 protein, were obtained from Dr. Teddy Fung of the
21
Division of Hematology/Oncology of Childrens Hospital of Los
Angeles.
2.004 Transfection of Normal Human Fibroblasts and
Selection of Transfectants
A method using C a3(P 04)2 precipitation of DNA (Graham et al.,
1973) was adopted to transfect human foreskin fibroblasts with
plasmid DNA. Briefly, 1x10§ cells were seeded into each 60 mm dish
the day before transfection. On the day of transfection, the DNA
precipitate containing 5 pg of plasmid DNA was added into each dish.
Three hours later, 1 ml of 15% glycerol was added to each dish for
two minutes and then aspirated, and the cells were fed with fresh
medium. Forty-eight hours after transfection, the medium was
replaced with medium containing 800 pg/ml G418. The transfected
cells that were able to grow and form colonies in medium containing
G418 were isolated with glass cloning rings, subcultivated into a
new 60 mm dish, and then into T-75 flask, and passaged up to
approximately 75% of confluence until cells passed through the
crisis (equivalent to senescence in normal cells) stage and became
immortal.
2.005 Plating Efficiency Determination Assays
Plating efficiency (PE) determinations were performed by seeding
500 cells per 60 mm dish, five dishes for cells of each passage
number (Landolph et al., 1979; Biedermann and Landolph, 1987,
22
1990). Two weeks after seeding cells, the colonies were fixed with
methanol and stained with Giemsa. Colonies containing more than
twenty cells were counted with a dissecting microscope by standand
methods established in our laboratory (Landolph et al., 1979;
Biedermann et al.,1987, 1990). This assay measures the cell's
ability to attach to and form colonies on the dishes.
2.006 Calculation of the Number of Population
Doublings
To measure the number of population doublings (Shay et al.,
1989), we seeded 1x10§ cells into each T-75 flask each time we
passaged the transfected fibroblasts and cultured the cells for
approximately one week until they reached subconfluence. The cells
were then harvested and counted electronically with a Coulter
Counter model ZF (Coulter Electronics, Hialeah, FL). The number of
population doublings the cells had undergone were calculated
according to the following formulae:
a) (Number of Cells recovered /number of cells seeded x plating
efficiency) = 2 t^o
b) Log2 (Number of Cells recovered /number of cells seeded x
plating efficiency)-t/tQ-# of Population Doublings
23
2.007 Determination Of the Survival Curves of Normal
Human Fibroblasts in G418
Cytotoxicity assays (Landolph and Heidelberger, 1979) were used
to determine the concentrations of the selective agent, G418, that
were sufficient to kill the untransfected normal human fibroblast
cells (HFC) down to approximate mutation frequency for
spontaneously G418 resistant variants (survival fraction of about
10~6 ). To do this, normal human fibroblast cells were seeded at
densities of 1x102 , 5x102 , 1x103 , 5x103 , 1x104 , 5x104 , 1x105 ,
5x105 , 1x106 per 60 mm dish, five dishes per group, and each group
was fed with medium containing 0, 200, 400, 800, 1,200, 1,600,
2,000, 2,400 |ig/ml of G418, respectively, begining one day after
seeding and for a total of two weeks. The cells on the dishes were
then fixed with methanol and stained with Giemsa, and the surviving
colonies containing more than twenty cells were counted with a
dissecting microscope by standand methods established in our
laboratory (Landolph and Heidelberger, 1979; Landolph, 1985;
Biedermann and Landolph,1987 ).
2.008 Biological Characterization of Immortal Human
Fibroblast Cell Lines
The biological characterization of the immortal human fibroblast
cell lines was conducted using the following assays:
24
1) Saturation Density Assays
To determine the saturation density of an immortal cell clone, 1 X
10 5 cells were seeded into 60 Tnm dishes in 5 ml of medium with a
biweekly medium change. Three dishes per clone were trypsinized,
and the numbers of cells were counted electronically on a Coulter
counter model ZF, every three days for six weeks. Cell counts were
reported as the mean ± standard deviation of determinations from
three dishes (Patierno, et al., 1988; Miura et al., 1989).
2) Anchorage Independence Assays
Assays were conducted according to methods previously
developed in our laboratory (Biedermann and Landolph, 1987, 1990)
which are a modification of previous methods (Milo et al., 1978;
Silinskas, et al., 1981), in which the earlier protocols of
MacPherson(1973) have been suitably modified for use in human
cells. Assays for plating efficiency to measure the fraction of
replicatively viable cells were conducted in parallel with assays
measuring induction of anchorage independence. Briefly, 1x105 cells
were suspended in 3 ml of 0.3% agar noble supplemented with
growth medium. Growth medium consisted of 2xH-MEM containing 2x
nonessential amino acids, hydrocortisone (50 pg/ml), insulin (0.15
units/ml), 10% fetal bovine serum, and 10% tryptose phosphate
broth. The mixture containing agar, growth medium, and cells at
370C was allowed to solidify on top of 5 ml of 0.5% agar base layer
containing growth medium in a 60 mm dish, and the dishes were then
25
placed in a 5% CO2 incubator. Cells were fed every six days with 1
ml H-MEM containing 10% fetal calf serum. Four weeks later,
colonies greater than 0.1 mm in diameter were scored by
microscopic examination. At the time cells were seeded into soft
agar for measurement of anchorage independence, an aliquot of the
cell suspension containing 1x10$ cells was also seeded onto each
plastic 60 mm dish, three dishes per group, to determine replicative
viability as measured by ability of cells to attach to plastic dishes
and form actively growing colonies. The surviving cell number per
dish was determined by trypisinizing and counting each dish twenty-
four hours later on a Coulter counter model ZF. The fraction of cells
that attached to the dishes (Seeding Efficiency) was calculated by
dividing the number of surviving cells by the number of cells seeded.
3) Focus Reconstruction Assay
Reconstruction assays were performed to determine whether
cells could form foci, the morphology and frequency of foci formed,
and the stability of the morphologically transformed phenotype
produced by the immortal cell clones under study using methods
derived in our laboratory (Patierno et al., 1988; Miura et al., 1989).
In this assay, 500 cells from each immortal clone were mixed with
1x104 normal fibroblasts. This mixture was then seeded into each of
five 60 mm dishes for each immortal clone. The medium was changed
every five days, and the cells were fixed with methanol, stained
with Giemsa, and counted by microscope three weeks after seeding
2 6
or when monolayers of the normal fibroblasts were confluent and
the foci of the immortal clone were readily apparent by visual and
microscopic inspection.
4 ) Assays to Determine Serum Requirements
for Cell Growth
Normal human cells typically require fetal bovine serum for
growth, while the growth of human cancer cells is usually much less
dependent upon serum factors. When normal cells are placed in
medium containing reduced levels (1%) of serum, they become
arrested in the Go phase of the cell growth cycle and stop dividing.
Tumor cells often will continue to grow under conditions of reduced
serum levels (Morgan et al., 1989). By monitoring the growth status
of cells in medium containing low concentrations of serum, we can
identify whether a clone has been transformed. To perform this
assay, 1 X 10® cells from each immortal clone were seeded into 60
mm dishes in 5 ml of medium containing 0, 1%, and 10% fetal calf
serum respectively. The medium was changed at the same frequency
as in the saturation density assay. Three dishes per clone were
trypsinized, and each dish was counted electronically on a Coulter
counter model ZF every three days for six weeks. Cell counts were
reported as the mean ± standard deviation of determinations from
three dishes (Patierno, et al., 1988; Miura et al., 1989).
27
2.009 Cytotoxicity Assays
After obtaining the immortal human fibroblast cell lines, we next
treated those cell lines with a direct-acting chemical carcinogen,
N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) in an attempt to
transform the immortal cell lines obtained. The cytotoxicity assay
is a test for determining the effectiveness of specific chemical in
killing cells. The cytotoxicity was determined by calculating the
seeding efficiency of each group of cells treated with different
concentrations of MNNG. Briefly, 1x10$ cells from individual
immortal clones were seeded into each 60 mm dish the day before
treatment, three dishes per treatment group. Each group was then
treated with acetone, or 0.125, 0.25, 0.50, 0.75, 1.0 or 2.0 pg/ml of
MNNG dissolved in acetone, respectively. One group of three dishes
without any treatment was included as a control. Acetone was added
to the cells as 25 pg/5 ml of tissue culture medium or 0.5% final
concentration in the medium. The cell number from three dishes per
treatment concentration was determined by counting the cell number
electronically using a Coulter counter twenty four hours after MNNG
treatment. This cell number was used to calculate the cytotoxicity
(relative plating efficiency) after MNNG treatment. The cytotoxicity
was calculated by dividing the surviving cell number in each
treatment group by that of the control (Landolph et al., 1979). This
cell-counting method was used because the plating of the cell was
too low to measure accurately (<10%).
28
2.010 Assays to Detect Chemically Induced
Morphological and Anchorage-Independent
Transformation
We used three assays to determine whether MNNG-treated cells
had acquired specific transformed phenotypes: anchorage
independence assays; focus formation assays; and saturation density
assays. Briefly, 1x10$ cells from individual immortal clones were
seeded into each 60 mm dish the day before treatment, twenty
dishes per treatment group. Each group was then treated with 0.125,
0.25, 0.50, 0.75, 1.0 or 2.0 pg/ml MNNG, respectively for 24 hours.
The control group was treated with acetone at a concentration of
0.25% final volume in the medium which itself caused no
cytotoxicity. The dishes of each treatment group were cultured for
two weeks, and the medium was changed every five days. Then, the
cells from twenty dishes of each treatment group were trypisinized
and counted electronically on a Coulter counter model ZF. 1x105
cells were again seeded into each 60 mm dish, ten dishes for each
treatment group, and the cells were then assayed for their ability to
form foci, to grow in soft agar (anchorage independence), and to
reach elevated saturation densities according to the methods
described previously (Landolph and Heidelberger, 1979; Patierno et
al., 1989; Miura et al., 1989; Biedermann and Landolph, 1987, 1990;
reviewed in Landolph, 1985). The cells to be assayed for focus
formation and anchorage independence were cultured for another
four weeks, and then the cells were either fixed with methanol and
29
stained with Giemsa for detecting foci, or examined for anchorage-
independent colonies with a dissecting microscope. For the
saturation density assay, the cells were cultured for six weeks, and
the cell numbers were determined by trypisinizing the dishes and
counting the cells electronically with a Coulter counter (Landolph et
al., 1979).
2.011 Immunostaining to Detect Specific Nuclear
Proteins (SV40 Large T Antigen)
The entire procedure was performed using an ABC Kit according to
the manufacturer's directions (ABC Kit, Vector Labs, Burlingame,
CA): 1x104 cells were seeded into each well of a Falcon 24-well
Multiwell Tissue Culture Plate (Bacton Dickinson Labware, Oxnard,
CA) to assay the expression of SV40 large T antigen. The cells were
fixed for ten minutes in 5% acetic acid in ethanol at 0 °C , and then
the fixed cells were treated with blocking solution (3% BSA, 1.5%
normal horse serum, 0.2% Triton X-100, 0.02% NaN3). The cells were
then incubated with a 1:100 dilution of the mouse monoclonal anti
SV40 Large T antigen antibody, Pab101, in PBS containing 1% BSA
and 0.02% NaN3 at 4°C overnight. Cells were then washed at room
temperature with PBS. Next, they were incubated with biotinylated
secondary rabbit anti-mouse IgG antibody and the Vectastain ABC
reagent (Vector Labs, Burlingame, CA). The cells were again washed
at room temperature with PBS. Stained microplates were
photographed at 100x using an Olympus IMT-2 inverted microscope
30
and Kodak Ectachrome 400 film. Cells were scored positive for the
presence of SV40 large T antigen if a yellowish-brown color was
observed inside the cells.
2.012 Western Blotting Analysis
To determine the size and relative amount of the SV40 large T
antigen protein in transfected cells, different immortalized clones
were grown on ten 100 mm dishes to subconfluence (approximately
75% of confluence), rinsed twice with ice-cold PBS, and removed
from the dishes by scraping them off with a rubber policeman. The
entire procedure was performed at 4°C. Cells suspended in PBS were
then centrifuged at 2,000xg and resuspended in 2 ml NP-40 lysis
buffer (10 nM Tris, pH 7.4; 10 mM NaCI; 3 mM MgCl2I 0.05% NP-40).
We then centrifuged the suspension at 3,000xg, resuspended the
pellets in 0.3 ml of Laemmli lysis buffer (0.1 M Tris, pH 6.8; 25%
glycerol; 2.0% SDS; 10% 2-mercaptoethanol; 0.01% bromphenol
blues), and centrifuged the resultant nuclear extracts at 430,000xg.
The supernatant, containing the soluble nuclear proteins, was
collected, and 60 nl of each cell extract was loaded onto gels and
analysed by SDS-polyacrylamide gel electrophoresis using a 5-20%
linear gradient. After electrophoresis, the protein was transfered to
a nitrocellulose transfer and immobilization membrane (0.45-jim
pore size, lot no. 2012/3, Schleicher and Schuell, Keene, NH) at 4 °C
overnight at 10 mV in transfer buffer (25 mM Tris-HCL, pH 8.3, 192
mM glycine, and 20% methanol). The blots were then incubated for
31
one hour at room temperature in PBS, with 5% blocking agent, and
then washed with PBS containing 0.05% Tween 20 for five times.
100 ul of Pab 101 was then added, and the mixture incubated for one
hour at room temperature with gentle shockings. After washing
with PBS containing 0.05% Tween-20, the blots were incubated with
peroxidase-linked goat-anti-mouse IgG (Amersham Life Science,
Buckinghamshire, England) for one hour at room temperature. The
membrane was washed with PBS containing 0.05% Tween 20 five
times, and then a detection agent (Amersham Life Science,
Buckinghamshire, England) was added onto the membrane for one
minute. The transfer membranes were then exposed to ECL-
Hyperfilm (Amersham Life Science, Buckinghamshire, England) for at
least twenty-four hours. For each experiment, normal HFC and HFC
transfected with the Zip vector were used as negative controls. The
blots were scanned on a densitometer to measure the relative
quantity of SV40 large T antigen in each nuclear extract. The entire
procedure was performed according to the manufacturer's directions
(Amersham Life Science, Buckinghamshire, England).
2.013 Measurement of Protein Concentrations
The concentration of the total protein in the nuclear extract was
determined by the Bio-Rad Protein Assay Kit according to the
protocol provided by the manufacturer (Bio-Rad Laboratories,
Richmond, CA). Briefly, 200 pi of Dye reagent was mixed with 800pl
of doubly deionized H2 O, and then 2pl of nuclear protein extract was
32
added to the mixture in an Eppendorf tube. The OD at 595 nm was
determined for each sample. The protein concentration in the nuclear
extract sample was calculated according to standard calibration
curves.
33
CHAPTER 3
RESULTS
3.100 Polyoma Large T Antigen
3.101 Introduction
The immortalizing and transforming ability of polyoma virus
was first discovered when cultures of hamster embryo cells
infected with polyoma virus demonstrated characteristics of
transformed cells, such as infinite growth potential in vitro, and the
ability to give rise to progressively growing tumors when they were
inoculated subcutaneously into adult hamsters (Vogt and Dulbecco,
1963). It was then found that the ability of polyoma virus to
transform mammalian cells was due to three distinct proteins
encoded by the early region of its genome, named large T, middle T,
and small T antigens (Cherington et al., 1986). Polyoma large T
antigen is a nuclear protein essential for viral DNA replication and
repression of the transcription of viral "early" gene during viral
infection (Tooze, 1981).
Transfection of rodent embryo fibroblasts in primary cultures
with a plasmid encoding only the large T protein has led to the
establishment of immortal cell lines at a frequency equal to that of
transformation by wild-type polyoma virus (Rassoulzadegan et al.,
1986; Asselin, et al., 1985; Cowie et al., 1986; Jat and Sharp, 1986;
Rassoulzadegan et al., 1983). Immortalization of rodent embryo
34
fibroblasts was also obtained after transfer of recombinant
plasmids encoding only the amino-terminal 40% of the polyoma large
T antigen, suggesting that this "immortalization" function
corresponds to the activity of an amino-terminal domain of the
protein (Rassoulzadegan et al., 1983). The amino terminal domain
was later found to bind a growth-suppressing protein, the
retinoblastoma gene product, p105-Rb (Dyson et al., 1990). This
suggested that the ability of polyoma large T antigen to immortalize
rodent cells was probably due to inactivation of this growth-
suppressing Rb protein by the polyoma large T antigen. The polyoma
large T antigen-immortalized rodent fibroblasts exhibited an
otherwise normal phenotype, i.e., they grew to a low saturation
density and were anchorage-dependent for cell growth. Polyoma
large T antigen was shown to act in concert with other oncogenes
such as polyoma middle T antigen, mutated H-ras, to transform
primary cells, and the transformed cells were able to induce tumors
upon inoculation into animal hosts (Asselin et al., 1983, 1986; Land
et al., 1983; Rassoulzadegan et al., 1982).
Polyoma middle T antigen is a cytoplasmic-membrane bound
protein which has tyrosine protein kinase activity (Courtneidge and
Smith, 1983). It can transform immortalized cell lines to form foci
in monolayer cultures, to form colonies in soft agar, and to become
tumorigenic (Treisman et al., 1981). Unlike large T antigen and
middle T antigen, polyoma small T antigen has no known intrinsic or
associated biochemical activity, except that it increases the
35
saturation density of established rodent cell lines (Liang et al.,
1984).
Since polyoma large T antigen confers immortality upon rodent
cells without changing their cellular morphology, it has been used on
many occasions to establish rodent cell lines for the purpose of
studying the multistep process of neoplastic transformation in vitro
(Asselin et al., 1986; Rassoulzadegan et al., 1982). However, its
effects on human cells are not well established. It is important to
determine whether this rodent "immortalizing agent" can also
immortalize human fibroblasts without changing their cellular
morphology.
3.102 Survival Curve Determination to Define
Concentrations of G418 to Be Used in
Transfection Assays
Survival curves were first constructed to detect the growth of
normal diploid human fibroblasts in G418> the selecting drug. The
survival fraction for normal human diploid fibroblasts in MEM
containing 200 ng/ml G418 was less than 1.5 x 1 0 '6 , which
indicates that 200 n.g/ml of G418 killed all the nontransfected cells
seeded in the dish down to spontaneous mutation frequencies. No
spontaneous G418 resistant mutants were observed when
transfected fibroblasts were treated with from 200-2500 ng/ml of
G418. The concentration of G418 we chose for selecting transfected
HFC clones was 800 pg/ml, which ensured that all the wild-type
36
cells died and only the transfectant cells survived. To prove this, we
showed that a cell line derived from one of the transfected clones,
from our first experiment, HFC/SV1, grew in MEM containing up to
2400 |ig/ml G418, indicating that it was a true transfectant (Figure
1), harboring the G418 resistant gene, and that this selection
protocol worked well.
3.103 Transfection of Normal HFC and Selection of
Transfectants
We next transfected a plasmid containing polyoma large T antigen
and G418 resistance into cultured normal human diploid fibroblasts
and studied whether the transformed properties arose. Five 60 mm
dishes were seeded with normal human diploid fibroblasts at a
density of 1x10$ cells/dish and then transfected by the calcium
phosphate method (Graham et al., 1973) with either the pNEOPLTMLV
plasmid expressing polyoma large T antigen and a neomycin
resistant marker or the vector pNEOMLV containing only the
neomycin resistant marker. Both plasmids carry a selectable marker
coding for neomycin phosphotransferase, which confers the
resistance to antibiotic G418. Dishes were fed with MEM medium
containing G418 (800 pg/ml) every week for two to three weeks
until drug-resistant colonies were visible. A second control group
was treated in the same manner as the two transfection groups,
except that this control group was not transfected with plasmid
DNA. The efficiency of transfection in each experiment was
37
c
o
u
CD
' 10- 3 >
>
k
a
C O 10
10
10
- 4
- 5
- 6
HFC
HFC/SV1
=ZSL
5 0 0 1000 1500 2000 2500
G418 Concentration (ug/ml)
Figurel Survival Curve for Normal HFC Cells and a
Neo-SV40 Large T antigen Transfected Clone
(HFC/SV1)
The survival fraction of normal human fibroblast cells (HFC) in
medium containing 200 pg/ml G418 is less than 1.5x1 O'6 ,
while a transfectant clone (HFC/SV1) grew in medium
containing G418 up to 2400 pg/ml.
38
Table 1
Summary of Transfection Results with Polyoma Large T Antigen
Group # of Cells
Transfected
Per Dish
Total #
of G418
Colonies
Observed
Total #
of
Colonies
Cloned
# of G418
Resistant
Colonies Per
Dish
Total # of
Colonies
Sub
cultured
Immortal
Cells
pNEOPLT
MLV
1 x 105
(5 dishes)
1 9 1 2 4 ± 2 6 0
pNEOMLV 1 x 105
(5 dishes)
16 6 3±1 3 0
Control 1 x 105
(5 dishes)
0 0 0 0 0
39
determined by transfecting the cells as above, then fixing them with
methanol, and then staining them with Giemsa three weeks after
transfection. Colonies containing more than twenty cells were
counted as G418 resistant transfectants.
Individual transfectant colonies that arose were cloned with a
glass cloning ring (Nianjun et al., 1994) and expanded into cell
strains. Nineteen G418-resistant colonies were obtained from five
dishes of pNEOPLTMLV-transfected human fibroblast cells, and
sixteen G418-resistant colonies were obtained from five dishes of
pNEOMLV transfected cells, suggesting that we obtained similar
transfection efficiencies with both plasmids (Table 1). No surviving
G418-resistant colonies were observed in the control group. This
suggested that the G418-resistant colonies contained the plasmids
they were transfected with (Table 1).
Tw elve G 418-resistant colonies were cloned from the
pNEOPLTMLV-transfected group, and six colonies were cloned from
the pNEOMLV-transfected group. The resultant cell strains were
designated PLT Clone 1 through 12 for transfectants harboring the
gene encoding polyoma large T antigen, and MLV 1 through 6 for
transfectants containing only the control vector. All clones
multiplied to large enough cell numbers to be passaged.
40
3.104 Western Blotting Analysis of Polyoma Large T
Antigen
The relative quantity and size of polyoma large T antigen from
nuclear extracts of each polyoma large T antigen transfected HFC
clone were determined at the precrisis stage by Western blotting
techniques described in the Materials and Methods section. As shown
in Figure 2, a band with molecular weight of 97 Kd (Rassoulzadegan
et al., 1983) was detected in the nuclear extracts of polyoma large T
antigen transfected HFC clones but not from those extracted from
MLV transfected and normal HFC cells, indicating that polyoma large
T antigen was expressed in those transfected HFC clones.
3.105 Subcultivation of Polyoma Large T Antigen
Transfected HFC Clones
Six clones containing polyoma large T antigen and three control
clones containing pNEOMLV were subcultured in medium containing
400 |ig/ml G418 until all of them entered crisis. The plating
efficiencies and number of population doublings were measured each
time the clones were passaged, according to the procedures
described in the Methods and Materials section (Figures 3 and 4). We
observed that the transfected clones from both groups initially had a
high plating efficiency (5-15%), which then decreased and eventually
reached zero at passages 6-8 when the transfected clones from both
groups entered crisis. Although the polyoma large T antigen
transfected clones underwent slightly more passages (1-2) than the
41
1
WESTERN BLOT ANALYSIS OF POLYOMA LARGE T ANTIGEN
1 - C M C O ^ in C D
UJ H I L U L L I LU L L J
z z z z z i
O O O O O Q
- J - J »J —J ^ 7^
O O O O O O ^ £3
h h h h h b —i — J i l l
,CL 0 . 0 . 0— CL CL 2 2 5 T
^ N l l M
97 K D
50 K D
Figure 2
Western Blot Analysis of Polyoma Large T Antigen
A band with molecular weight of about 97 Kd which is similar to the
molecular weight of polyoma large T antigen (Rassoulzadegan et al.,
1983) was detected from the nuclear extracts of polyoma large T
antigen transfected HFC clones but not from those extracted from
normal HFC cells and HFC cells transfected with a plasmid vector,
MLV.
42
0 2 4 6 8
Passage Number
Figure 3
Plating Efficiency of Polyoma Large T Antigen
Transfected HFC Clones
The plating efficiency of precrisis transfected ceils decreased
as they were being subcultured and reached zero when they
were in crisis. The plating efficiency never increased again,
indicating that no immortal cells had appeared.
43
50 n
40 -
o >
c
A
3
O
Q
30-
e
o
2 0 -
3
a
o
a
10 -
100 200 0 300
plt1
plt2
plt3
plt4
plt5
plt6
mlvl
mlv2
mlv3
Days after Transfection
Figure 4
Population Doubling Curve of Polyoma Large T Antigen
Transfected HFC Clones
After transfection, the polyoma large T antigen transfected cells
grew for about 40±6 population doublings and MLV transfected cells
underwent 36±3 population doublings. The cultures then ceased to
proliferate and entered crisis. No rapidly growing cells appeared
after they had been in crisis for additional six months.
44
MLV-transfected clones, no fast-growing cells were observed after
the cultures were in the crisis stage for six months. Population
doubling curves (Figure 4) indicated that after transfection, the
MLV-transfected HFC clones underwent 30-40 (36±3) population
doublings, and the polyoma large T antigen transfected clones grew
for 40±6 population doublings. The cultures then ceased to
proliferate and entered crisis. No rapidly growing cells appeared
after the cultures had been maintained in the crisis stage for an
additional six months. Therefore, the polyoma large T antigen
conferred only a slightly increased life span, an extra four
population doublings, to diploid human fibroblast cells. However,
this increase was not statistically significant (P>0.05).
3.106 Biological Characterization of Polyoma Large T
Antigen Transfected HFC Clones
All six clones transfected with the polyoma large T antigen, and
three MLV clones, were examined for their morphology, their ability
to grow in soft agar, their ability to form foci against normal human
fibroblasts in reconstruction experiments, and their saturation
density. These properties were compared to those of HT1080 and
normal HFC cells according to the methods described in Methods and
Materials. The data are summarized in Table 2 and Figures 5 and 6.
We found firstly that all the polyoma large T antigen-transfected
HFC clones had a normal flat morphology. Secondly, none of the
polyoma large T antigen-transfected HFC clones formed foci against
45
Table 2
Summary of Biological Characterization of Polyoma Large T
Antigen Transfected Human Fibrobalst Cells_________
Clones Morphology Focus Reconstruction
against normal HFC
Anchorage Independent
Growth
HFC flat 0 311
PLTCI1 flat 0 0
PLTCI2 flat 0 0
PLT Cl 3 flat 0 0
PLTCI4 flat 0 0
PLT Cl 5 flat 0 0
PLT Cl 6 flat 0 0
MLV1 flat 0 0
MLV2 flat 0 0
MLV3 flat 0 0
H T 1080 piled-up 3 5 1 4 31251413
Focus reconstruction assay was counted as "foci/dish/200 cells seeded”. Anchorage
Independence was counted as "Al colonies/105 survivors.
46
HT1080
«
o
E
E
o
< 0
Q .
o
O
*
HFC AND
PLT 3
HT1080
Days after Seeding
Figure 5
Saturation Density Curve of Polyoma Large T Antigen
Transfected HFC Clones
1x1 ()5 cells were seeded per 60 mm dish, and the cell numbers
in three separate dishes were determined every three days for
24 days. HT1080 is a human fibrosarcoma derived cell line and
was used as a positve control. HFC are normal human fibroblast
cells.
47
> »
Q
c
o
£
u i
o
U L
«
3
o
o
1:PLTCai
2: PLT CI2
3: PLT 0 3
4: PLT 04
5: PLT CIS
6: PLT a 6
7: MLV 1
8: MLV 2
9: MLV 3
Figure 6
Focus Forming Efficiency of HT1080 against Polyoma Large
T Antigen and MLV Transfected HFC Ceils
500 HT 1080 cells were mixed with 5x104 cells from polyoma large
T antigen and MLV transfected HFC cells, and the mixture was then
seeded into each of five 60 mm dishes. Three weeks later, the dishes
were fixed and stained and examined for foci under a dissecting
microscope.
48
normal HFC cells in reconstruction assays for focus formation.
Thirdly, none of the polyoma large T antigen-transfected HFC clones
formed anchorage-independent colonies in soft agar. Fourthly, all the
polyoma large T antigen transfected HFC clones grew to lower
saturation densities than the tumorigenic HT1080 human
fibrosarcoma cell line and even to lower saturation densities than
normal human fibroblast cells (Figure 4). Finally, The human
fibrosarcoma cell line, HT1080, formed foci against each polyoma
large T antigen transfected clone (Figure 5).
Hence, by all these criteria, these transfectants are not
transformed.
3.107 Conclusions:
The conclusions of these studies are firstly that polyoma large T
antigen did not significantly change the phenotype of normal human
fibroblasts. Secondly, most polyoma large T antigen transfected
clones had undergone 40 population doublings and multiplied to a
population of about 1012 cells before they entered crisis. Since no
immortal cells appeared, we concluded that either polyoma large T
antigen could not immortalize human fibroblasts, or the
immortalization frequency of human fibroblasts by polyoma large T
antigen was less than 10~1 2 . Finally, we concluded that polyoma
large T antigen slightly extended the life span of normal human
fibroblasts, by about four population doublings, although this
extension was not statistically significant.
49
3.200 Transfection of the Mutated p53 Gene into
Normal Diploid Human Fibroblasts
3.201 Introduction
The P53 protein was discovered initially through its association
with the SV40 large T antigen in SV40 virus transformed cells (Lane
and Crawford, 1979) and as an overexpressed protein in chemically
transformed sarcoma cells (Deleo et al., 1979). It was regarded as a
dominantly acting oncogene at that time because of its nuclear
localization and overexpression in transformed cells (McCormick and
Harlow, 1980; Linzer and Levine, 1979; Eliyahu et al., 1985; Tuck et
al., 1989; Parada et al., 1984). It soon became clear that many of the
cloned p53 genes that had been first studied were actually mutated
versions of the normal p53 gene. The biological function of the wild-
type p53 gene is now known to suppress or inihibit the
transformation of cells in culture (Finlay et al., 1989; Eliyahu et al.,
1989, Merlo, 1994). The normal form of p53 within the cell
assembles into homotetramers and higher order homo-oligomeric
structures (Longnecker et al., 1988). Therefore, the p53 protein acts
in a dominant-negative fashion. Defective subunits of such an
oligomerizing protein (e.g. a mutant p53 molecule) participate in
forming a multi-subunit complex together with wild-type monomers
and, therefore, destroy the function of the complex as a whole
(Block, 1991).
50
An immortalizing potential of the mutated p53 gene was
demonstrated by its ability to extend the life span of rat
chondrocytes (Jenkins et al., 1984; Jenkins et al., 1985) and its
ability to immortalize primary rat embryo fibroblast cells (Eliyahu
et al., 1984; Hinds et al., 1987; Parada et al., 1984; Rovinski et al.,
1988). However, the effect of mutated p53 on the growth of normal
human cells has so far not been reported. It is therefore important
to determine whether this rodent "immortalizing gene" can also
immortalize human fibroblasts. In addition, it is important to know
whether a mutated p53 gene can immortalize human fibroblasts
without inducing morphological transformation or anchorage
independence. Our strategy was therefore to transfect a mutated
human p53 gene into normal diploid human fibroblast cells. Our goal
was to disturb the function of the wild-type p53 inside human
fibroblast cells and, therefore, possibly to immortalize them.
3.202 Transfection of Normal HFC with a Mutated p53
Gene and Selection of Transfectants
Five 60 mm dishes of normal human diploid fibroblasts at a
density of 1x10$ each were transfected by the calcium phosphate
method (Graham et al., 1973) with a P53-143 plasmid expressing a
mutated p53 gene and a neomycin resistant marker. This p53 gene
carries a single point mutation that results in substitution of a
alanine for valine at codon 143 of the p53 gene (Baker et al., 1990).
We also transfected as a control the vector pCMV-Neo-Bam, which is
51
a retroviral vector under the control of the cytomegalovirus
promoter. Both plasmids carry a selectable marker coding for
neomycin phosphotransferase, which confers resistance to the
antibiotic G418 (Sourhern and Berg, 1982). Dishes were fed with
MEM medium containing 800 pg/ml of G418 every week for two to
three weeks until drug-resistant colonies were visible. The control
group was treated in the same manner as the two transfection
groups except that the control group was not transfected with
plasmid DNA. Individual transfectant clonies were selected with a
glass cloning ring and expanded into cell strains (Nianjun et al.,
1994). The efficiency of transfection was determined by fixing the
G418-resistant colonies with methanol and staining them with
Giemsa three weeks after transfection. Colonies containing more
than twenty cells were counted under a dissecting microscope.
In total, twenty-four G418-resistant colonies were obtained
from five dishes of P53-143 transfected HFC cells, and eleven
G 418-resistant colonies were obtained from five dishes of PCMV-
neo-Bam transfected cells (Table 3). The transfection efficiency
was 4.8±1.6 colonies/dish for the p53-143 transfected cells and
2.2±0.8 colonies/dish for the PCMV transfected cells (Table 3). This
suggested that either the P53-143 vector had a higher transfection
frequency, or that it stimulated the growth of the transfectants so
that more colonies appeared in this group. No surviving colonies
were observed in the control group. This suggested that the G418-
resistant colonies contained the plasmids with which they were
52
Table 3
Summary of Transfection Results with a Mutated P53 Gene and a Control Vector
Group # of Cells
Transfected
Per Dish
Total #
of G418
Colonies
Obseived
Total #
of
Colonies
Cloned
# of G418
Resistant
Colonies Per
Dish
Total # of
Colonies
Subcultur
ed
Immortal
Cells
P 5 3 -1 43 1 x 105
(5 dishes)
24 1 2 4.8± 1.6 6 0
PCMV-neo-
Bam
1 x 10s
(5 dishes)
11 6 2.2±0.8 3 0
Control 1 x 105
(5 dishes)
0 0 0 0 0
53
0 2 4 6 8 10
Passage Number
Figure 7
Plating Efficiency of Mutated p53 and PCMV
transfected HFC Clones
The plating efficiency of precrisis transfected cells decreased
as they were being subcultured and reached zero when they
were in crisis. The plating efficiency never increased again,
indicating that no immortal cells had appeared.
54
60 -i
CM
Cl 2
50-
CI3
o
c
Cl 4
40-
- O
3
0
a
CIS
30-
CI6 c
o
«
3
a
o
a
PCMV1
2 0 -
PCMV2
PCMV3
IQ -
100 200 300 0
Days after Transfection
Figure 8
Population Doubling Curve of Mutated p53 and PCMV
Transfected HFC Clones
After transfection, the mutated p53 gene transfected cells grew for
about 48±8 population doublings and MLV transfected HFC clones
underwent 33±3 population doublings. The cultures then ceased to
proliferate and entered crisis. No rapidly growing cells appeared
after the cultures had been in crisis for an additional six months.
55
transfected (Table 3). Twelve G418-resistant colonies were cloned
from the P53-143 transfected group, and six colonies were cloned
from the PCMV-neo-Bam group. They were designated P53 Clone 1
through 12 for transfectants harboring the mutated p53 gene, and
PCMV 1 through 6 for transfectants containing the control vector.
All of them multiplied to large enough cell numbers to be passaged.
Six clones transfected with p53 and three PCMV-transfected
control clones were passaged in medium containing 400 pg/ml G418
until eventually all of them became senescent. The plating
efficiency and number of population doublings were determined each
time the clones were passaged according to the methods described
in the Methods and Materials (Figures 7 and 8). As shown in Figure 7,
the transfected clones from both groups first had a high plating
efficiency (10-30%) which decreased and eventually reached zero
when transfected clones from both groups entered crisis. Clones
arising from cells transfected with the mutated p53 gene did
undergo fourteen more population doublings than PCMV transfected
clones. However, no fast growing colonies were observed after the
cells transfected with the mutated p53 gene were in the crisis
stage for six more months post-transfection. The population
doubling curve (Figure 8) showed that after transfection, the PCMV
transfected HFC clones had undergone 33±3 population doublings, and
the mutated p53 gene transfected clones grew for 48±8 population
doublings. The cultures then ceased to proliferate and entered crisis.
The population doubling cun/e (Figure 8) showed that the mutated
56
P53 gene extended the life span of human fibroblast cells by
fourteen population doublings (P<0.05).
3.203 Biological Characterization of Mutated p53 Gene
Transfected HFC Clones
All six clones transfected with the mutated p53 gene and three
PCMV transfectants, were examined for their morphology, their
ability to grow in soft agar, and their ability to form foci against
normal human fibroblasts according to the procedures described in
the Methods and Materials section.
Firstly, we found that all the mutated p53 gene and PCMV
transfected HFC clones had a normal flat morphology (Table 4), and
Secondly, none of the mutated p53 gene and PCMV transfected HFC
clones formed foci against normal HFC cells (Table 4). Thirdly, none
of the mutated p53 gene and PCMV transfected HFC clones formed
anchorage independent colonies. Fourthly, the human fibrosarcoma
cell line, HT1080, formed foci against each mutated p53 and PCMV
transfected clones (Figure 9). Finally, the mutated p53 gene
extended the life span of normal human fibroblast cells by about
fourteen population doublings (Figure 8).
3.304 Conclusions
We therefore concluded that the mutated p53 gene did not change
the phenotype of normal human fibroblasts in regard to standard
transformation parameters.
57
Table 4
Summary of Biological Characterization of Mutated p53 Gene
Transfected Human Fibrobalst Cel s
Clones Morphology Focus Reconstruction
against normal HFC
Anchorage Independent
Growth
HFC flat 0 2.510.8
mP53 Cl 1 flat 0 0
mP53 Cl 2 flat 0 0
mP53 Cl 3 flat 0 0
mP53 Cl 4 flat 0 0
mP53 Cl 5 flat 0 0
mPS3 Cl 6 flat 0 0
PCMV1 flat 0 0
PCMV2 flat 0 0
PCMV3 flat 0 0
HT1080 piling-up 35 14 31251413
Focus reconstruction assay was counted as "foci/dish/200 cells seeded”. Anchorage
Independence was counted as "Al colonies/105 survivors.
58
40 -i
>.
u
e
•
o
3 =
U l
3
O
o
Ik
1: mp53 CI1
2: mpS3 CI2
3: mp53 CI3
4: mp53 CI4
5: mp53 CI5
6: mp53 CIS
7: pCMV 1
8: pCMV 2
9: pCMV 3
Figure 9
Focus Forming Efficiency of HT1080 against HFC Cells
Transfected with the mutated p53 gene or PCMV
500 HT 1080 cells were mixed with 5x104 cells from mutated p53
gene and PCMV transfected HFC cells, and the mixture was then
seeded into each of five 60 mm dishes. Three weeks later, the dishes
were fixed and stained and examined for foci.
59
Further, most clones transfected with the mutated p53 gene had
undergone about 50 population doublings and multiplied to a
population of about 1015 cells before they entered crisis. The
mutated p53 gene did extend the life span of normal human
fibroblasts by fourteen population doublings. However, no immortal
cells appeared. Therefore, either the mutated p53 gene could not
immortalize human fibroblasts on its own, or the immortalization
frequency of human fibroblasts following transfection by the
mutated p53 gene was less than 10*17. It may be that one copy of a
mutated p53 gene is eventually degraded, and that when it is present
together with two copies of the wild-type p53 gene, this can only
transiently extend life span, whereas an endogenously mutated p53
gene and only one wild-type could confer immortality.
60
3.300 SV40 Large T Antigen
3.301 Introduction
The DNA tumor virus, SV40, has been reported to immortalize
human fibroblast cells, although at a very low frequency (Ide et al.,
1984; Lomax et al., 1978; Stein, 1985). Infection of human
fibroblasts with SV40 virus frequently results in an extension of
their life span by about 20 population doublings. The cells then enter
what has been termed a crisis stage (Girardi et al., 1965). Crisis in
SV40-infected human cells represents a period in which cell growth
and cell death are initially balanced, and the number of cells in a
cell culture as a whole stops increasing. This is followed by a period
in which the number of surviving cells progressively declines. At
low frequencies, immortal cells then appear, and these can then be
subcultivated to give rise to established human cell lines (Shay et
al., 1989).
The immortalizing function of SV40 virus has been localized to
the SV40 large T antigen. Human cells transfected with a plasmid
that expresses only the SV40 early region immortalize at a much
higher frequency than cells transfected with the intact SV40 virus
(Mayne et al., 1986; Chang et al., 1986). The SV40 large T antigen is
a 94 Kd protein that has a wide variety of functions, including the
ability to induce the synthesis or activation of enzymes involved in
DNA metabolism, the stimulation of RNA synthesis, and the induction
of DNA synthesis (Stahl and Knippers, 1987). The mechanism by
61
which SV40 large T antigen immortalizes human cells, however, is
not yet understood. Since polyoma large T antigen and a mutated p53
gene did not immortalize diploid human fibroblasts, we next tried
transfecting SV40 large T antigen, which is believed to be a better
immortalizing agent in rodent cells, into human diploid fibroblasts.
3.302 Tansfection of Normal HFC, Selection of
Transfectants, and Subcultivation of
Transfectant Human Fibroblast Clones
Normal human diploid fibroblasts were next transfected by the
calcium phosphate precipitation method (Grahm et al., 1973) with
the pSV3neo plasmid, which encodes the SV40 T antigen and a
selectable marker encoding neomycin phosphotransferase, which
confers the resistance to antibiotic G418.
Three transfection experiments were conducted in which the
pSV3neo plasmids were transfected into human diploid foreskin
fibroblasts at different passage numbers, and neomycin-resistant
transfectant clones were selected (Table 5). The transfected cells
were selected by culturing them in MEM containing 800 ng/ml G4 1 8 ,
which kills wild type human fibroblasts down to a surviving fraction
of less than 1.5 x 10‘6 (Figure 1). All three experiments yielded
G 4 I 8 resistant transfectant colonies, indicating that the
transfection procedure was reproducible (Table 5). Individual
healthy-looking transfected colonies were cloned with glass cloning
ring and expanded (Nianjun et al., 1994). The working model for
62
immortalizing normal human fibroblasts using SV40 large T antigen
is that SV40 large T antigen only extends the life span of normal
cells by 20 population doublings. A second rare genetic event must
then occur in order for SV40 large T expressing cells to become
immortalized (Shay et al., 1989). W e maintained a minimum
population size of at least 1x10& cells when we passaged the
individual clones. This involved expanding the cells so that at least
twenty T-75 flasks (20x75 cm2 ) of cells were available for each
clone when they were being subcultured and when they entered
crisis. We then maintained the cells for at least an additional six
months after the clones went into crisis. During the subcultivation
of precrisis human fibroblasts, because of the limitations of the
availability of incubator space, a portion of the precrisis cells were
often frozen down and later thawed to continue passage. Not all
transfected clones multiplied significantly before or following their
initial cloning. These poorly growing clones most likely represented
unhealthy, degenerating, and senescent clones and were thus
discarded.
From the first SV40 large T antigen transfection experiment, five
drug-resistant colonies were obtained, and they were all cloned by
the glass cylinder cloning ring method (Nianjun et al., 1994). These
five clones were designated HFC/SV1 through HFC/SV5. Only two of
these five, namely HFC/SVI and HFC/SV4, grew sufficiently well to
support subsequent passaging and subculturing in medium containing
G418- The survival curve for HFC/SV1 (Figure 1) shows that
63
Table 5
Transfection of SV40 Large T Antigen Encoding Gene
Experiment
#
# of cells
Transfected
Per Dish
Total #
of G418
Colonies
Observed
Total #
of
Colonies
Cloned
Total # of
Colonies
Subcultured
Total # of
Immortal
Cell Lines
1 1 x 105
(5 dishes)
5 5 2
HFC/SV1
HFC/SV4
2
2 1 x 105
(5 dishes)
3 3 1
HFC/SV6
0
3 1 x 105
(5 dishes)
5 3 3
HFC/SV9
HFC/SV10
HFC/SV11
1 3
200 - |
H FC /S V 10-2
HFC/SV0-1
HFC/3V1-1
HFC/SV10-3
HFC/SV10-4
HFC/SV1-2
HFC/3V4
H FC /S V S
H FC /S V 11
0 H 1 ------------1 ------------1 ------------ 1 ------------ 1 ------------1 ------------ 1
0 200 400 600 800
DAYS AFTER TRANSFECTION
Figure 10 Population Doubling Curve of SV40 Large T
Antigen Transfected HFC Clones
After transfection and selection in G418, and cloning, the
transfected cells grew for about 40-100 population doublings.
The cultures then ceased to proliferate and entered crisis.
After about 2-3 months, rapidly growing cells began to appear,
and the cells were routinely subcultured.
65
£
>
o
c
•
U l
O )
e
0
a
40
HFC/SVn
HFC/Sw)
' HFC/SV6
30
HFC/SV10
20
HFC/SV9
10
o
30 o 10 20 40 50
Passage Number
Figure 11 Plating Efficiency of Passaged SV40 Large T
Antigen Transfected HFC Clones
The plating efficiency of precrisis transfected cells decreased
as they were being subcultured and reached zero when they
were in crisis. The plating efficiency increased again after
immortal postcrisis cells appeared.
66
HFC/SV1 was able to grow in medium containing up to 2,400 pg/ml
of G418. This indicates that it is likely a transfectant, rather than a
spontaneous mutant, because spontaneous neomycin-resistant
mutants were not observed in our assays (frequency > 10'®, Figure
I). These two clones (HFC/SV1 and HFC/SV4) were cultured in MEM
with 800 pg/ml G 418- They grew well until they reached passage
number seven and population doublings of forty-five and forty-three,
respectively, then stopped dividing and entered a crisis stage
(FigurelO and 11). During this period, the total cell number
decreased, and cells from different flasks of the same clone were
pooled. The plating efficiency of these two cell strains derived from
the transfection experiments was zero by passage number 9 (Figure
I I ).
During crisis, focal clonal growth in a few areas of the dishes
could be seen occasionally against the background of dying cells.
One surviving colony that could be seen under the microscope after
about two months postcrisis stage was isolated, designated
HFC/SV1-1, and grew much faster than the pre-crisis HFC/SV1
cells. It was expanded, and a portion of the cells were kept for
subcultivating and for assaying for immortality determination, for
biological characterization, and for chemical transformation
experiments. The rest of the cells were frozen down for future use.
To date, HFC/SV1-1 has been passaged over fifty times in two years
after emerging from the crisis period, and it is still actively
growing. Another clone, HFC/SV1-2 emerged after having been in
67
crisis stage for a six month period (Figure 10). This clone has also
been passaged over forty times post-crisis and assayed for cellular
immortality, biologically characterized, and tested for its
suitability to use for chemical transformation assay. Both these
clones have been found to possess transformed phenotypes when
assayed by in vitro transformation characterization assays (see
below). Other fast growing colonies were also found, but because of
their similarity in morphology to the first two clones, we did not
characterize those colonies. No fast-growing colonies have been
seen from HFC/SV4 cells after one year period in crisis. This
precrisis clone was finally not passaged further. Plating efficiency
assays and population doubling calculations were conducted each
time the clones were passaged (Figures 10 and 11).
The second transfection experiment yielded three drug-resistant
colonies which were also isolated and named HFC/SV6 through
HFC/SV8. Only one clone, HFC/SV6, proliferated sufficiently to be
passaged. This clone entered crisis at about thirty population
doublings (passage number six), and no rapidly growing colonies have
emerged from this cell preparation after one year period in culture
(Table 5; Figures 10 and 11).
Five transfectant colonies were obtained in a third experiment
(Table 5). Three of these colonies grew fast enough to be cloned and
subcultivated and were designated HFC/SV9, HFC/SV10, and
HFC/SV11. HFC/SV11 entered crisis after forty population
68
doublings. The other two colonies multiplied for 90-100 population
doublings before they entered the crisis stage. One fast growing
clone emerged from the HFC/SV10 cells 60 days after being in
crisis. This clone was named HFC/SV10-1. Unfortunately it was
lost due to an incubator accident after being passaged for another
three times, and no cells from this clone survived. Additional fast-
growing colonies were ocasionally seen. One colony usually emerged
from three to four flasks of pooled precrisis cells, which contained
about 2x10? cells. Therefore, the immortalization frequency is
approximately 1-2/10? precrisis cells. From our experience with
the first two clones obtained from HFC/SV1, that cells from most of
the postcrisis colonies were transformed, we picked the colonies
with less transformed phenotype (eg. less piling-up, less retractile
colonies under microscopic examination). Another four clones with
less transformed phenotypes were picked at about 150, 250, 300,
and 400 days after the precrisis clone (HFC/SV10) went into crisis
respectively. These four clones were named HFC/SV10-2 through
HFC/SV10-5. They were also tested for immortality, biologically
characterized, and tested for suitability for in vitro chemical
transformation assays. Plating efficiency assays and population
doubling calculations were conducted each time the clones were
passaged (Figures 10 and 11).
The precrisis HFC/SV9 had the least transformed phenotypes
of all the precrisis transfected HFC cell clones (see below), and it
entered the crisis stage at population doubling of about 90. The first
69
postcrisis fast growing clone appeared about two months after
having been in crisis. Colonies derived from this HFC/SV9 showed a
flatter morphology and less transformed phenotype on in vitro
transformation characterization assays (see below). Nine post
crisis clones were picked from HFC/SV9 and assayed for
immortality, biological characterization, and suitability for in vitro
chemical transformation assays.
3.303 Detection of Immortal Human Fibroblast Clones
We used two methods to document the emergence of immortal
human fibroblast cells. One was the population doubling calculation.
The rationale was that normal human fibroblast cells usually
undergo 50 to 80 population doublings. Transfection of the SV40
large T antigen encoding gene can confer another 20 population
doublings. If a clone has undergone more than 100 population
doublings and is still growing, it is probably immortalized.
Interestingly, the results we obtained (shown in Figure 10) suggest
that SV40 large T antigen extended the life span of normal
fibroblasts but could not immortalize normal human fibroblasts on
its own, because all the SV40 large T antigen transfectants entered
crisis first. Presumably, some additional mutational events needed
to occur after SV40 large T antigen transfection, which enabled the
transfected human fibroblasts to escape the control of senescent
genes in the cell genome.
70
The other method we used was to determine the colony-forming
efficiency for each passage of the individual clones. If a clone
maintained a stable colony forming efficiency with cells of each
subsequent passage, it was probably also immortalized. However,
we observed that all the transfectant HFC cells first had a high
colony-forming efficiency (10-30% ), which then decreased and
eventually reached zero. Some time later, fast-growing cells
appeared. The colony forming efficiency of these newly emerged
clones was approximately 5%, not as high as in the precrisis SV 40
large T antigen transfected cells (Figure 11).
3.304 Assay for Immortality of Postcrisis Clones
Altogether, fifteen postcrisis clones were isolated. These
postcrisis clones were tested to determine whether they were
immortal by continuously subculturing a small portion of cells in
vitro and calculating the cumulative number of population doublings
for each individual clone. The first isolated immortal clone has
undergone more than 300 population doublings postcrisis and is still
actively growing. The cumulative population doublings to date for all
the postcrisis HFC clones are shown in Figure 12. All these cell
strains are still growing.
71
HFC/SV1-1
HFC/SV1-2
HFC/SV10-2
HFC/3V10-3
HFC/SV10-4
HFC/SV10-5
HFC/SV9-1
HFC/3V9-2
HFC/SV9-3
HFC/SV9-4
HFC/SV9-5
HFC/SV9-7
HFC/SV9-8
HFC/SV9-9
HFC/SV9-10
1 2 3 4 5 6 7 8 9 101112131415
SV40 LARGE T ANTIGEN IMMORTALIZED
HFC CLONES
Figure 12
Cumulative Population Doubling Curve for SV40 Large T
antigen Immortalized HFC Clones
The total number of population doublings beyond the life span of
normal human fibroblasts which each postcrisis immortal clone has
undergone is listed on this graph.
72
3.305 Biological Characterization of Precrisis and
Postcrisis Human Fibroblasts
There are many assays to characterize the phenotype of cells in
vitro. These are mostly based on unique properties that tumor cells
have. The tumor cells have a different morphology from normal cells,
lose contact-inhibition, grow to a higher saturation density, and
form foci when cultured with normal cells, which are contact
inhibited. Many tumor cells can also grow in suspension and form
colonies in soft agar. The transformed cells have a lower
requirement for serum (serum independence). The more an immortal
clone resembles tumor cells on these in vitro transformation
characterization assays, the more this clone will be tumorigenic in
vivo.
1) Morphological Changes
Normal human fibroblasts have the spindle-shaped morphology of
a typical fibroblast. Both the precrisis and the immortal postcrisis,
SV40 large T antigen transfectants showed a morphology different
from that of normal fibroblasts. Most of the transfected HFC cells
increased in size. Their nucleus was larger than that of normal
fibroblasts and contained more nucleoli, which suggested that they
were dividing more actively (Milo et al., 1988). Most of the immortal
clones had a morphology like that of HT1080, a human fibrosarcoma
cell line. The transfected human fibroblasts, both precrisis and most
of the immortal postcrisis clones, appeared more retractile than
73
Table 6
Morphology of SV40 Large T Antigen Immortalized Human
Fibroblast Clones
Moroholoav Number(%) Cell Lines
Spindle-shape 6
(3 7 .5 % )
HFC/SV1-1, HFC/SV1-2, HFC/SV10-4,
HFC/SV10-5. HFC/SV9-3. HFC/SV9-10
Spindle-Cuboidal 6
(3 7 .5 % )
HFC/SV1-2CI6, HFC/SV10-2, HFC/SV9-
4. HFC/SV9-5. HFC/SV9-6. HFC/SV9-7
Cuboidal 4
(2 5 % )
HFC/SV9-1, HFC/SV10-3, HFC/SV9-2,
HFC/SV9-9
74
normal fibroblasts under the microscope. The piled-up growth
pattern, one of the relatively common characteristics of
transformed cells, was observed for most SV40 large T antigen
transfected precrisis and postcrisis fibroblast clones. Three types
of morphology (Table 6) have been observed among the postcrisis
immortal clones: 1) The first type had spindle-shaped morphology
but marked piling up of cells on top of each other. HFC/SV1-1,
HFC/SV1-2, HFC/SV10-4, HFC/SV10-5, HFC/SV9-3, HFC/SV9-10 had
this morphology. These clones had the most distinctive transformed
phenotypes on in vitro characterization assays (focus
reconstruction, anchorage independence, serum dependece,
saturation density assays). 2) The second morphology was
intermediate between spindle-cuboidal shaped, somewhat rounded.
These cells still kept the spindle morphology but also showed some
rounded-shape or cuboidal-shape, which made their morphology
intermediate between fibroblast and epithelial cells. These clones
had a moderately transformed phenotype on in vitro transformation
characterization assays. These include HFC/SV1-2 Subclone 6,
HFC/SV10-2, HFC/SV9-4, HFC/SV9-5, HFC/SV9-6, HFC/SV9-7. 3)
The third type of morphology of the immortal clones was cuboidal,
which most epithelial cells possess. These clones had the least
transformed phenotypes as measured in in vitro transformation
characterization assays, and included cell lines H FC /SV9-1,
HFC/SV10-3, HFC/SV9-2 and HFC/SV9-9.
75
2) Saturation Density Assays
To determine cell growth curves, 1 X 105 cells were seeded per
60 mm dish for both pre-crisis and postcrisis transfected cells, and
the cell numbers in three dishes was determined every three days
for six weeks. Normal HFC had the lowest saturation density.
HT1080 was used as a positive control, and had the highest
saturation density compared to other immortalized HFC clones
(Figures 13 and 14). The precrisis transfected clones had
approximately two-to four-fold increases in saturation density
compared to normal primary diploid human fibroblasts, but the
saturation density curve or growth curve of these precrisis SV40
transfected clones was affected by the inability of precrisis cells
to proliferate continuously, so the data obtained was difficult to
interpret and are not presented here. Immortal, postcrisis, SV40
large T antigen transfected clones had higher saturation densities,
ranging from 1.3 to 7.8 times higher than that of normal fibroblasts
(9x1 ()5 cells/60 mm dish) after being cultured for six weeks
(Figures 13 and 14). These data imply, however, that both pre- and
post-crisis cells possess a transformed phenotype of elevated
saturation densities, even if the extent of transformation acquired
by different clones varies.
3) Reconstruction Assay for Focus Formation
in the reconstruction assay for focus formation, 5 x 104 normal
fibroblasts were mixed with 500 precrisis or post-crisis
76
s
s
< - HT1080
HFC
105
0 10 20 40 30 50
Days After Seeding
Figure 13
Saturation Density Curve of SV40 Large T Antigen
Immortalized HFC Clones
1x105 cells were seeded per 60 mm dish and the cell numbers
in three dishes was determined every three days for six weeks.
HT1080 is a human fibrosarcoma derived cell line and used as
a positve control. HFC: normal human fibroblast cell. Fifteen
clones and one subclone were tested. These included HFC/SV1-
1, HFC/SV1-2, HFC/SV1-2 Cl 6, HFC/SV10-2, HFC/SV10-3,
HFC/SV10-4, HFC/SV10-5, HFC/SV9-1, HFC/SV9-2, HFC/SV9-
3, HFC/SV9-4, HFC/SV9-5, HFC/SV9-6, HFC/SV9-7, HFC/SV9-
9, HFC/SV9-10.
77
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m
0
<
1
s
o
£
01:
02:
03:
04:
05:
06:
07:
0 8 :
09:
10:
11:
12:
13:
14:
15:
16:
17:
18:
HFC
HFC/SV1-1
HFC/SV1-2
HFC/SV1-2 CL6
HFC/SV10-2
HFC/SV10-3
HFC/SV10-4
HFC/SV10-5
HFC/SVB-1
HFC/SV9-2
HFC/SV9-3
HFC/SV9-4
HFC/SV9-5
HFC/SV9-7
HFC/SV9-8
HFC/SV9-9
HFC/SV9-10
HT1080
1 01 11 21 31 41 51 61 71 8
SV40 LARGE T ANTIGEN IMMORTALIZED HFC CLONES
Figure 14
Fold Increase in Saturation Density of SV40 Large T
Antigen Immortalized HFC Clones Compared to That of
Normal HFC
1x105 cells were seeded per 60 mm dish and the cell numbers
in three dishes was determined every three days for six weeks.
The saturation density of each SV40 large T antigen
immortalized HFC clones at end of six week culture was
compared to that of Normal HFC cells. HT1080 is a human
fibrosarcoma derived cell line and used as a positve control.
HFC: normal human fibroblast cell.
78
30 i
01: HFC
02: HFC/SV1-1
03: HFC/SV1-2
04: SV1-2CL6
05-.HFC/8V10-2
06: HFC/SV10-3
07: HFC/SV10-4
O S: HFC/SV10-5
09: HFC/SV9-1
10: HFOSV9-2
11:HFC/3V9-3
12: HFC/SV9-4
13: HFC/SV9-5
14: HFC/SV9-7
15: HFOSV9-S
16: HFC/SV9-9
17: HFC/SV9-10
18: HT1080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 15 Plating Efficiency of SV40 Large T antigen
Immortalized HFC Clones
500 cells from individual immortal clone were seeded into
each 60 mm dish, five dishes for each clone. Two weeks after
seeding, dishes were fixed and stained. The colonies containing
more than twenty cells were counted. Plating efficiency
represents the fraction of surviving cells of all the cells
seeded.
79
o 10 -
HFC
HFC/SV1-1
HFC/SV1-2
SV1-2 CL6
HFC/SV10-2
HFC/SV10-3
HFC/SV10-4
HFC/SV10-5
HFC/8V9-1
HFC/SV9-2
HFC/SV9-3
HFC/SV9-4
HFC/SV9-S
HFC/SV9-7
HFC/SV9-8
HFC/SV9-9
HFC/SV9-10
HT1080
a 9 10 1 1 12 13 14 1 5 16 1 7 18
Figure 16
Focus Forming Efficiency of SV40 Large T Antigen
immortalized HFC Clones
500 cells from each immortal clone were mixed with 5x104 normal
fibroblasts, and the mixture was then seeded into each of five 60
mm dishes. Three weeks later, the dishes were fixed and stained and
examined for focus.
80
Table 7
Focus Reconstruction Assay of HFC/SV Cells against Normal HFC
Cell Length in Culture
(Week)
Number of Dishes Total # of Foci
Formed
HFC 6 20 0
Precrisis
HFC/SV1 (P#3)
6 20 9 2 8
(46±1 7/dish)
Precrisis
HFC/SV1 (P#8)
6 20 0
Postcrisis
HFC/SV1-1
6 20 2 1 4
(10+ 4/dish)
81
Table 8
Assay of Precrisis Focus Forming Cells for Immortality
Transfected Cell
Strain
(precrisis)
Passage
Number After
Cloning
Time of Focus
Appearance
After Seeding
(Months)
# of Foci Cloned Immortal
Clones
HFC/SV9 3 3 7 0
HFC/SV10 5 3 1 8 0
82
transfected cells, and the mixed cultures were grown for three
weeks. Because more normal cells were seeded into each dish, they
formed a flat monolayer, and since the transformed cells have lost
contact-inhibition, they piled up on themselves and formed foci. A
number of foci were observed with the early passage (passage #3)
precrisis HFC/SV1 cells of the transfectant (46±17 foci/dish). With
the late passage (passage #8) pre-crisis HFC/SV1 cells, no foci
were observed (Table 7). This is probably due to the mortality of the
late passage precrisis cells, which limits their ability to
proliferate and thus, their ability to form foci. Therefore, the
inability of late passage (passage #8) precrisis SV40 large T
antigen transfected fibroblasts to form foci does not imply that
SV40 large T antigen is not able to induce transformed phenotype in
normal human fibroblasts. Postcrisis immortal clones of the SV40
large T antigen transfectants formed foci (Figure 16, Table 7). This
suggests that in vitro morphological cell transformation and
immortalization probably are genetically independent events in
diploid human fibroblasts.
The results from figure 16 showed that 15/16 or 94% of the
immortal postcrisis clones formed foci against normal HFC cells.
The efficiency of focus formation varied from clone to clone, from
2-10%. This variation probably represented both the variation in the
ability of individual clones to form foci against normal fibroblasts
and variations in the plating efficiency of those clones (from 2-10%)
when they were first seeded (Figure 15). One clone, HFC/SV9-1, did
83
Table 9
The Ability of HT1080 to Form Foci against SV40 Large T Antigen
mmortalized HFC Clones
Clone Morphology of
HT1080 Foci
Focus Forming
Efficiency(%)
Clone Morphology of
HT 1080 Foci
Focus Forming
Efficlency(%)
HFC/SV1 -1 Not seen 0 HFC/SV10-4 very weak 4 .2 ± 2 .4
HFC/SV1-2 Not seen 0 HFC/SV10-5 barely seen 0
SV1-2 CL6 Weak foci 9.2±2.6 HFC/SV9-1 distinct 19 .3 ± 6 .3
HFC/SV10-2 Weak foci 7.5±2.7 HFC/SV9-2 distinct 16.4± 4.1
HFC/SV10-3 Weak foci 8.8±3.1 HFC/SV9-9 distinct 1 7 .2 ± 5 .8
84
not form foci against normal HFC cells after three weeks of culture,
although we found that this clone formed weak foci against normal
fibroblasts after six weeks of culture. This is probably correlated
with the slow growth rate of this clone. Figure 13 shows that at
three weeks this clone had a saturation density similar to that of
normal HFC cells, while at six weeks, it had a 1.3 fold-higher
saturation density compared to that of normal fibroblasts.
Many foci (focal piling up) were observed with the pre-crisis
cells after they reached confluence. More than twenty-five foci from
the pre-crisis HFC/SV9 and HFC/SV10 were cloned and subcultured,
but cells from all those cloned foci entered crisis. No isolated focus
has survived repeated passaging for the precrisis state, again
suggesting that in vitro morphological cell transformation and
immortalization probably are not necessary correlated in diploid
human fibroblasts (Table 8).
We also tested the ability of HT1080 fibrosarcoma cells to form
foci against SV40 large T antigen immortalized HFC clones by
mixing 500 HT1080 cells with 1x104 cells from the immortalized
clones and seeding them into 60 mm dishes. Five dishes per group
were cultured for three weeks, and then, the dishes were fixed and
stained for focus screening (Table 9). The rationale of this assay is
that the more a clone is transformed, the less possible HT1080 cells
will form foci against it. Table 9 shows that HT1080 cells could
form foci against 7 out of 10 SV40 large T antigen immortalized
85
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>
3
(0
i n
u >
o
o
o
o
«*-
O
*
4000
3000
2000
1000
0
1 2 3 4 S e 7 8 9 10 11 12 13 14 15 18 17 18
01: HFC
02: HFC/SV1-1
03: HFC/SV1-2
04: SV1-2 CL6
05: HFC/SV10-2
06: HFC/SV10-3
07: HFC/SV10-4
08: HFC/SV10-5
09: HFC/8V9-1
10: HFC/SV9-2
11: HFC/SV9-3
12: HFC/SV9-4
13: HFC/SV9-5
14: HFC/SV9-7
15: HFC/SV9-8
16: HFC/SV9-9
17: HFC/SV9-10
18: HT1080
Figure 17
Anchorage Independent Growth of SV40 Large T Antigen
Immortalized HFC Clones
1x105 cells were suspended in 3 ml of 0.3% agar noble. The mixture
was allowed to solidify on the top of 5 ml 0.5% agar base layer in 60
mm dish, five dishes per immortal clone. Four weeks later, colonies
greater than 0.1 mm in diameter were scored by microscopic
examination.
86
clone, but not against clones HFC/SV1-1, H FC/SV1-2, and
HFC/SV10-5. When HT1080 did form foci against an immortal clone,
the morphology of foci it formed varied from strong (HFC/SV9-1,
HFC/SV9-2 and HFC/SV9-3) to weak (HFC/SV1-2 CL6, HFC/SV10-2,
HFC/SV10-3, HFC/SV10-4). This was due to the difference in
saturation density between the HT1080 and the clone of cells tested.
If HT1080 cells had a much higher saturation density than the cells
from the clone tested, it could form distinctive foci. Otherwise, it
would only form weak foci or no foci. The results from table 9 are
well correlated with the results obtained from the saturation
density assay (Figure 13 and 14). These results further suggest that
three out of ten SV40 large T antigen immortalized HFC clones were
as strongly morphologically transformed as HT1080 cells, but the
phenotypes and degree of transformation acquired by different
clones varied.
4) Anchorage Independence Assays
To assay for anchorage independence, 1 x 10^ cells were seeded
into 0.3% agar noble as described in Materials and Methods. The
surviving cell number was determined by seeding equal number of
cells into 60 mm dishes and counting the number of cells attached to
dishes twenty-four hours later. This number was used to adjust the
soft agar colony forming efficiency for each immortal clone. The
cells in agar were fed once a week with 1 ml MEM for four weeks,
and the colonies with a diameter larger than 0.1 mm were counted as
87
anchorage independent (Al) colonies. As shown in Figure 17, most of
the immortal clones were anchorage-independent and grew in soft
agar. However, two immortal clones, derived from HFC/SV9,
HFC/SV9-1 and HFC/9-9, had vrey low anchorage-independent colony
forming efficiencies in soft agar, similar to that of normal human
fibroblast cells. These results suggest that not all the SV40 large T
antigen immortalized clones have acquired this transformed
phenotype because HFC/SV9-1 and HFC/SV9-9 did not grow in soft
agar. As expected, the positive control cell line, HT 1080
fibrosarcoma cells, had the highest soft agar colony forming
efficiency compared to the SV40 immortalized HFC clones.
5) Serum Dependence Assays
To determine whether an immortal clone was dependent on serum
for growth, 1 X 10$ cells from each immortal clone were seeded into
60 mm dishes in 5 ml of medium containing 0, 1%, and 10% fetal
bovine serum respectively, the medium was changed twice a week,
and the number of cells from each of three dishes per serum
concentration was determined every three days for six weeks. HT
1080 fibrosarcoma cells were used as a positive control. Figure 18a
shows that HT1080 grew a little better in higher serum
concentration (10%) than in 1% FBS. However, cells cultured in 1%
FBS could reach the same saturation density as cells cultured in 10%
FBS, but they had to be cultured for six weeks. Serum deficiency
depressed the growth of HT 1080 cells by 100-fold, so they did not
88
E
E
o
< o
o.
o
o
*
106
0 20 30 40 10 50
-O 10%
- • 1%
-« 0%
Days after Seeding
Figure 18a
Growth of HT1080 Cells in Medium Containing Different
Concentrations of FBS
1x105 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
89
«
a
E
E
o
< o
o
o
*
106
10 20 0 30 40 50
-a— 10%
1%
0%
Days after Seeding
Figure 18b
Growth Property of Normal HFC Cells in Medium Containing
Different Concentrations of FBS
1x1 o5 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
90
0 10 20 30 40 50
Days after Seeding
Figure 18c
Growth Property of HFC/SV1-1 Cells in Medium Containing
Different Concentrations of FBS
1x10® cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
91
10 4 -|------------- 1 --- « -----------1 -----1 -----------1 ---------« i-----------1 --1 -------- 1 -------- 1
0 10 20 30 40 50 60
Days after Seeding
Figure 18d
Growth Property of HFC/SV1-2 Cells in Medium Containing
Different Concentrations of FBS
1x10® cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
92
1 0 -| 1 --------- 1 1 -------1 1 ------ 1 1 ------ 1 1
0 10 20 30 40 50
Days after Seeding
Figure 18e
Growth Property of HFC/SV1-2 Cl 6 Cells in Medium
Containing Different Concentrations of FBS
1 x 1 0 5 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
93
w
Q
E
E
o
< 0
O
o
tt
107
106
105
10 20 30 40 0 50
-a — 10%
1%
-■— o%
Days after Seeding
Figure 18f
Growth Property of HFC/SV10-2 Cells in Medium Containing
Different Concentrations of FBS
1x10® cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
94
£
«
E
E
o
< 0
a
o
*
107
20 40 10 30 0 50
- 1 3 ----- 10%
1%
-■ 0%
Days after Seeding
Figure 18g
Growth Property of HFC/SV10-3 Cells in Medium Containing
Different Concentrations of FBS
1x10® cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
95
«
s
a
o
o
*
106
30 10 20 40 50 0
10%
1%
0%
Days attar Seeding
Figure 18h
Growth Property of HFC/SV9-1 Cells in Medium Containing
Different Concentrations of FBS
1x105 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
96
E
E
o
< 0
a
o
o
*
105
30 0 10 20 40 50
-O 10%
- * 1%
-e 0%
Days attar Seeding
Figure 18i
Growth Property of HFC/SV9-9 Cells in Medium Containing
different Concentrations of FBS
1x105 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively. Three
dishes were trypsinized and counted on a Coulter counter every three
days for six weeks. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes.
97
■ 10%
■ 1%
□ 0%
01: HFC
02: HFC/SV1-1
03: HFC/SV1-2
04: 3V1-2 CL6
05: HFC/SV10-2
06: HFC/SV10-3
07: HFC/SV10-4
08: HFC/SV10-5
09: HT1080
Figure 19a
Growth Property of SV40 Large T Antigen Immortalized HFC
Clones in Medium Containing Different Concentrations of
Fetal Bovine Serum
1x105 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively and
cultured for six weeks. Then, cell number from three 60 mm dishes
per clone was determined by trypsinizing and counting cells from
each dish on a Coulter counter. Cell counts were reported here as the
mean ± standard deviation of determinations.
98
■ 10%
■ 1%
□ 0%
01: HFC
02: HFC/SV9-1
03: HFC/SV9-2
04: HFC/SV9-3
05: HFC/SV9-4
06: HFC/SV9-5
07: HFC/SV9-7
08: HFC/SV9-8
09: HFC/SV9-9
10: HFC/3V9-10
11: HT1080
a a 1 0 1 1
Figure 19b
Growth Property of SV40 Large T Antigen Immortalized HFC
Clones in Medium Containing Different Concentrations of
Fetal Bovine Serum
1x105 cells were seeded into 60 mm dish in 5 ml of medium
containing 0, 1%, and 10% fetal bovine serum respectively and
cultured for six weeks. Then, cell number from three 60 mm dishes
per clone was determined by trypsinizing and counting cells from
each dish on a Coulter counter. Cell counts were reported here as the
mean ± standard deviation of determinations.
99
grow beyond the initial seeding density but cell growth was indeed
observed although at very slow rate. Therefore, HT1080 cells have
the property of serum-independent growth. This is consistent with
the fact that HT1080 is derived from a human fibrosarcoma and has
the properties of tumor cells (Morgan et al., 1991). This also
conforms to the theory that tumor cells can synthesize the growth
factors they require (autocrine mechanism, Michalopoulos, 1989).
Our finding that HT1080 cells can grow slightly in medium without
serum also supports this theory (Figure 18a), although HT1080 cells
grow in serum-free medium at a much slower rate. Unlike cells
seeded in 10% or 1% serum medium, the number of HT1080 cells
seeded in 0% was seen first to drop and then to increase slowly
(Figure 18a). We also found under microscopic examination that cell
growth on the dishes in this group was not evenly distributed, and
most of the cell growth was concentrated in focal areas. This
suggests that HT1080 is probably a heterogenous population, and the
increasing number in the later part of the growth curve probably
represents the multiplication of the sub-population which has
serum-independent growth.
Normal HFC grew better in medium containing 10% serum than
that in 1% serum (Figure 18b). The final saturation density normal
HFC cultured in 10% serum reached is 3.6 times higher at end of six
week culture than for HFC cells cultured in 1% serum. Normal HFC
cells do not grow at all in medium without serum. In this group, the
cells kept detaching from the dishes and finally, less than 1% of
100
those cells could be observed on the dishes (Figure 18b). This
indicated that normal human fibroblast cells have the property of
strongly serum dependent growth.
Among the SV40 large T antigen Transfectants, HFC/SV1-1 grew
almost equally well in MEM containing either 1% or 10% serum,
indicating that it has the property of serum-independent growth and
has been transformed. Furthermore, this clone grew much better
than H T1080 in medium without serum, increasing the cell
innoculumm 20-fold, and the cells were evenly distributed over the
dish, suggesting that they were more resistant to serum depletion
and more homogenous than HT1080 cells (Figure 18c). For all the
other immortal HFC clones derived from SV40 large T antigen
transfected HFC clones, various degrees of cell growth was observed
when they were cultured in medium without serum, indicating that
these clones have acquired this transformed phenotype, but to
different degrees (Figure 18c through 18g; Figure 19 a and b).
HFC/SV1-1 behaved as a serum-independent clone (Figure 18c),
HFC/SV1-2 behaved like serum-independent HT1080 (Figure 18d), as
did HFC/SV10-2 (Figure 18f), and HFC/SV10-3 (Figure 18g).
All the immortal clones derived from HFC/SV9 did not grow in
medium without serum (Figure 18h and i; Figure 19b). In those
clones, no cell division was observed on the dishes under
microscopic examination when they were cultured in medium free of
serum. Except for HFC/SV9-1 and 9-9, the saturation density of all
101
the HFC/SV9 clones reached in 10% FBS-containing medium at the
end of six week culture was less than 3-fold higher compared to
what they could be reached in 1% serum medium. HFC/SV9-1 and
HFC/SV9-9, like normal HFC, grew much better in 10% serum-
containing medium than in 1% serum and did not grow in medium
without serum. The final saturation density they obtained in 10%
serum was more than 3.6 times higher than in 1% serum medium
after six week culture. This indicates that they still retained the
normal cell phenotype for serum-dependent growth (Figure 18h and i;
Figure 19b). The final cell density of each clone in 60 mm dish after
six weeks of culturing in medium containing different
concentrations of serum is shown in Figures 19a and 19b. Except for
HFC/SV1-1 and HT1080, which show no difference in saturation
density at the end of six weeks culturing (Figure 18a and c; Figure
19a) irrespective of serum concentration, all the other immortal
clones grew less favorably in low serum (1%) medium than in higher
(10%) serum medium, suggesting that they are not as transformed as
HFC/SV1-1 and HT1080 on this in vitro assay. If the 3.6-fold
increase in saturation density observed between culturing normal
HFC cells in 1% and 10% serum could be used as a standard for serum
dependent growth of the immortal clones, only two clones,
HFC/SV9-1 and HFC/SV9-9, derived from HFC/SV9 could be
considered to be as serum-dependent as normal HFC. All the other
clones show a difference of less than 3-fold increase in cell
102
Table 10
Growth of SV40 Large T Antigen Immortalized HFC Clones in
Medium without Serum
Chanae in Saturation Density Immortal Cell Clone
Saturation Density Decreases Normal HFC, HFC/SV01-5, HFC/SV9-1,
HFC/SV9-2, HFC/SV9-3, HFC/SV9-4,
HFC/SV9-5, HFC/SV9-7, HFC/SV9-8,
HFC/SV9-9. HFC/SV9-10
Saturation Density stays the Same HFC/SV1 -2 CL 6. HFC/SV10-2
Saturation Density Increases HT1080, H F C /S V 1-1, HFC/SV1 -2,
HFC/SV10-3. HFC/SV10-4
103
saturation density when being cultured in 10% than in 1% serum
(Figure 18a through i; Figure 19a and b).
3.306 Western Blotting Analysis of SV40 Large T
Antigen in Immortal HFC Clones
The relative quantity and size of the SV40 large T antigen from
nuclear extracts of each SV40 large T antigen immortalized clone
were determined by Western blotting techniques. The total protein
concentration in each nuclear extract sample was determined and
used to adjust the relative quantity of SV40 large T antigen obtained
on Western blotting analysis to eliminate the effects of variations
in the concentration of SV40 large T antigen among individual
nuclear extract samples. As shown in Figure 20, a band with
molecular weight of about 90 Kd was detected from the nuclear
extracts of all SV40 immortalized HFC clones, but not from those
extracted from normal HFC cells nor from HFC cells transfected
with a plasmid vector, Zip. The relative quantity of SV40 large T
antigen was determined on a densitometer and adjusted by the total
protein concentration in each individual sample. As shown in Figure
21, HFC/SV9-1 and HFC/SV9-9 had the lowest relative amount of
SV40 large T antigen protein in their cells.
104
3.307 Immunostaining of different SV40 Immortalized
Clones for SV40 Large T antigen
Different SV40 large T antigen immortalized clones were next
immunostained to determine the expression of SV40 large T antigen
inside those cells with a C-terminal specific antibody, Pab101,
according the procedure described in the Materials and Methods
Section. Cells from each clone shown showed positive nuclear
staining, indicating the presence of SV40 large T antigen in the
nucleus of the cells of the transfectant clones.
3.308 Correlation Analysis of the Relative Amounts
Intracellular SV40 Large T Antigen and specific
Transformed Phenotypes
We next asked whether the amount of SV40 large T antigen of
each immortal HFC clone was correlated with its transformed
phenotypes. The correlation between intracellular SV40 large T
antigen and transformed phenotypes was determined by the Pearson
Correlation Analysis (Rosner, 1990). As shown in Figures 22a, b and
c. the intracellular levels of SV40 large T antigen in each clone was
significantly correlated with the saturation density (r=0.6, P<0.05)
that each individual clone could reach (Figure 22a). It was even
better correlated with anchorage independent growth (r=0.7,
P<0.025) (Figure 22b) and focus forming efficiency (r=0.8, P<0.01)
(Figure 22c) of each individual clone. This suggests that the more
SV40 large T antigen each immortal clone contains, the greater the
105
WESTON BLOT ANALYSIS OF SV40 LARGE T ANTGBI
Figure 20
Western Blot Analysis of SV40 Large T Antigen
A band with molecular weight of about 90 Kd was detected in the
nuclear extracts of SV40 large T antigen immortalized HFC clones
but not from those extracted from normal HFC cells and HFC cells
transfected with a plasmid vector, Zip.
106
HFC/SV1-1
HFC/SV1-2
SV 1-2 CL6
HFC/SV10-2
HFC/3V10-4
HFC/SV10-5
HFC/3V9-1
HFC/SV9-2
HFC/SV9-3
HFC/SV9-7
HFC/SV9-8
HFC/3V9-9
10 11 12
Figure 21
Relative Quantity of SV40 Large T Antigen per pg Total
Protein in Nuclear Extracts from Different Immortal HFC
Clones
The relative quantity of SV40 large T antigen was determined by
scanning the blots on a densitometer and total protein concentration
in each nuclear extract sample was determined to adjust the
variation caused by the difference in SV40 large T antigen
concentration from each individual sample.
107
8
y = 0.53197 + 0.36227X RA 2 = 0.306
6
4
2
0
2 4 6 0 8 10
Relative Amount of SV40 Large T Antigen
(ug Nuclear Protein)
Figure 22a
Correlation Between Saturation Density and Amount of
Intracellular SV40 Large T Antigen in SV40 Large T Antigen
Immortalized Human Fibroblast Clones
Correlation between saturation density each individual immortal
clone could reach after six week culture and amount of their
intracellular SV40 large T antigen in SV40 large T was determined
by Pearson correlation analysis and found to be statistically
significant (P<0.05)
108
10 n
- 1.1628 + 0.77582X RA 2 = 0.551
E
k .
o
u.
w
3
o
o
II.
0 &
0 2 8 4 6 10
Relative Amount of SV40 Large T Antigen
(per ug Nuclear Protein)
Figure 22b
Correlation Between Focus-Forming Efficiency and Amount
of Intracellular SV40 Large T Antigen in SV40 Large T
Antigen Immortalized Human Fibroblast Clones
Correlation between focus-Forming efficiency of each individual
SV40 large T antigen immortalized human fibroblast clone and
amount of their intracellular SV40 large T antigen was determined
by Pearson correlation analysis and found to be statistically
significant (P<0.01)
109
2000-1
«
w
O
»
>
3
C O
- 207.53 + 128.61 x RA 2 = 0.489
■A
lU
O
c
o
o
o
<
o
*
2 6 0 4 8 10
Relative Amount of SV40 Large T Antigen
(per ug Nuclear Protein)
Figure 22c
Correlation Between Anchorage-Independent Growth and
Amount of Intracellular SV40 Large T Antigen in SV40
Large T Antigen Immortalized Human Fibroblast Clones
Correlation between anchorage-independent growth of each
individual SV40 large T antigen immortalized human fibroblast clone
and the amount of their intracellular SV40 large T antigen was
determined by Pearson correlation analysis and found to be
statistically significant (P<0.025)
110
quantitative inactivation of Rb and P53 tumor suppressing protein
products, hence the more transformed the cell.
3.309 Subcloning of Immortalized Clone with Less
Transformed Phenotypes
To obtain immortal but nontransformed cell clones from SV40
large T antigen immortalized HFC clones, we first further subcloned
HFC/SV1-2, because this clone had heterogeneous uneven cell layers
on culture dishes after being fixed and stained, and we thought we
might clone out flat, immortalized clones from this clone. Parental
HFC/SV1-2 cells were therefore trypsinized and seeded at a low
density (1000 cells/60 mm dish, which would yield about ? viable
colonies/dish) into the four dishes and cultured for three weeks.
When the colonies became large enough ( 5 mm in diameter), fifteen
clones which looked flat were isolated, subcultivated, and
biologically characterized in saturation density (Figure 23a), focus
formation (Figure 23b), and anchorage independent assays (Figure
23c) according to the methods described in Material and Methods.
One subclone, HFC/SV1-2 clone 6 showed the least transformed
phenotype on these in vitro transformation assays. It had only a 1.6-
fold higher saturation density than normal HFC cells have after six
weeks in vitro culture. However, it had a low but reasonable focus
forming efficiency, about 2%, and it also acquired the property of
anchorage-independent growth, so this clone was still transformed
111
E
E
o
1 0
a
o
• * -
o
*
01: HFC
02: SUBCLONE 1
03: SUBCLONE
04: SUBCLONE
OS: SUBCLONE
06: SUBCLONE
07: SUBCLONE
08: SUBCLONE
09: SUBCLONE 8
10: SUBCLONE 9
11: SUBCLONE
12: SUBCLONE
13: SUBCLONE
14: SUBCLONE
IS: SUBCLONE
16: SUBCLONE
17: HT1080
10
11
12
13
14
1 5
2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7
Figure 23a
Saturation Density Curve of HFC/SV1-2 Subclones
1x1 ()5 cells each were seeded into three 60 mm dishes for each
individual immortal HFC/SV1-2 subclones and the cell numbers in
each dishes were determined at end of six week culture.
112
> »
o
c
•
0
5:
ui
O )
c
1
o
0 )
3
O
o
u.
20
10
0
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7
01: HFC
02: SUBCLONE 1
03: SUBCLONE 2
04: SUBCLONE 3
05: SUBCLONE 4
06: SUBCLONE 5
07: SUBCLONE 6
06: SUBCLONE 7
09: SUBCLONE 8
10: SUBCLONE 9
11: SUBCLONE 10
12: SUBCLONE 11
13: SUBCLONE 12
14: SUBCLONE 13
15: SUBCLONE 14
16: SUBCLONE 15
17: HT1060
Figure 23b
Focus Forming Efficiency of HFC/SV1-2 Subclones
500 cells from each subclone were mixed with 1x104 normal
fibroblast, and the mixture was then seeded into each of five 60 mm
dishes. Three weeks later, the dishes were fixed and stained and
examined for foci.
113
o
>
e
3
C D
in
ui
o
o
o
o
o
*
3000
2000 -
1000
1 2 3 4 5 6 7 8 9 101112 13 14 1516 17
01: HFC
02: SUBCLONE 1
03: SUBCLONE 2
04: SUBCLONE 3
05: SUBCLONE 4
06: SUBCLONE 5
07: SUBCLONE 6
08: SUBCLONE 7
09: SUBCLONE 8
10: SUBCLONE 9
11: SUBCLONE 10
12: SUBCLONE 11
13: SUBCLONE 12
14: SUBCLONE 13
15: SUBCLONE 14
16: SUBCLONE 15
17: HT1080
Figure 23c
Anchorage Independent Growth of HFC/SV1-2 Subclones
1x105 cells were suspended in 3 ml of 0.3% noble agar. The mixture
was allowed to solidify on the top of 5 ml 0.5% agar base layer in 60
mm dishes. Four weeks later, colonies larger than 0.1 mm in
diameter were scored by microscopic examination.
114
TABLE 11
SUMMARY OF PHENOTYPES OF SV40 LARGE T ANTIGEN IMMORTALIZED HFC CLONES
ONiNvrr
l
9
RMATION CHARACTER IZATION ASSAYS
Cell Clone Relative
Amount of
SV40 Large
T Antigen Per
Cel Line
Focus-
Forming
Efficiency in
Reconstruct!
on Assays
<%)
Anchorage
Independent
Colonies/
10s
Surviving
Cells
Saturation
Density
(Number of
Cells
x10s /6 0
mm dish)
Serum In
dependence
Plating
Efficiency on
Plastic (%)
Normal
HFC
0 0 1.211.2 9.511.2 - 1412 •
HFC/SV
1-1
8.6 2.210.8 1 8241194 72X12.8 + 3.211.7
HFC/SV
1-2
7.3 3.110.7 6 5 5115 9 4711.2 + 3.110.7
HFC/SV
1-2 Cl 6
5.9 1.510.5 503176 1512.1 + 1.010.3
HFC/SV
10-2
4.3 4.211.1 740197 2411.3 + 3.110.7
HFC/SV
10-3
ND 2.210.8 109311 04 2111.8 + 1.010.4
HFC/SV
1 0 -4
8.7 5.011.1 594150 3611.6 + 5.910.9
115
Table 11 (continued)
HFC/SV
10 -5
8.8 9 .4 ± 1 .8 1 2 2 1 1 1 1 6 4517.2 + 6.611.8
HFC/SV
9-1
3.1 0 6 1 4 1311.2 - 3.310.7
HFC/SV
9 -2
5.3 2.210.5 2 2 6 1 6 6 1311.1 + 3.510.7
HFC/SV
9-3
7.9 4 .2 ± 1 .0 6 2 2 1 5 8 1215.3 + 5.511.1
HFC/SV
9-4
NO 4.011.0 2 5 2 1 3 2 1711.2 + 4.611.2
HFC/SV
9 -5
NO 6.410.9 3 5 2 1 2 2 2 0 11.5 + 7.311.1
HFC/SV
9 -7
8.3 6.610.8 6 6 2181 2.011.7 + 8.511.3
HFC/SV
9 -8
8.3 7 .4 ± 1 .2 5 0 0 1 6 8 25 11.7 + 9.611.4
HFC/SV
9 -9
3.4 1.0±0.4 1 2 1 3 2611.8 - 2.210.8
HFC/SV
9 -1 0
ND 6.511.1 8 9 1 1 6 6 3211.9 + 7.810.6
HT1080 NO 18± 4 3 1 2 8 1 4 1 2 8314.2 + 2 5 1 6
116
Note: * P.E. was somewhat low because cells were at passage #8 (population doubling
of 35). The amount of SV40 large T antigen was determined as the relative amount of
SV40 large T antigen per pg nuclear protein. The saturation density was determined as
number of total cells per 60 mm dish after six weeks of culture. Anchorage-independent
growth was measured as number of Al colonies/105 survivors. Normal HFC cells did not
have transformed phenotypes in any of the above assays, while the human fibrosarcoma
cell line, HT1080, used as a positive control, had the most transformed phenotypes and
thus is strongly positive in all the above transformation charaterization assays. As can
be seen above, the SV40 large T antigen immortalized clones possess different degrees of
transformed phenotypes.
117
and therefore not suitable for use in cell transformation assays
(Figures 23 a, b and c).
3.310 Conclusions
The results of in vitro transformation characterization assay for
each of the SV40 large T antigen immortalized clones are
summarized in Table 11. From these results we have obtained so far,
we first concluded that: firstly, SV40 large T antigen could
immortalize human diploid fibroblast cells, but not on its own. It
likely triggered some genetic instability, which led to a second
spontaneous genetic change that resulted in immortality in human
fibroblast cells. However, the presence of the SV40 large T antigen
in the cells was necessary, although not sufficient on its own, to
induce cellular immortality. Secondly, we found that different SV40
large T antigen immortalized clones possessed different degrees of
various transformed phenotypes in in vitro transformation
characterization assays. Thirdly, we found that the ability of each
immortal clone to form foci, to grow in soft agar, and to reach
higher saturation density, and to grow serum independently is
crudely correlated. Fourthly, we concluded that the phenotype of
SV40 large T antigen immortalized human fibroblast clones is
correlated with the amount of SV40 large T antigen inside the cells
of each individual clone. The more SV40 large T antigen the
individual clone expresses, the greater the degree of transformed
phenotypes it possesses.
118
These data taken together imply that the process of
transformation or carcinogenesis of the normal cells is a multistep,
cumulative process. By selecting the immortal clone(s) expressing
very low amounts of SV40 large T antigen, we should be able to
obtain immortal clones with nontransformed phentypes. Table 11
shows that HFC/SV9-1 is the clone with the least transformed
phentype in these in vitro transformation characterization assays.
3.311 Chemical Transformation Assays in Immortalized
HFC Clones
We first conducted in vitro chemical transformation assays with
the precrisis SV40 large T antigen transfected cells. However,
because of the mortality of those precrisis clones, they started to
senesce soon after MNNG treatment, and most of the time they did
not grow sufficiently well to form an intact cell layer. Our chemical
transformation assays with the precrisis cells were therefore not
successful, consistent with previous data from this laboratory
showing that chemical carcinogen could not induce foci in primary
human fibroblasts (Biedermann and Landolph, 1987, 1990). Our next
chemical transformation were conducted on the immortal HFC
clones we established. The cytotoxicity and transformation assays
were conducted as described in Material and Methods, and the results
are presented below and summarized in Tab!e12.
119
1) Cytotoxicity Assays
We first used the chemical carcinogen N-methyl-N’-nitro-N-
nitroso-guanidine (MNNG). This is an activated alkylating agent
which reacts with numerous nucleophilic sites in DNA to form DNA
adducts such as 0 6 -methylguanine, causes various mutations,
including point mutations and rearrangements (Maher, 1990), is
carcinogenic in animal models, and induces cell transformation in
vitro mammalian cell culture assays (Milo et al., 1978). Ten SV40
large T antigen immortalized clones were therefore treated with
MNNG and then assayed for survival and their ability to undergo
chemically induced morphological and anchorage-independent
transformation. For each clone, MNNG treatment over the range of
0.067 pg/ml to 2.0 pg/ml consistently caused dose-dependent
cytotoxicity (Figure 24a through 24j, Table 12), demonstrating that
MNNG was cytotoxic to these cells. Because acetone was used to
dissolve MNNG, an acetone control group was also included. The
L C 50 (concentration that killed 50% of the cells) varies from 0.5
pg/ml for SV1 and SV10 clones and 0.1 pg/ml for SV9 clones.
2) Focus Formation Assays
In focus formation assays, we did obtain foci growing on the top
of a cell monolayer for these MNNG treated clones. However, the cell
monolayers were not intact, while the control and acetone treatment
groups showed relatively thick cell monolayers. We believe that
formation of "foci" was due to the cytotoxicity caused by MNNG
120
c
o
o
«
w
IL
«
>
>
3
(0
0 2 1 3
MNNG Concentration (ug/ml)
Figure 24a
Survival Curve of HFC/SV1-1 Cells Treated with MNNG
1 x 1 ()5 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 jig/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
121
10
c
o
o
a
w
IL
0
>
>
w
3
C O
.01
0 1 2 3
MNNG Concentration (ug/ml)
Figure 24b
Survival Curve of HFC/SV1-2 Cells Treated with MNNG
1x105 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
122
10 -a
c
o
+ +
o
m
t m
IL
m
>
>
3
< 0
2 3 1 0
MNNG Concentration (ug/ml)
Figure 24c
Survival Curve of HFC/SV1-2 Cl 6 Cells Treated with MNNG
1x10® cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
123
10
1
.1
0 1 2 3
MNNG Concentration (ug/ml)
Figure 24d
Survival Curve of HFC/SV10-2 Cells Treated with MNNG
1x 105 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
124
10 -q
e
o
♦ *
o
«
w
IL
a
>
.01
1 2 3 0
MNNG Concentration (ug/ml)
Figure 24e
Survival Curve of HFC/SV10-3 Cells Treated with MNNG
1x1 o5 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
125
10 1
c
o
o
<0
k
U L
a
>
a
C O
1 2 3 0
MNNG Concentration (ug/ml)
Figure 24f
Survival Curve of HFC/SV10-4 Cells Treated with MNNG
1x105 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
126
0 1 2 3
MNNG Concentration (ug/ml)
Figure 24g
Survival Curve of HFC/SV10-5 Cells Treated with MNNG
1x1 ()5 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
127
1 0 -
c
o
o
m
w
Ik
>
>
3
< 0
.01
0.2 0.0 0.4 0.6 0.8
MNNG Concentration (ug/ml)
Figure 24h
Survival Curve of HFC/SV9-1 Cells Treated with MNNG
1x1 ()5 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
128
10 1
e
o
o
a
w
u .
■
>
>
w
3
C O
.01
0.2 0.0 0.4 0.8 0.6
MNNG Concentration (ug/ml)
Figure 24i
Survival Curve of HFC/SV9-2 Cells Treated with MNNG
1x105 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
129
c
o
o
«
u.
«
>
>
w
3
u >
.01
0.2 0.0 0.6 0.8 0.4
MNNG Concentration (ug/ml)
Figure 24j
Survival Curve of HFC/SV9-9 Cells Treated with MNNG
1x 1 05 cells were seeded into each 60 mm dish, three dishes per
group. Twenty four hours later, each group was treated with acetone,
and with 0.25, 0.50, 0.75, 1.0, 2.0 pg/ml MNNG respectively. The
surviving cell number per dish for each group was determined by
trypisinizing and counting the cell number from each dish on a
Coulter counter model ZF 24 hours later. The % surviving cells was
calculated by dividing the surviving cell number in each treatment
group by that of the control group. This method was used because the
plating efficiency of the cells was too low (<10%) to conduct plating
efficiency assays.
130
Table 12
Phenotypic Changes of Different HFC Immortal Clones after being
Treated with MNNG
Clone Cytotoxicity Focus Formation Increase In Anchorage
Independent Growth
Increase In
Saturation Density
HFC/SV1-1 YES NO NO NO
HFC/SV1-2 YES NO NO NO
SV1-2 CL6 YES NO NO NO
HFC/SV10-2 YES NO NO NO
HFC/SV10-3 YES NO NO NO
HFC/SV10-4 YES NO NO NO
HFC/SV10-5 YES NO NO NO
HFC/SV9-1 YES NO NO NO
HFC/SV9-2 YES NO NO NO
HFC/SV9-9 YES ND ND ND
ND: Not determined; The results presented in this table do not include the the results
of transformation of MNNG-treated HFC/SV1-1, HFC/SV1-2 CI6, and HFC/SV9-1 after
continuous subculture.
131
treatment rather than the appearance of true transformants which
were able to grow to higher saturation density and form foci. We
observed a similar response in each of the ten clones we tested
(Table 12).
3) Anchorage Independence and Saturation
Density Assays
The results from anchorage independence and saturation density
assays also did not show the presence of transformed cells. We
actually observed a dose-dependent decrease in the ability to form
Al colonies of each clone (Figures 25a, Table 12), and similarly, a
dose-dependent decrease of saturation density in direct proportion
to concentration of MNNG we used was observed (Figures 25b, Table
12). We believe the decreased ability of immortal HFC clones to
grow in soft agar and decreased saturation density after MNNG
treatment was probably due to the cytotoxicity caused by MNNG, and
that these cells in the assays had not recovered from the
cytotoxicity of MNNG treatment. Another possibility is that the
expression time for the transformed phenotypes to appear is longer
among these immortal clones, and the more transformed phenotypes
might not be expressed at this stage. We therefore subcultivated
these MNNG-treated cells, and during the process of subcultivation,
we tested their ability to form foci and to grow in soft agar, and
determined their saturation density.
132
3.312 Early Attempts to Induce Transformation in SV40
Large T Antigen Immortalized Cell Lines and
Expression Time for the Appearance of
Transformed Phenotype
In prelimary experiments, we first chose two clones, HFC/SV1-1
and HFC/SV1-2 Cl 6, to determine the expression time of
transformed phenoytpes by subcultivating the MNNG-treated clones
continuously and assaying for transformed phenotypes. HFC/SV1-1,
was the first clone we derived, and HFC/SV1-2 Cl 6 was a subclone
derived from HFC/SV1-2 which had a low saturation density and low
focus-forming efficiency. We seeded 5x10$ cells into each 75 cm2
T flask, two flasks for each treatment concentration, and then
treated the cells with different concentrations of MNNG. The cells
in the flasks were passaged every two weeks. Every two to three
passages, we assayed focus formation, saturation density, and
anchorage independent growth according to procedures described in
Materials and Methods.
We did not observe any focus formation during the process of
subcultivating these two clones, presumably because both clones
could already form sufficiently thick cell layers on the dishes so
that obvious foci could not be observed. However, as we passaged the
cells treated with different concentrations of MNNG, the saturation
density and soft agar colony forming efficiency assays did show
significant changes. The saturation density and anchorage-
independent colony forming efficiency remained stable in the control
133
and acetone treatment group. In the MNNG treatment groups, the Al
colony forming efficiency was below the high background of the
control cells, so this experiment was not successful. However, these
two clones had a high background of soft agar colony forming
efficiency and high saturation density. They therefore already
possessed the transformed phenotype and were therefore not
suitable for in vitro chemical transformation assays. We suspected,
however, that if we could obtain an immortal clone with a normal or
less transformed phenotype, we would be able to transform the
immortal human cells and measure transformation with these two in
vitro transformation assays.
3.313 In Vitro Chemical Transformation of HFC/SV9-1
HFC/SV9-1 is an immortal clone with the least transformed
phenotype among our SV40 large T antigen immortalized human
fibroblast cell lines, as determined by its having the lowest
saturation density and growth in soft agar. When we treated this
clone with MNNG, we observed a dose-dependent decrease in plating
efficiency (Figure 24h), indicating that we used cytotoxic
concentrations of MNNG. At early passage, we actually observed a
dose-dependent decrease in anchorage independent colony forming
efficiency, and in the saturation density of these cells (Figure 25h;
Figures 25a and b). However, because of the experience obtained
from the subcultivation of the two previous
134
80-1
«
w
O
>
£
a 60 -
C O
i n
U J
o
40-
c
o
o
o
<
« • »
o
2 8 4 6 10 0
— B----
• Control
«----
- Acetone
----- m ----
- 0.067 ug/ml
----- 9----
' 0.125 ug/ml
' 0.025 ug/ml
■ 0.5 ug/ml
Passage Number
Figure 25a
Effects of Passage on the Efficiency of Anchorage
Independent Growth in MNNG Treated HFC/SV9-1 Cells
Cells treated by different concentrations of MNNG were passaged.
The anchorage independent colony forming efficiency of each
treatment group was determined every two to three passages
according to the method described previously. The Al colony forming
efficiency increased in MNNG treated groups as they were being
passaged while it stayed stable in control and acetone treated group.
135
m
O
E
E
o
C O
w
•
&
O
o
%
107 i
1 0 °-;
105 i
104 -i
10;
-o— Control
• Acetone
Hi 0.067
-«---- 0.125
v " ~ s s s 0.25
— O 0.5
i
1 0
Passage Number
Figure 25b
Effects of Passage on Saturation Density in MNNG Treated
HFC/SV9-1 Cells
Cells treated by different concentrations of MNNG were passaged.
The saturation density of each treatment group was determined
every two to three passages according to the method described
previously. The saturation density increased in MNNG treated groups
as they were being passaged while it stayed stable in control and
acetone treated group.
136
1: Control
2: Aeotono
3: 0.067 ug/ml
4: 0.125 ug/ml
5: 0.25 ug/ml
6: 0.5 ug/ml
Figure 25c
Saturation Density Curve of HFC/SV9-1 after MNNG
Treatment (P8)
After MNNG treated cells had been passaged for eight times,
1x105 cells from each treatment group were seeded per 60 mm
dish and cultured for six weeks. The cell numbers in three
dishes was determined at the end of six weeks. Cell counts
were reported as mean ± sd. MNNG treatment caused a dose-
dependent increase in saturation.
137
MNNG-treated clones, we continued to passage MNNG-treated
HFC/SV9-1 cells, and during the process of subcultivation we tested
their ability to form foci, to grow in soft agar, and measured their
saturation densities. As shown in Figures 25a, 25b, 25c, and Table
13, after being passaged eight times in a four month period, the
MNNG-treated HFC/SV9-1 cells formed anchorage independent
colonies in a dose-dependent manner (Table 13). Interestingly, the
frequency of A.I. colonies/10^ survivors increased in MNNG treated
group from passage 1 to passage 8. This indicated that the
expression time of the MNNG-induced A.I. phenotype took many
passages to reach optimal values. More work will be conducted to
determine the contribution of expression time of the A.I. phenotype
vs whether the A.I. cells had a selection advantage over the non-A.I.
cells during passaging and growth of the MNNG-treated populations.
The saturation density of the MNNG treated cells was originally
significantly less (2-100 fold less) than that of the non-MNNG
treated cells at passage 1. However, this value increased with
passage so that at passage 8, the saturation density of MNNG treated
cells was 1.3 to 2-fold that of nontreated cells (Figures 25b and c).
The MNNG-treated cells also showed microscopic focal areas of cell
overgrowth even though this clone did not form obvious foci.
138
Table 13
Induction of Anchorage Independence of HFC/SV9-1 by MNNG
Treatment(P#8)
Group Cytotoxicity (%) # of Al
colonies/dish
# of Al
colonies/105
Survivors
HFC 2.211.48 3.412.3
Control 100±14 9.612.6 5.511.5
Acetone 99.8±15.2 9.211.5 5.310.9
MNNG 0.067
ug/ml
81.4±10.6 21.415.9 1514.0
MNNG 0.125
ug/ml
55.617.2 41.017.31 2915
MNNG 0.25
ua/ml
35.513.5 92.2113.8 79112
MNNG 0.5
ug/ml
18.212.6 13.213.56 1815
139
3.314 Characterization of Cells Derived from Anchorage
Independent Colonies of MNNG treated HFC/SV9-1
Cells
Three anchorage-independent colonies with a diameter of more
than 0.35 mm arising from MNNG-treated HFC/SV9-1 cells were
cloned from soft agar and expanded into cloned cell lines. These A.I.
cell lines, named HFC/SV9-1 Al M1, M2, and M3, along with the
parental HFC/SV9-1 and acetone-treated HFC/SV9-1 cells, were
assayed for anchorage-independent growth, saturation density, and
serum independence of growth (Figures 26 a through e; Figure 27a
and b; Figure 28; Figure 29).
All three clones Al M1, Al M2, and Al M3 grew to higher saturation
densities than HFC/SV9-1 cells from the control and acetone-
treated groups in medium containing either 1% or 10% serum after
sixteen days of in vitro culture (Figure 26 a through e; Figure 27a).
These three anchorage independent colonies either maintained their
cell number (M1, M2) or could grow in medium without serum,
although at very slow rate (M3) (Figure 27b; Figure 28), indicating
that unlike their progenitor HFC/SV9-1 cells, they have acquired the
property of serum independence, which is a phenotype of
transformed cells. Cells derived from those three clones were also
tested for their ability to form soft agar colonies according to the
methods described in Materials and Methods, as shown in Figure 29,
the ability of these three clones to form anchorage independent
140
«
o
E
E
a
o
*
106
20 0 10
n a — 10%
- • — 1%
n a — o %
Days after Seeding
Figure 26a
Growth Curve of HFC/SV9-1 (Control)
1x105 cells were seeded per 60 mm dish and the cell numbers in
three dishes was determined every three days for sixteen days. Cell
counts were reported as mean ± sd. This clone did not grow in
medium without serum.
141
0 10 20
Days attar Seeding
Figure 26b
Growth Curve of HFC/SV9-1 (Acetone)
1x105 cells were seeded per 60 mm dish and the cell numbers in
three dishes was determined every three days for sixteen days. Cell
counts were reported as mean ± sd. This clone did not grow in
medium without serum.
142
*
10 4 -J --------------- .------------------1 -----------------.---------------- 1
0 10 20
Days after Seeding
Figure 26c
Growth Curve of HFC/SV9-1 (Al M-1)
1x105 cells were seeded per 60 mm dish and the cell numbers in
three dishes was determined every three days for sixteen days. Cell
counts were reported as mean ± sd. This clone grew in medium
without serum.
143
N
a
E
E
o
< 0
o
o
*
106
10 20 0
-O 0%
- * 1%
-u 10%
Days after Seeding
Figure 26d
Growth Curve of HFC/SV9-1 (Al M-2)
1x105 cells were seeded per 60 mm dish and the cell numbers in
three dishes was determined every three days for sixteen days. Cell
counts were reported as mean ± sd. This clone grew in medium
without serum.
144
o
*
104- i i " —
0 10 20
Days after Seeding
Figure 26e
Growth Curve of HFC/SV9-1 (Al M-3)
1x105 cells were seeded per 60 mm dish and the cell numbers in
three dishes was determined every three days for sixteen days. Cell
counts were reported as mean ± sd. This clone grew in medium
without serum.
145
E
E
o
< 0
o
o
*
107
106
10®
0 10 20
AIM-1 10%
AIM-2 10%
AIM-3 10%
Acetone 10%
Days after Seeding
Figure 27a
Comparison of the Growth Properties of Anchorage-
Independent Cells Derived from MNNG Treated HFC/SV9-1 in
Medium Containing 10% Fetal Bovine Serum
1x105 cells derived from different Al colonies were seeded into 60
mm dish in 5 ml of medium containing 10% fetal bovine serum. After
sixteen days of culture, the number of cells per 60 mm dish was
determined by trypsinizing and counting the number of cells from
each dish on a Coulter counter. Cell counts were reported here as the
mean ± standard deviation of determinations from three dishes. All
three anchorage-independent-colony-derived clones could grow to
higher saturation density than the acetone treated cells.
146
1000000 1
E
E
o
to
100000
w
a
o
o
*
10000
0 20 10
Al M -1 0%
Al M-2 0%
Al M-3 0%
Acetone 0%
Days after Seeding
Figure 27b
Comparison of the Growth Properties of Anchorage-
Independent Cells Derived from MNNG Treated HFC/SV9-1 in
Medium Containing 0% Fetal Bovine Serum
1x1 ()5 cells derived from different Al colonies were seeded into 60
mm dish in 5 ml of medium containing 0% fetal bovine serum. After
sixteen days of culture, the number of cells per 60 mm dish was
determined by trypsinizing and counting the number of cells from
each dish on a Coulter counter. Cell counts were reported here as the
mean ± standard deviation of determinations from three dishes. All
three anchorage-independent-colony-derived clones could grow in
medium without serum, while the acetone treated cells could not.
147
□ 0%
■ 1%
10%
1 :A c « to n «
2: Al M-1
3: Al M-2
4: Al M-3
Figure 28
Comparison of the Growth Properties of Anchorage-
Independent Cells Derived from MNNG Treated HFC/SV9-1 in
Medium Containing Different Concentrations of Fetal Bovine
Serum
1x 105 cells derived from different Al colonies were seeded into 60
mm dish in 5 ml of medium containing 0, 1%, and 10% fetal bovine
serum respectively. After sixteen days of culture in medium
containing the above mentioned concentrations of fetal bovine
serum, the number of cells per 60 mm dish was determined by
trypsinizing and counting the number of cells from each dish on a
Coulter counter. Cell counts were reported here as the mean ±
standard deviation of determinations from three dishes. All three
anchorage-independent-colony-derived clones could grow in medium
without serum, while the acetone treated cells could not.
148
1 2 3 4 5
Figure 29
Anchorage Independent Assay of Al Cells Derived from
MNNG Treated HFC/SV9-1
1 x105 cells derived from each Al colony were suspended in 3 ml of
0.3% noble agar. The mixture was allowed to solidify on the top of 5
ml 0.5% agar base layer in 60 mm dish. Four weeks later, colonies
greater than 0.1 mm in diameter were scored by microscopic
examination. Cells from control and acetone-treated groups did not
show an obvious increase in anchorage-independent growth
properties while all three clones derived from anchorage
independent colonies showed anchorage-independent growth,
suggesting the stability of this phenotype.
149
colonies was much higher (25 fold to 50 fold) than for HFC/SV9-1
cells from control and acetone treated groups, indicating that this
MNNG-induced anchorage-independence is stable upon passage.
3.315 Transfection of Immortal SV40 Large T Antigen
Clones with Oncogenes
To determine whether the immortal HFC clones could be further
transformed, we transfected the activated oncogenes, ras, myc and
ras+myc into HFC/SV1-1 and HFC/SV1-2 Cl 6 cells by the calcium
phosphate precipitation method (Graham et al., 1973), and then
cultured the dishes for four weeks. The cells on the dishes were
then fixed with methanol and stained with Giemsa. No foci were
observed in any transfection group (Tables 14 and 15). We believe
this was because both of these two clones already had the phenotype
of morphological transformation (high saturation density).
We then transfected both the ras and myc genes into HFC/SV9-1
cells, which had a lower saturation density, and after two weeks
culture, the cells were trypisinized and seeded again into 60 mm
dishes to assay for focus formation, and into soft agar to assay for
anchorage independence. After four weeks the cells on the dishes
were either fixed with methanol and stained with Giemsa to identify
foci, or they were examined under a dissecting microscope to count
anchorage independent colonies. We did not observe obvious
macroscopic foci on the dishes (data not shown), but
150
Table 14
Focus Formation Assay o HFC/SV1-1 after Oncogene Transfection
Oncogene Amount of
Oncogene
Containing
Plasmid
(ua/dish)
# of Dishes Length in Culture
(weeks)
Results
Control 0 5 4 TL*. No Foci
ras 15 5 4 TL, No Foci
mvc 15 5 4 TL, No Foci
ras+myc 15 + 15 5 4 TL, No Foci
* TL, thick cell layer was formed; 1x105 cells were seeded into each 60 mm dish the
day before transfection. On the day of transfection, the DNA precipitate containing 15 pg
of plasmid DNA was added into each dish. Three hours later, 1 ml of 15% glycerol was
added to each dish for two minutes and then aspirated, and the cells were fed with fresh
medium. The transfected cells were then cultured for another four weeks, and then the
cells were fixed with methanol and stained with Giemsa.
151
Table 15
Focus Formation Assay of HFC/SV1-2 Cl 6 after Oncogene
Transfection
Onoogene Amount of
Onoogene
Containing
Plasmid
(ua/dish)
# of Dishes Length in Culture
(weeks)
Results
Control 0 5 4 TL*. No Foci
ras 1 5 5 4 TL. No Foci
mvc 1 5 5 4 TL. No Foci
ras+myc 15 + 15 5 4 TL. No Foci
* TL, thick cell layer was formed; 1x105 cells were seeded into each 60 mm dish the
day before transfection. On the day of transfection, the DNA precipitate containing 15 pg
of plasmid DNA was added into each dish. Three hours later, 1 ml of 15% glycerol was
added to each dish for two minutes and then aspirated, and the cells were fed with fresh
medium. The transfected cells were then cultured for another four weeks, and then they
were fixed with methanol and stained with Giemsa.
152
Table 16
Induction of Anchorage Independence in HFC/SV9-1 by Transfection
of H-ras and myc Oncogenes
Group Seeding Efficiency
(% >
# of Al colonies
/ Dish
# of Colony /105
Survivors
HFC 64.0 2±1 3 1 2
Control (HFC/SV9-
1 )
86.4 9±2 9 1 2
ras
(HFC/SV9-11
105.7 158143 184149
myc
(HFC/SV9-11
91.7 135134 14713 7
1 x105 cells were seeded into each 60 mm dish the day before transfection. On the
day of transfection, the DNA precipitate containing 15 pg of plasmid DNA was added into
each dish. Three hours later, 1 ml of 15% glycerol was added to each dish for two
minutes and then aspirated, and the cells were fed with fresh medium. The transfected
cells were then cultured for another four weeks before being fixed with methanol and
stained with Giemsa. The control group went through the same treatment as the two
transfection groups, except that it was not transfected with plasmid DNA.
153
these cells did form focal areas of cell overgrowth under
microscopic examination. Furthermore, those transfected cells did
form distinct anchorage independent colonies in soft agar (Table 16).
Transfection of ras caused a 22-fold increase and transfection of
myc caused a 19-fold increase in the frequency of A.I. colonies
(Table 16).
154
3.400 Effects of the C-Myc Gene on Human Fibroblasts
3.401 Introduction
The protein product of the c-myc gene is one of the nuclear
proteins involved in the control of cellular proliferation and
differentiation (Cole, 1986). Mutation of the c-m yc gene in the cell
contributes to a wide range of neoplasias such as myelocytomatosis,
endotheliomas, liver and kidney carcinomas, and the in vitro
transformation of both rodent and human fibroblasts (Graf and Beug,
1978; Alexander et al., 1979; Duesberg and Vogt, 1979; Graf et al.,
1981; Hayman, 1983; Bishop and Varmus, 1985). It was reported that
constitutive expression of the c-m yc gene could immortalize
primary rodent fibroblast cells in culture, and in cooperation with
an activated c-Ha-ras gene, this established a fully transformed
phenotype in rodent fibroblastic cells (Land et al., 1983; Ruley,
1990). V-m yc has been utilized to immortalize human fibroblastic
cells (Morgan et al., 1991), but the effects of a constitutively
expressed c-myc gene on the growth of human fibroblast cells have
not been reported. This motivated us to determine whether an
overexpressed c-m yc gene would confer immortality but not the
transformed phenotype on human fibroblastic cells.
155
3.402 Transfection of Normal HFC and Selection of
Transfectants
We obtained a c-myc plasmid construct, which encodes exons 2
and 3 of the myc gene under the control of the SV40 promoter (Land
et al., 1983). This ensured the overexpression of the myc gene once
it was transfected into human fibroblasts. However, this m y c
plasmid construct did not carry a selectable marker like our other
plasmids did. Therefore, our strategy was to transfect the c-m yc
gene into senescing fibroblast cells, and then to observe whether
immortal fibroblast clones would appear. The fibroblast cells were
subcultured to a point close to the end of their life span (senscence),
(passage # 13), and then they were seeded into five 60 mm dishes at
a density of 5x10^ per dish. Those cells were then transfected by
the calcium phosphate method (Graham et al., 1973) with the c-myc
plasmid. Cells were fed with MEM medium every week for three to
four weeks until fast growing colonies appeared. Three control
groups were also cultured. The first control group was transfected
with an H-ras plasmid which carried a mutated c-Ha*ras gene and
was reported capable of transforming both rodent and human cells
(Land et al., 1983; Kinsella et al., 1990). The second control group
was transfected with a plasmid vector, Zip, which carries the SV40
promoter but no gene fragments encoding functional proteins like
myc or ras. The third control group underwent calcium phosphate
precipitate treatment, but without any plasmid present (Table 17).
156
3.403 Biological Characterization of c-myc Gene
Transfected HFC Clones
Three weeks after transfection, three fast-growing colonies
were observed in five dishes of the myc transfected group but not in
the H-ras or Zip transfected groups, nor in the control group. This
suggested that the appearance of these fast growing colonies was
correlated with transfection of the myc gene. These fast growing
colonies were isolated by the glass cylinder cloning ring technique
(Nianjun et al., 1994), expanded into cell strains, and designated myc
1, myc 2, and myc 3. They were characterized by in vitro
transformation characterization assays according to the procedures
described in Materials and Methods.
We found that firstly that cell lines derived from all three m yc
transfected clones demonstrated anchorage-independent growth in
soft agar (10-40 A.I. colonies/105 survivors), while normal human
fibroblast cells did not grow in soft agar (Figure 30). Secondly, we
found that clone myc 1 showed a two-fold higher saturation density
than that of normal fibroblast cells. The other two c-myc
transfected human fibroblast cells did not demonstrate an increased
saturation density at fourteen days after seeding compared to
normal human fibroblast cells (Figure 31). Thirdly, focus
reconstruction assays of myc transfected clones did not show
obvious focus formation by c-m yc transfectants against normal
human fibroblast cells, although we did observe focal areas of
157
Table 17
Results of Transfection of c-myc and an activated ras Oncogenes
into Senescing Human Fibroblasts
Group # of Cells
Transfected
Per Dish
Total #
of Fast
Growing
Colonies
Total #
of
Colonies
Cloned
# of Fast
Growing
Colonies Per
Dish
Total # of
Colonies
Subcultur
ed
Immortal
Cell Lines
Derived
c-myc 5 x 105
(5 dishes)
3 3 0.6±0.9 3 3
H-ras 5 x 105
(5 dishes)
0 0 0 0 0
Zip 5 x 105
(5 dishes)
0 0 0 0 0
Control 5 x 105
(5 dishes)
0 0 0 0 0
158
Table 18
Results of Reconstruction Assays to Detect Focus Formation of c-
myc Immortalized Human Fibroblast Clones against Normal HFC Cells
Clone Plating Efficiency Strength of Foci* in
the Original Dishes
# of Foci per Dish
HFC 14±4 weak 0
Mvc 1 22±7 weak 0
Mvc 2 1 7 ± 6 weak 0
Mvc 3 20±7 weak 0
* Degree of piling-up into multilayers.
159
1 2 3 4
Figure 30
Anchorage-Independent Growth of c-myc Immortalized HFC
Clones
1x105 cells were suspended in 3 ml of 0.3% agar noble. The mixture
was allowed to solidify on the top of 5 ml 0.5% agar base layer in 60
mm dish, five dishes per immortal clone. Four weeks later, colonies
greater than 0.1 mm in diameter were scored by microscopic
examination.
160
1 2 3 4
Figure 31
Saturation Density Curve of c-myc Immortalized HFC
Clones
1x1 ()5 cells were seeded per 60 mm dish, and the cell numbers in
each of three dishes for each clone was determined electronically at
end of two week culture by use of a Coulter.
161
piling-up in cultures of c-myc transfected cells, and the c-myc
transfected cells appeared more retractile than normal fibroblasts,
by microscopic examination (Table 18).
3.404 Conclusions
W e therefore concluded first that transfection of an
overexpressed c-myc plasmid construct into senescing human
fibroblasts could extend their life span, and studies are in progress
to determine whether the myc gene actually immortalized human
fibroblasts. Secondly, we concluded that these myc-transfected
human fibroblast clones have gained the property of anchorage-
independent growth (Figure 30). thirdly, we observed focal piling-up
of cells in the cultures of c-myc transfected human fibroblast
cells, but this property was not strong enough to yield macroscopic
in focus reconstruction assays (Table 18).
162
CHAPTER 4
DISCUSSION
4.100 Cellular Immortality Studies
Cellular senescence has been considered a specific type of
terminal cell differentiation whose biological function is to
counteract uncontrolled cell proliferation (Monti et al., 1992). In
this sense, cellular senescence can be considered one of the most
important mechanisms in avoiding the continuous onset of tumors,
by causing cells with many acquired mutations to die. Therefore,
overcoming senescence is a crucial requirement for a normal cell to
become tumorigenic. Overcoming senescence is therefore also
crucial for studying the process of carcinogenesis with in vitro cell
culture systems (Monti et al., 1992).
For the past twenty years, researchers have attempted to
immortalize normal human cells with various chemical, physical, or
biological agents (McCormick 1994). However, the only agents that
are known to rarely but consistently immortalize diploid mammalian
fibroblasts are DNA tumor viruses, such as simian virus 40 (Shay et
al., 1989), adenovirus (Shay et al, 1993b), and human papilloma virus
(Shay et al., 1993b), activated oncogenes like v-myc (Morgan et al.,
1991), and mutated tumor suppressor genes such as the mutated p53
gene (Rovinski et al., 1988). The immortalizing ability of DNA tumor
viruses has been localized to specific DNA fragments in the viral
163
genome, such as the genes encoding polyoma large T antigen and
SV40 large T antigen (Cherington, 1986; Shay et al., 1989).
To establish an immortal human fibroblast cell line for studying
the process of in vitro chemical transformation in normal human
diploid fibroblasts, we transfected four genes into the human
fibroblastic cells. These included two DNA virus genes encoding
polyoma large T antigen and SV40 large T antigen, an overexpressed
cellular oncogene (c-myc), and one mutated tumor suppressor gene
(p53). Our studies showed that transfection of polyoma large T
antigen, which presumably inactivated one of the two growth
suppressing proteins inside the cell, the retinoblastoma protein (Rb)
(Dyson et al., 1990), extended the life span of normal human
fibroblast cells by four population doublings but did not immortalize
the human fibroblast cells (Table 19). Transfection of a mutated
copy of the p53 tumor suppressor gene into human fibroblast cells
(which disturbs the function of normal p53 inside the cell) extended
the life span of normal human fibroblast cell by fourteen population
doublings, but again did not immortalize the human fibroblasts
(Table 19). Transfection of either of these two genes did not change
the morphology of normal human fibroblasts, and did not change the
phenotype of human fibroblast cells in in vitro transformation
characterization assays. These results are consistent with those of
Hara et al. (1991) who transfected antisense rb oligomer into human
fibroblast cells. This extended the life span of the cells slightly, but
failed to immortalize human fibroblasts. Our findings are also
164
consistent with the results of Shay et al. (1991a and 1991b), who
reported that human fibroblasts could only be immortalized by
eliminating the activity of both P53 and Rb. Inactivation of p53
alone (by viral proteins HPV E6, adenovirus E1b, or a mutant form of
SV40 large T antigen defective in either p53 or Rb binding ) had no
effect on the growth property of human fibroblasts.
P53 and Rb are two major growth suppressing proteins which
probably exert their growth suppressing effects by different
mechanisms (Huppi et al., 1994; Fagen et al., 1994). The Rb protein
inhibits cell cycle progression at the G1/S boundary by inhibiting
E2F transcription factors. E2F transcription factors are involved in
the transcription of myc, myb, thymidine kinase, and other genes
whose products are implicated in the induction of S-phase. This
suggests that the Rb protein may be responsible for causing cells to
traverse the G1/S restriction point of the cell cycle. It has been
shown that E2F overexpression is sufficient to promote DNA
synthesis in growth-arrested cells (Johnson et al., 1993). Therefore,
inactivation of Rb could potentially immortalize human cells.
The p53 gene is the most commonly mutated gene in human cancer
(Hollstein, et al. 1991). Recent data suggest that one function of the
P53 protein is to induce apoptosis. Introduction of the wild-type p53
gene into different tumor cell lines leads to either inhibition of the
growth potential of tumor cell lines and/or a reduction of their
transformed phenotype (Merlo et al., 1994), or to a rapid loss of cell
165
viability and induced apoptosis. (Yonish-Rouache et al, 1991). The
P53 protein can activate transcription of a cell cycle-dependent
kinase inhibitor (CdKI, WAF1), which inhibits several cell cycle-
dependent kinases (Cdk), and therefore cell cycle progression (El-
Deiry et al., 1993; Harper et al., 1993; Noda et al., 1994; Xiong et al.,
1993). Therefore, inactivation of P53 protein could potentially also
lead to immortalization of human cells. In fact, it has been reported
that inactivation of P53 by transfecting a mutated copy of the p53
gene or a viral protein encoding gene such as HPV E6, which
abrogates the function of normal cellular P53 protein, into human
mammary epithelial cells leads to immortalization of epithelial
cells (Sedman et al., 1992; Shay et al., 1993a, 1993b, 1993c).
However, in our study, transfection of a mutated p53 gene, which
presumably disrrupted the function of the normal P53 protein inside
the cells, failed to immortalize human fibroblasts. This may
indicate that the mechanisms regulating senescence are easier to
overcome in epithelial cells than in fibroblasts. Additional
constraints on cellular growth may also exist in fibroblasts that are
not found in epithelial cells. This is consistent with epidemiological
findings which indicate that the total annual incidence of soft
tissue sarcomas (which include sarcomas derived from fibroblast
like cells) in the United States is at least 100 times less common
than cancers of epithelial cell origin such as breast carcinoma
(Pollock, 1992). Of course, the epithelial cells are also the first
cells exposed to carcinogens, particularly in skin and lung.
166
Based our results and the work of Hara et al.(1991) and Shay et al.
(1991a and 1991b), we hypothesize that Rb and P53 together can
induce cell senescence (or apoptosis) in human fibroblasts, and that
these genes function through different mechanisms. If the function
of one protein is disturbed, the other protein can individually induce
an apoptotic response in the fibroblasts. Therefore, elimination of
the function of one of the two growth suppressing proteins, Rb and
P53, was not able to overcome the mechanism that mediates
senescence of human fibroblasts.
The results we obtained from transfecting polyoma large T
antigen and a mutated p53 gene into human fibroblasts suggested
that transfection of a more powerful agent which disrrupts the
function of both growth suppressing genes may give rise to
immortalized human fibroblast cell lines. We therefore transfected
the SV40 large T antigen, which should bind to and inactivate both
Rb and P53 proteins, into the human fibroblasts. Our results showed
that the SV40 large T antigen immortalized human fibroblast cells
(Table 19). It first conferred an increased life span and abnormal
morphology, but most of the cells derived from transfectant
colonies in the cultures eventually entered the senescent (or crisis)
stage. During crisis, some areas of rapidly growing cells appeared
among the senescent cells, and subcultivation of these rapidly
growing clones led to the production of immortal cell lines. This
implies that SV40 large T antigen alone is necessary but not
sufficient to immortalize human diploid fibroblasts. A further
167
spontaneous mutation, in an unknown gene(s), probably has to
cooperate with the genetic changes caused by SV40 large T antigen
to immortalize human fibroblast cells.
Altogether, we obtained fifteen SV40 large T antigen
immortalized cell lines. We observed that one immortal postcrisis
colony appeared from among about 10? precrisis cells in culture,
which is consistent with the mutation frequency at a single gene
locus. This suggests that the secondary genetic change which leads
to the immortalization of SV40 large T antigen transfected human
fibroblasts is due to an inactivating mutation in one allele of
specific gene. This could be caused by mutational inactivation of
that gene or deletion of that gene by chromosome loss due to a non-
disjunctional event during cell division.
The exact mechanism involved in the immortalization of human
fibroblasts by SV40 large T has not been elucidated. The ability of
SV40 large T antigen to bind to and inactivate two cellular
antiproliferative proteins, Rb and P53, (Decaprio et al, 1988; Linzer
et al, 1979) almost certainly plays an important role in the
immortalization process. We propose that binding and subsequent
inactivation of Rb and P53 protein by SV40 large T antigen extends
the life span of human fibroblasts. However, not all the Rb or P53
cellular proteins are bound by the SV40 large T antigen, so the SV40
large T antigen transfected human fibroblast cells still enter
senescence (crisis). In the senescent or crisis stage, one or both
168
copies of chromosomes carrying the p53 or Rb genes may have been
lost or mutationally inactivated during cell division in some cells,
causing a decrease in the amount of p53 or Rb proteins or
elimination of these two proteins inside these cells. These cells
then escaped the senescent state, and became immortal.
Pereira-Smith, et al (1983, 1987) found that when normal human
fibroblast cells with a finite life span were fused with cells
immortalized with simian virus(SV) 40, the great majority of hybrid
cells showed the senescent phenotype, i.e. a highly reduced
proliferative capability. This suggests that the immortal phenotype
of SV40 large T antigen immortalized human fibroblasts is
recessive. This recessive nature of immortality explains why
immortal cells appeared only after the SV40 large T antigen
transfected human cell populations had gone into crisis. Even if the
potential immortal cells had appeared before the whole cell
population went into crisis, intercellular communication between
immortal cell and its surrounding precrisis mortal cells may have
transferred the Rb and P53 proteins from mortal precrisis cells to
the immortal cells and therefore Killed potential immortal cells.
Therefore, no matter how early such infinite-life span cells could
arise in the cell population, the only opportunity for infinite life
span cells to survive may be in the crisis stage, where cell-to-cell
contact was absent.
Our results showed that the phenotypes of SV40 large T antigen
169
immortalized human fibroblasts varied among individual clones.
Most of the immortal clones had various degrees of transformed
phenotypes. The exact mechanism why each individual human
fibroblast clone immortalized by SV40 large T antigen had different
combinations and degrees of transformed phenotypes is not clear.
The degree of transformation did correlate well with the levels of
SV40 large T antigen inside the cell. The clone which has the least
transformed phenotype, HFC/SV9-1, also had the least amount of
SV40 large T antigen among all immortal clones.
SV40 large T antigen appears to be vital for maintaining the
immortality of human fibroblasts (Tanaka et al., 1992; Price et al.,
1994) and responsible for the transformed phenotype of human
fibroblasts. Transfection of antisense SV40 large T antigen into
human fibroblast cells immortalized by SV40 large T antigen caused
repression of the SV40 large T antigen, which resulted in
senescence of immortal fibroblast cells (Tanaka et al., 1992). Price
et al. (1994), using an inducible SV40 large T antigen expression
vector, found that high expression of the SV40 large T antigen in
human fibroblasts resulted in a high rate of proliferation, an
extended in vitro life span, loss of contact inhibition of growth, and
a morphology of characteristic of transformed cells. Reduction of
SV40 large T antigen expression using a low level of inducing agent
was accompanied by a reduction in proliferative rate and restoration
of normal cell morphology and contact inhibition, similar to that
found in nontransformed cells (Price et al., 1994). The rate of
170
proliferation of the cell, transformed morphology and loss of
contact-inhibition were dependent upon the amount of SV40 large T
antigen present in the cell. This suggests that the phenotypes of
SV40 large T antigen immortalized human fibroblasts could be
regulated by the quantatity of SV40 large T antigen present inside
the cell.
Our results supported this hypothesis. We showed that the
transformed phenotype correlates with the quantity of SV40 large T
antigen inside the cells. Immortal clones with more transformed
phenotypes tended to have higher amounts of intracellular SV40
large T antigen, while the immortal clones with the least
transformed phenotype had the least amount of SV40 large T antigen.
This also implies that the processes of both immortalization and
transformation are multistep processes and the transformed
phenotypes are separate from immortality in human fibroblasts.
4.200 In Vitro Chemical Transformation Studies
Once we obtained the immortal but morphologically near normal
human cell line, HFC/SV9-1, we then treated this cell line with a
chem ical carcinogen, N -m ethyl-N ’-nitro-N -nitroso-guanidine
(MNNG), an alkylating agent which has been demonstrated to be
carcinogenic in animal models and induces cell transformation in
vitro in mammalian cell culture assays (Milo et al., 1978). We also
transfected this clone with a mutated H-ras oncogene and a c-myc
construct under the control of the SV40 large T promoter (Land et
171
al., 1983). We used three quantitative assays to select transformed
cells from untransformed cell populations - focus formation,
anchorage independence, and saturation density assays. However, of
all three in vitro quantitative transformation assays we used, only
the soft agar assay showed positive results. The focus formation
assay was positive for foci, but we did not obtain good monolayers
in this assay. The saturation density assay did not show obvious
changes after MNNG treatment, even after long term passage. This
suggested that the saturation density was not very sensitive. It is
also possible that human fibroblast cells may have their own unique
features of transformation which are different from that of mouse
fibroblastic cells, so that the above two assays could not detect
those unique transformed phenotypes in human fibroblasts.
We did successfully transform immortal human fibroblast cells
to anchorage independence with MNNG treatment in a dose-dependent
manner and with oncogene transfection. However, contrary to ras and
myc oncogene transfected cells, which demonstrated anchorage
independence soon after introduction of the oncogene into the cells,
MNNG treated immortal cells had to be subcultured for four to six
months before a dose-dependent anchorage independence could be
induced in these cells. This suggests that MNNG is not as strong an
agent in transforming human fibroblasts as other biological agents
like ras and m yc. It also implies that the process of chemical
carcinogenesis is a multistep process, and that multiple genetic
mutations need to be accumulated in the cell genome for the
172
chemical carcinogen treated cells to demonstrate this transformed
phenotype.
The anchorage independence assay has been proved to be a very
useful quantitative assay to permit the selective growth of
transformed human cells by inhibiting the growth of normal cells
(Milo et al, 1981, Borek, et al., 1980). However, the exact changes
responsible for anchorage independence are not fully understood.
There are many suggestions from other studies that anchorage-
independence relates to the endogenous synthesis of protein growth
factors and other modifications of growth factor pathways
(McCormick et al., 1987). The fact that high concentrations of serum
(Peehl and Stanbridge 1981; McCormick et al., 1987), and platelet-
derived growth factor (Palmer et al., 1987), causes human
fibroblasts to form colonies in soft agar is consistent with this
hypothesis. We also found that cells derived from anchorage-
independent colonies cloned from MNNG treated HFC/SV9-1 could
grow in medium without fetal bovine serum. This suggests that they
are able to synthesize their own growth factors. These soft agar-
derived cells could form large numbers of colonies when they were
reseeded into soft agar. This implies that they have acquired the
anchorage-independent phentype stably. It suggests that the
property of anchorage independence is probably caused by genetic
changes, which is consistent with our previous findings that
carcinogenic metal salts or MNNG-induced anchorage-independent
173
cell strains stably retained their anchorage-independent phenotype
even after successive passages in vitro (Biedermann and Landolph,
1987)
4.300 C-myc Oncogene
The cellular proto-oncogene c-myc is involved in regulating cell
proliferation, transformation, and apoptosis (Marcu et al., 1992;
Evan et al., 1992). Induction of myc is sufficient to drive quiescent
cells into the cell cycle (Eilers, 1991), suggesting its role in
regulating cell cycle progression. So far, the exact mechanism by
which myc regulates cellular proliferation is not clear. In mouse
fibroblasts, c-myc is dominant over p53-mediated growth arrest and
can drive cells from the G1 into S phase despite large amounts of
wild-type p53 protein (Hermeking and Eick, 1994).
By transfecting a c-myc plasmid construct under the SV40
promoter control into senescing human fibroblasts, we obtained
three myc-transfected clones that grew post the crisis period. This
suggests that transfection of this overexpressed c-myc plasmid
could immortalize or at least extend the life span of normal human
fibroblasts (Table 19). The transformation characterization assays
showed that the myc-transfectants grew in soft agar. Microscopic
examination showed that myc transfectants exhibited transformed
morphologies, such as enlarged cell size, prominant nuclei, and a
more refractile appearance, but these transfectants grew to only a
slightly higher saturation density than normal human fibroblasts,
174
and did not form obvious macroscopic foci (Table 19). . For these
m yc clones, the expression status of the m yc gene should be
determined to establish the correlation between c-myc expression
and immortality of human fibroblasts. The immortality and extended
life span of these myc immortalized clones should also be assayed
as we have done with the SV40 large T antigen immortalized human
fibroblast cell lines. N evertheless, we demonstrated that
transfection of an overexpressed c-myc plasmid construct into
human fibroblasts extended the life span of human fibroblastic cells
(Table 19). All the c-myc transfectants exhibited a less transformed
phenotype than SV40 large T antigen transfected human fibroblasts.
These m yc transfected clones are therefore provided another
promising cell source for studying the multistep process of in vitro
chemical transformation.
4.400 Summary and Prospectives
We established an immortal and otherwise phenotypically near
normal human fibroblast cell line by selecting the SV40 large T
antigen immortalized human fibroblast clone which had the lowest
levels of SV40 large T antigen in the cells. We also showed that this
cell line could be transformed to dose-dependent anchorage
independent growth, which is one of the properties of tumor derived
cells by MNNG treatment. The anchorage-independent cells showed
serum independence and a stable anchorage-independent phenotype.
This cell clone therefore provided a tool to study the process of in
175
Table 19
Summary of the Effects of Various Genes on the Phenotypes of
Human Diploid Fibroblasts
Gene
Transfect
ed
Extended
Life Span
Immortaliza
-tion
Morphologi
-cal
Transforma
-tion
Anchorage
In
dependence
Focus
Induction in
Recon
struction
Assays
Increased
Saturation
Density
Polyoma
Large T
Antiqen
yes
4 pd
no - - - -
Mutated
p53 Gene
yes
14 od
no - - - -
C-myc
Gene
yes
(20 Dd)
+?
In Progress
+
(Weak Foci)
+ + / - + /-
H-ras Gene no no ND ND ND ND
SV40 Large
T Antigen
yes yes + + + +
ND: not determined.
176
vitro chemical transformation. We now need to determine finally
whether these anchorage-independent cells have acquired the ability
to cause tumors in vivo to ensure the effectiveness of this in vitro
chemical transformation assay. We also need to determine which
genes are attacked and the changes in these gene that are
responsible for anchorage-independent growth after MNNG treatment
in this cell clone. This could be achieved by comparing differences in
oncogene or tumor suppressor gene between the anchorage-
independent cells and their parental cells which have the anchorage-
dependent growth property.
W e observed under microscopic examination morphological
changes in MNNG-treated cells, such as focal areas of cell
overgrowth, but no obvious macroscopic foci were observed.
Therefore, further studies need to be conducted to delineate the
relationship between this microscopic view of focal areas of cell
overgrowth and the dosage of MNNG treatment so that a quantatitive
assay suitable for measuring transforming ability of MNNG could be
established.
It is also possible that human fibroblast cells have their own
unique features of chemical transformation which are different
from those of the mouse fibroblastic cells. It is therefore important
to discover other features correlating best with chemical
transformation in human fibroblast cells. This could be achieved by
observing closely the morphology of chemical carcinogen-treated
177
immortal human fibroblast cells or assaying the phenotypes of these
chemical carcinogen-treated immortal human fibroblast cells with
other transformation characterization assays we have not tried.
The c-m yc immortalized human fibroblast clones did not form
obvious foci on focus reconstruction assay and did not grow to
higher saturation density than human fibroblasts. They are also
potential candidate cell lines for use for in vitro chem ical
transformation assays. However, those cell lines need to be further
characterized to determine their suitability for in vitro chemical
transformation assays, because we observed obviously under
microscope that these myc transfectants exhibited a transformed
morphology, such as enlarged cell size, prominant nucleus, and a
more refractile appearance. The extended life span should also be
determined by measuring the popualtion doublings each individual
myc clone could undergo to ensure they can divide enough population
doublings for in vitro chemical transformation assays.
We believe that the immortal cell clones we have generated will
be useful for studying the process of in vitro chem ical
transformation in human cells. We also project that with these cell
culture systems we could derive an overall view of the molecular
events which are responsible for converting a normal human cell
into a malignant cell after chemical carcinogen treatment. We
further expect that these cell culture systems will provide not only
insight into the mechanisms of chemical carcinogen-induced
178
carcinogenesis, but also information on potential genes that can be
targeted for intervention in the prevention, detection, and treatment
of chemical carcinogen induced cancer.
179
BIBLIOGRAPHY
Albino, A.P., Le, S.R., Oliff A.I., Furth.M.E. and Old, L.J. (1984):
Transforming ras genes from human melanoma: A manifestation of of
tumor heterogeity? Nature 308: 69-72.
Alderson, M.(1986): Epidemiological method, in Alderson, M. (eds):
Occupational Cancer., Butterworthy, London, pp 1-24.
Alexander, R. W., Moscovici, C. and Vogt, P.K. (1979): Avian oncovirus
Mill Hill No2: Pathogenicity in chickens. J. Natl. Cancer Inst. 62, 359-
366.
Allsopp, R.C., Vaziri, H, Patterson, C. Goldstein, S., Younglai, E.V.,
Futcher, A.B., Greider C.W., Harley, C.B. (1992): Telomere length
predicts replicative capacity of human fibroblast. Proc Natl Acad.
Sci USA 89: 10114-10118.
Asselin, C., Gelinas, C., and Bastin, M. (1983): Role of the three
polyoma virus early proteins in tumorigenesis. Mol. Cell. Biol. 3,
1 451-1459.
Asselin, C., and Bastin, M. (1985): Sequences from polyoma virus and
simian virus 40 large T genes capable of immortalizing primary rat
embryo fiblasts. J. Virol. 56, 958-968.
Asselin, C., Vass-Marengo, J., and Bastin, M. (1986): Mutation in the
polyoma virus genome that activates the properties of large T
associated with neoplastic transformation. J. Virol. 57, 165-172.
Baker, S.J., Markowitz, S., Fearon, E.R., Wilson, J.K.V., Vogelstein, B.
(1990): Suppression of human colorectal carcinoma cell growth by
wild-type p53. Science, 249, 912-915.
180
Bellett, A.J.D. and Younghusband, H.B. (1979): Spontaneous mutagen-
induced and adenovirus-induced anchorage-independent tumorigenic
variants of mouse cells. J. Cell Physiol. 101, 33-48.
Berwald, Y, Sachs L (1963): In vitro transformation with chemical
carcinogens. Nature 200: 1182-1184.
Berwald, Y, Sachs, L (1965): In vitro transformation of normal cells
to tumor cells by carcinogenic hydrocarbons. J Natl Cancer Inst 35:
6 41-657.
Bettger, W.J.,S.T.Boyce, B.J. Walthall and R.G. Ham (1981): Rapid
clonal growth and serial passage of human diploid fibroblasts in a
lipid-enchriched synthetic medium supplemented with epidermal
growth factor, insulin, and dexamethasone, Proc. Natl Acad. Sci USA
78:5588-5592.
Biedermann, K.A. and Landolph, J.R. (1987): Induction of anchorage
independence in human diploid forskin fibroblasts by carrcinogenic
metal salts. Cancer Res. 47: 3815-3823.
Biedermann, K.A. and Landolph, J.R. (1990): Role of valence state and
solubility of chromium compounds on induction of cytotoxicity,
mutagenesis, and anchorage independence in diploid human
fibroblasts. Cancer Res. 50, 7835-7842.
Bishop, J.M. and Varmus, H. (1985): Molecular Biology of Tumor
Viruses: RNA Tumor Viruses. Supplements and Appendixes. Weiss, R.,
Teich, N., Varmus, H. and Coffin, J. (eds) Cold Spring Harbor
Laboratory, Cold Spring Harbor: New York, pp 249-356.
Blackburn, E.H. (1991): Telomeres. Trends in Biochem 16:378-381.
Blackburn, E.H. (1991): Structure and function of telomeres. Nature
350: 569-573.
181
Blackburn, E.H. (1992): Telomerase. Annu Rev Biochem 61:113-129.
Borek, C. (1980): X-ray-induced in vitro neoplastic transformation of
human diploid cells. Nature 283: 776-778.
Burbeck, S., Latter, G., Metz, E., Leavitt, J. (1984): Neoplastic human
fibroblast proteins are related to epidermal growth factor
precursor. Proc. Natl Acad Sci USA 81: 5360-5363.
Cepko, C.L.; Roberts, B.E., and Mulligan, R.C. (1984): Construction and
application of a highly transmissible murine retrovirus shuttle
vector. Cell 37, 1053-1057.
Chang, P.L., Gunby, J.L., Tomkins, D.J., Mak, I., Rosa, N.E. and Mak, S.
(1986): Transformation of human cultured fibroblasts with plasmids
carrying dominant selection marker and immortalizing potential.
Exp. Cell. Res. 167, 407-416.
Chen, T.R.(1977): In situ detection of mycoplasma contamination in
cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res 104,
255-262.
Chen, P.L., Chen, Y.M., and Bookstein, R. (1990): Genetic mechanisms
of tumor suppression by the human p53 gene. Science 250, 1576-
1580.
Cherington, V., Morgan, B., Spiegelman, B.M., and Roberts, T.M. (1986):
Recombinant retroviruses that transduce induvidual polyoma tumor
antigens: Effects on growth and differentiation. Proc. Natl. Acad. Sci.
USA. 83, 4307-4311.
Counter, C.M., Avilion, A.A., LeFeuvre, C.E., Stewart, N.G., Greider,
C.W., Harley, C.B., Bacchetti, S (1992): Telomere shortening
associated with chromosome instability is arrested in immortal
cells which express telomerase activity. EMBO J 11: 1921-1929.
182
Courtneidge, S.A.and Smith, A.E. (1983): Polyoma virus transforming
protein associated with the product of the c-src cellular gene
Nature 303, 435-439.
Cowie, A., Villiers, J.D., and Kamen, R. (1986): Immortalization of rat
embryo fibroblasts by mutant polyoma virus large T antigens
deficienct in DNA binding. Mol. Cell. Biol. 6, 4344-4352.
Curatolo, L., Erba, E. and Morasca, L. (1984): Culture conditions
induce the appearance of immortalized C2H mouse cell lines, In
Vitro, 28, 597-601.
Daly, M.B. (1993): Epidemiology of Cancer in Clinical Oncology. Weiss,
G. R. (eds), Appleton & Lange, East Norwalk, CT, pp 19-29.
Decaprio, J.A., Ludlow, J.W., Figge, J., Shew, J-Y, Huang, C-M, Lee, W-
H, Marsilio, E, Paucha, E, Livingston, D.M. (1988): SV40-large tumor
antigen forms a specific complex with the product of the
retinoblastoma susceptibility gene. Cell 54: 275-283.
Deleo, A.B., Jay, G., Appeila, E., Dubois, G.C., Law, L.W., and Olds, L.J.
(1979): Detection of a transformation related antigen in chemically
induced sarcomas and other transformed cells of the mouse. Proc.
Natl. Acad. Sci. USA. 76, 2420-2424.
Duesberg, P.H. and Vogt, P.K. (1979): Avian acute leukemia virus
MC29 and MH2 share specific RNA sequences: evidence for a second
class of transforming genes. Proc. Natl. Acad. Sci. USA 76, 1633-
1637.
Dyson, N., Bernards, R., Friend, S.H., Gooding, L.R., Hassell, J.A., Major,
E.O., Pipas, J.M., Vandyke, T., and Harlow, E. (1990): Large T antigens
of many polyoma virus are able to form complex with the
retinoblastoma protein. J. Virol. 64, 1353-1356.
El-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., Vogelstein, B.
183
(1992): Human genomic sequences define a consensus binding site
for p53. Nat Genet 1: 45-49.
El-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., Vogelstein, B.
(1992): Cell 75, 805-816, 1993
Eliyahu, D., Raz, A., Gruss, P., Givol, D., and Oren, M. (1984):
Participation of p53 cellular tumor antigen in transformation of
normal embryonic cells. Nature, 312, 646-649.
Eliyahu, D., Michalowitz, D., and Oren, M. (1985): Overproduction of
p53 antigen makes established cells highly tumorigenic. Nature 316,
158-160.
Eliyahu, D., Michalowitz, D., and Eliyahu, S. (1989): Wild type p53 can
inhibit oncogene mediated focus formation. Proc. Natl. Acad. Sci. USA
8 6 , 8763-8767.
Evan, G.l. et al. (1992): Induction of apoptosis in fibroblasts by c-
myc protein. Cell 69, 119-128.
Fagan, R., Flint, K.J., and Jones, N. (1994): Phosphorylation of E2F-1
modulates its interaction with the rb gene product and the
adenoviral E4 19 kDa protein. Cell 78: 799-811.
Finlay, C.A., Hinds, P.W., Levine, A. J. (1989): The p53 protooncogene
can act as a suppressor of transformation. Cell 57, 1083-1093.
Franceschi, C. (1989): Cell proliferation, cell death and aging. Aging
1:3-15.
Freedman, V.H., Shin, S.l. (1974): Cellular tumorigenicity in nude
mice: correlation with cell growth in semi-solid medium. Cell 3,
3 5 5-3 5 9 .
184
Fry, D.G., Milam, L.D., Maher, V.M., and McCormick, J.J. (1986):
Transformation of diploid human fibroblasts by DNA transfection
with the v-sis oncogene. J Cell Physiol 128, 313-321.
Fry, D. G„ Hurlin, P. J., Maher , V. M., McCormick, J. J. (1988):
Transformation of diploid human fibroblats by transfection with the
v-sis, PDGF2/c-sis, or T24 H-ras genes. Mutat Res 199:341-351.
Funk, W.D., Pak, D.T., Karas, R.H., Wright, W.E., Shay, J.W. (1992): A
transcriptionally active DNA-binding site for human p53 protein
complex. Mol Cell Biol 12:2866-2871.
Girardi, A.J., Jensen, F.C. and Koprowski, H. (1965): SV40-induced
transformation of human dipliod cells: crisis and recovery. J. Cell.
Comp. Physiol. 65, 69-84.
Graf, T. and Beug, H. (1978): Avian leukemia viruses interaction with
their target cells in vivo and in vitro. Bichim. Biophys. Acta, 516,
2 6 9 -2 9 9 .
Graf, T., von Kirchbach, A and Beug, H. (1981): Characterization of
the hematopoietic target cells of AEV, MC29 and AMV Avian
leukemia virus. Exp. Cell Res., 131, 331-343.
Grahm, F.L. and Van der Eb, A.J. (1973): A new technique for the assay
of infectivity of the human adenovirus 5 DNA. Virology 52, 456-467.
Gregory, C. D., C. Dive, S. Henderson, C. A. Smith, G. T. Williams, J.
Gordon and A. B. Rickinson. (1991): Activation of Epstein-Barr virus
latent genes protects human B cells from death by apoptosis. Nature
349: 612-614.
Greider, C.W. (1990): Telomeres, telomerase and senescence.
Bioessays 12: 363-369.
185
Halbert, C.L., Demers, G.W., Galloway, D.A. (1991): The E7 gene of
human papillomavirus 16 is sufficient for immortalization of human
epithelial cells. J Virol 65: 473-478.
Hall, A., Marshall, C.J., Spurr, N.K., Weiss, R.A. (1983): Identification
of transforming gene in two human sarcoma cell lines as a new
member of the ras gene family located in chromosome I. Nature, 303:
3 9 6 -4 0 0 .
Hara, E., Tsurai, H., Shinozaki, A., Nakada, S., Oda, K.(1991):
Cooperative effect of antisense Rb and antisense p53 oligomers on
the extension of lifespan in human diploid fibroblasts, TIG-1.
Biochem Biophys Res Commun 179: 528-534.
Harley, C.B., Futcher, A. B. Greider, C.W. (1990): Telomeres shorten
during aging of human fibroblasts. Nature 345: 458-460.
Harley, C.B. (1991): Telomere loss: mitotic clock or genetic time
bomb? Mutation Res 256: 271-282.
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K. and Elledge, S. J.
(1993): The p21 CdK-interacting protein Cip 1 is a potent inhibitor
of G1 cyclin-dependent kinases. Cell 75, 805-816.
Noda, A., Ning, Y., Venable, S. F., Pereira-Smith, O. M. and Smith, J. R.
(1994): Cloning of senescent cell-derived inhibitors of DNA
synthesis using an expression screen. Exp. Cell Res. 211, 90-98.
Hartwell, L. (1992): Defects in a cell cycle checkpoint may be
responsible for the genomic instability of cancer cells. Cell 71: 543-
546.
Hayman, M.J. (1983): Avian acute leukemia viruses. Curr. Topics
Microbiol. Immunol., 103, 109-126.
186
Hinds, P.W., Finlay, C.A., Frey, A.B., and Levine, A.J. (1987):
Immunological evidence for the association of p53 with a heat shock
protein, hsc 70, in p53-plus-ras-transformed cell lines. Mol. Cell.
Biol., 7, 2863-2869.
Hinds, P.W., Finlay, C.A., and Levine, A.J. (1989): Mutation is required
to activate the p53 gene for cooperation with the ras gene and
transformation. J. Virol. 63, 739-746.
Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R. D., and
Korsmeyer, S. J. (1990): Bcl-2 is an inner mitocondrial membrane
protein that blocks programmed cell death. Nature 348: 334-336.
Hollstein, M, Sidransky, D., Vogelstein, B, Harris, C.C. (1991): p53
mutations in human cancer. Science 253: 49-52.
Huppi, K., Siwarski, D., Dosik, J., Michieli, P., Chedid, M., Reed, S.,
Mock, B., Givol, D., and Mushinski, J.F. (1994): Molecualr cloning,
sequencing, chromosomal localization and expression of mouse p21
(Waf1). Oncogene, 9(10): 3017-3020.
Hurlin, P. J., Fry, D. G., Maher, V. M., McCormick, J. J. (1987):
Morphological transformation, focus formation, and anchorage
independence induced in diploid human fibroblasts by expression of
of a transfected H-ras oncogene. Cancer Res 47:5752-5757.
Hurlin, P. J., Maher, V. M., and McCormick, J. J. (1989): Malignant
transformation of human fibroblasts caused by expression of
transfected T24 HRAS oncogene. Proc Natl Acad Sci USA 86: 187-
191.
Huschtscha, L.I., Holliday, R. (1983): The limited and unlimited
growth of SV40 transformed cells from human diploid M RC-5
fibroblast. J Cell Sci 63: 77-99.
187
Ide, T., Tsuji, Y., Nakashima, T. and Ishibashi, S. (1984): Progress of
aging in human diploid cells transformed with a tsA mutant of SV40.
Exp. Cell. Res. 150, 321-328.
Jat, P.S. and Sharp, P.A. (1986): Large T antigens of SV40 and
polyoma virus efficiently establish primary fibroblasts. J. Virol. 59,
746-750.
Jenkins, J.R., Rudge, K., and Currie, G.A. (1984): Cellular
immortalization by a cDNA clone encoding the transformation
associated phosphoprotein p53. Nature, 312, 651-654.
Jenkins, J.R., Rudge, K., Chumakov, P., and Currie, G.A. (1985): The
cellular oncogene p53 can be activated by mutagenesis. Nature, 317,
816-818.
Johnson, D. J., Schwartz, J. K., Cress, W. D., and Nevins, J. R. (1993):
Expression of transcription factor E2F1 induces quiescent cells to
enter S phase. Nature 365, 349-352.
Kakunaga, T.(1973): A quantitative system for assay of malignant
transformation by chemical carcinogens using a clone derived from
Balb C /3T3. Int J Cancer 12: 463-473.
Kakunaga, T. (1985): Critical review of the use of established cell
lines for in vitro cell transformation. In Kakunaga T, Yamasaki, H
(eds)"Transformtion Assay of Established cCell Lines: Mechanism and
Applications." Lyon, France: IARC, pp 55-73.
Kaplan, P.L., Simon, S., and Eckhart, W. (1985): Polyomavirus middle T
antigen protein encoded by a Retrovirus transforms nonestablished
chicken embryo cells. J Virol., 56, 1023-1026.
Kaplan, P.L, Simon, S., Cartweight, C., and Eckhart, W. (1987): cDNA
cloning with a retrovirus expression vector: generation of a pp60c ~
src cDNA clone. J Virol., 61, 1731-1734.
188
Kastan, M.B., Zhan, Q., El-Deiry, W.S., Carrier, F., Jacks, T., Walsh,
W.V., Plunkett, B.S., Vogelstein B, Fornace, A.J. (1992): A mammalian
cell cycle checkpoint pathway utilizing p53 and GADD45 is defective
in ataxia-telangiectassia. Cell 71: 587-597.
Kern, S.E., Kinzler, K.W., Bruskin, A., Jarosz, D., Friedman, P, Prives.,
C., Vogelstein, B. (1991): Identification of p53 as sequence-specific
DNA-binding protein. Science 252: 1708-1710.
Kern, S.E., Pietenpol, J.A., Thiagalingam, S., Seymour, A., Kinzler,
K.W., Vogelstein, B. (1992): Oncogenic forms of p53 inhibit p53-
regulated gene expression. Science 256: 827-829.
Klein-Szanto, A.J.P., lizasa, T., Momiki, S., Garcia-Palazzo, I.,
Caamano, J., Metcalf, R., Welsh, J., Harris, C.C. (1992): A tobacco-
specific N-nitrosamine or cigarette smoke condensate causes
neoplastic transformation of xenotransplanted human brochial
epithelial cells. Proc. Natl. Acad. Sci. USA 89, 6693-6697.
Land, H., Parada, L.F.and Weinburg, R.A. (1983): Tumorigenic
conversion of primary fibroblasts requires at least two co-operating
oncogenes. Nature, 304: 596-602.
Landolph, J.R., Heidelberger, C. (1979): Chemical carcinogens produce
mutations to ouabain resistance in transformable C3H10T1/2 CI8
mouse fibroblasts. Proc. Natl. Acad. Sci. USA, 76: 930-934.
Landolph, J.R. (1985): Chemical transformation in C3H10T1/2 CI8
mouse embryo fibroblasts: Historical background, assessment of the
transformation assay, and evolution and optimization of the
transformation assay protocol. In Kakunaga T, Yamasaki H
(eds)^Transformation Assay of the Established Cell Lines:
Mechanism ansd Applications." Lyon, France: IARC, pp185-198.
Landolph J.R. (1989): Molecular and cellular mechanisms of
transformation of C3H10T1/2 cl 8 and diploid human fibroblasts by
189
unique carcinogenic nonmutagenic metal compounds A review.
Biological Trace Element Res. 21, 459-467
Landolph, J. R. (1994) Molecular Mechanisms of Transformation of
C3H/10T1/2 Cl 8 Mouse Embryo Cells and Diploid Human Fibroblasts
by Carcinogenic Metal Salts. Environmental Health Perspectives.
102(Suppl 3): 119-125.
Lane, D., and Crawford, L.V. (1979): T antigen is bound to a host
protein in SV40 transformed cells. Nature 278, 261-263.
Lane, D., and Benchimol, S. (1990): Oncogene or anti-oncogene? Gene
Develop 4, 1-8.
Levine, A. J. (1990): Tumor suppressor genes. Bioassays 12: 60-67.
Levine, A. J. and J. Momand. (1990): Tumor suppressor genes: The p53
and retinoblastoma sensitivity genes and gene products. Biochem.
Biophys. Acta 1032: 119-136.
Levine, A. J. and J. Momand., Finlay, C.A. (1991): The p53 tumor
suppressor gene. Nature 351, 453-456.
Levy, M.Z., Allsopp, R.C., Futcher, A.B., Greider, C.W., Harley, C.B.
(1992): Telomere end-replication problem and cell aging. J Mol Biol
225: 951-960.
Liang, T.J., Carmichael, G.G., and Benjamin, T.L. (1984): A polyoma
mutant that encodes small T antigen but not middle T antigen
demonstrates uncoupling of cell surface and cytoskeletal changes
associated with cell transformation. Mol Cell. Biol.4, 2774-2783.
Linzer, D.I.H., and Levine, A.J. (1979): Characterization of a 54K
dalton cellular cellular SV40 tumor antigen present in SV40
transformed cells and uninfected embryonal carcinoma cells. Cell
17:43-52.
190
Lomax, C.A., Bradley, E., Weber, J. and Bourgaux, P. (1978):
Transformation of human cells by temperature sensitive mutants of
simian virus 40. Intervirology 9, 28-38.
Lu, X, Park, S.H., Thompson, T.C., Lane D.P. (1992): ras-induced
hyperplasia occurs with mutation of p53, but activated ras and myc
together induce carcinoma without p53 mutation. Cell 71: 543-546.
MacPherson (1973) I. Soft Agar Techniques. In: P.F. Kruse, Jr., and
M.K. Petterson, Jr., (eds), Tissue Culture: Methods and Applications,
New York: Academic Press, pp. 276-283.
Maher, V.M., Rowan, L.A., Silinskas, K.C., Kately, S.A., and McCormick
(1982): Frequency of UV-induced neoplastic transformation of
diploid human fibroblsts is higher in xeroderma pigmentosum cells
than in normal cells. Proc. Natl. Acad. Sci. USA. 79, 2613-2617.
Maher, V.M., Domoradzki, J., Bhattacharyya, N.P., Tsujimura, T.,
Corner, R.C., and McCormick, J.J. (1990): Alkylation damage, DNA
repair and mutagenesis in human cells. Mutation Research, 233, 235-
245.
Marcu, K.B., Bossone, S. A., Patel, A. J. (1992): Myc function and
regulation. Annu. Rev. Biochem. 61, 809-860.
Mayne, L.V., Priestly, A., James, M.R., and Burke, J.F. (1986): Efficient
immortalization and morphological transformation of human
fibroblasts by transfection with SV40 DNA linked to a dominant
marker. Exp. Cell. Res. 162, 530-538.
McCormick, F. and Harlow, E. (1980): Association of a murine 53000
dalton phosphoprotein with simian virus 40 large T antigen in
transformed cells. J. Virol. 34, 213-219.
McCormick, J. J., Silinskas, K. C. and Maher, V. M. (1980):
Transformation of of diploid human fibroblasts by chemical
191
carcinogens, In: Pullman, B., Ts'o, P.O.P. and Gelboin, H.(eds),
Carcinogenesis: Fundamental Mechanisms and Enviromental Effects,
Reidel, Dordrecht, pp. 491-498.
McCormick, J.J., Kateley-Kohler, S., Watanabe, M, Maher, V.M. (1986):
Abnormal sensitivity of fibroblasts from xeroderma pigmentosum
variants to transformaed to anchorage independence by ultraviolet
irradiation. Cancer Res., 7, 489-492.
McCormick, J.J., Kateley-Kohler, S., Maher, V.M.(1987): Methods for
quantifying carcinogen-induced transformation of diploid human
fibroblasts to anchorage independence. J. Tissue Cult. Meth., 10,
189-196.
McCormick, J.J. and Maher, V.M .(1994): Analysis of the Multistep
process of carcinogenesis using human fibroblasts. Risk Analysis, 14
(3), 257-263.
Merlo, G.R., Venesio, T., Taverna, D., Marte, B.M., Callahan, R., and
Hynes, N.E.(1994): Growth suppression of normal mammary epithelial
cells by wild-type p53. Oncogene, 9, 443-453.
Milo, G.E., Dipaolo, J.A.(1978): Neoplastic transformation of human
diploid cells in vitro after chemical carcinogen treatment. Nature
275: 130-132.
Milo, G.E., Oldham, J.W., Zimmerman, .R., Hatch, G.G., and Weisbrode,
S.(1981a): Characterization of human cells transformed by chemical
and physical carcinogens. In Vitro: 17: 719-729.
Milo, G.E., Weisbrode, S.A., Zimmerman R., McCloskey (1981b):
Ultraviolet radiation-induced neoplastic transformation of normal
human cells in vitro. Chem Biol Interact., 36, 45-59.
192
Milo, G.E., Casto, B., Ferrone, S.(1988): Comparison of features of the
carcinogen-transformed human cells in vitro with sarcoma-derived
cells, Mutation Res., 199, 399-410.
Miyaki, M., Akamatsu, N., Sato, C., Utsunomiya, J. (1988): Chemical
and viral transformation of cultured skin fibroblats from patients
with familial polyposis coli. Mutation Res., 199, 411-426.
Moll, U.M., Riou, G, Levine, A.J.(1992): Two distinct mechanisms alter
p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad
Sci USA 89: 7262-7266.
Momand, J. Zambetti, G.P., Olson, D.C., George, D., Levine, A.J. (1992):
The mdm-2 oncogene product forms a complex with the p53 protein
and inhibits p53-mediated transactivation. Cell 69: 1237-1245.
Monti, D., Grassilli, E., Troiano, L., and Franceschi, C. (1992):
Senescence, Immortalization, and Apoptosis. Annal New York Acad.
Sci., 673, 70-82.
Miura, T., Patierno, S., Sakuramoto, T., and Landolph, J.R. (1989).
Morphological and neoplastic transformation of C3H/110T1/2 Cl 8
mouse embryo cells by insoluble carcinogenic nickel compounds.
Environ. Mol. Mutagen. 14, 65-78.
Morgan, T.L., Yang, D., Fry, D.G., Hurlin, P.J., Kohler, S.K., Maher, V.M.
and McCormick, J.J. (1991): Characterization of an infinite life span
diploid human fibroblast cell strain and a near-diploid strain arising
from a clone of cells expressing a transfected v-myc oncogene. Exp.
Cell res. 197, 125-136.
Namba, M., Kusaka, T., Fukushima, F. and Kimoto T. (1984): Diferential
growth response of normal human diploid fibroblasts and in vitro
transformed human fibroblasts in serum-free defined culture
medium. Int J Cancer 34: 645-649.
193
Namba, M., Nishitani, K., Fukushima, F., Kimoto, T. (1988): Multistep
Carcinogenesis of Normal Human Fibroblasts. 8: 947-958.
Nianjun, H., Cerepnalkoski, L. Nwankwo, J. O., Dews, M., and Landolph,
J. R. (1994): Induction of Chromosomal Aberrations, Cytotoxicity,
and Morphological Transformation in Mammalian Cells by the Anti-
parasitic drug, Flubendazole, and the Anti-neoplastic Drug,
Harringtonine. Fundam. Appl. Toxicol. 22, 304-313.
Newbold, R.F. and Overell, R.W. and Connell, J.R. (1982): Induction of
immortality is an early event in malignant transformation of
mammalian cells by carcinogens. Nature, 299, 633-635.
Newbold, R.F. and Overell, R.W. (1983): Fibroblast immortality is a
prereququisite for transformiation by EJ c-Ha-ras oncogene. Nature
304: 648-651.
O'Brien, W., Stenman, G and Sager, R. (1986): Suppression of tumor
growth by senescence in virally transformed human fibroblasts.
Proc. Natl. Acad. Sci. USA 83: 8659-8663.
Palmer, H., Bennink, M., and McCormick, J.J. (1988): Platelet-derived
growth-factor (PDGF) or basic fibroblast growth factor induces
anchorage independent growth of human fibroblasts. J. Cell. Physiol.
137: 588-592
Pantazis, P.,Pellicci, P.G., Dalla-Favera, R. and Antoniades (1985):
Systhesis and secretion of protein resembling platelet-derived
growth factor by human glioblastoma and fibrosarcoma cell line in
culture. Proc Natl Acad Sci USA 82: 2404-2408.
Parada, L.F., Land., H., Weinberg, R.A., Wolf, D., and Rotter, V.(1984):
Cooperation between gene encoding p53 tumor antigen and ras in
cellular transformation. Nature, 312, 649-451.
194
Peehl, D.M., and Stanbridge, E.J. (1981): Anchorage-independent
growth of normal human fibroblast, Proc. Natl. Acad Sci USA: 78,
3 0 53 -3 0 57 .
Pereira-Smith, O.M. and Smith, J.R. Functional simian virus 40 T
antigen is Perez-Rodriguez, R., Chambard, J.C., Van Obberghen-
Schilling E, Franchi, A., Pouyssegur, J. (1981): Emergency of hamster
fibroblast tumors in nude mice-evidence for in vivo selection
leading to loss of growth factor requirement. J Cellul Physiol 109:
3 8 7 -3 9 6 .
Pereira-Smith, O.M. and Smith, J.R. (1983): Evidence for the
recessive nature of cellular immortality. Scienec 221, 964-966.
Pereira-Smith, O.M. and Smith, J.R. (1987): Functional simian virus
40 T antigen is expressed in hybrid cells havin gfinite proliferative
potential. Mol. Cell. Biol. 7, 1541-1544.
Pereira-Smith, O. M. and J. R. Smith. (1988): Genetic analysis of
indefinite division in human cells: Identification of four
complementation groups. Proc. Natl. Acad. Sci. USA 85: 6042-6046.
Perez-Rodriguez R, Franchi, A, Deys, B.F., Pouyssegur, J. (1982):
Evidence that hamster fibroblast tumors emerge in nude mice
through the process of two in vivo selection leading to growth
factor " relaxation " and to immune resistance. Int J Cancer 29: 309-
314.
Petierno, S., Banh, D., and Landolph, J.R. (1988): Transformation of
C3H10T1/2 mouse embryo cells to focus formation and anchorage
independence by insouble lead chromate but not souble calcium
chromate: Relationship to mutagenesis and internalization of lead
chromate particles. Cancer Res. 48, 5280-5288.
Peto, R. (1977): Epidemiology, multistage models, and short-term
mutagenicity tests. In Hiatt, H.H., Watson, J.D., Winsten, J.A. (eds):"
195
The origins of Human Cancer, Book C." Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory, pp 1403-1437.
Pollock, R.E. (1992): Evalution and treatment of soft-tissue sarcoma.
Cancer Bull 44: 268-274.
Pories, W.J., Murinson, D.S., Rubin, P. (1983): Soft tissue sarcoma, in :
P.Rubin(eds), Clinical Oncology, A Multidisciplinary Approach,
American Cancer Society, Washington, DC, pp. 308-324.
Rassoulzadegan, M., Cowie, A., Carr, A., Glaichenhaus, N., Kamen, R.
and Cuzin, F. (1982): The roles of individual polyoma virus early
proteins in oncogenic transformation. Nature, 300, 713-718.
Rassoulzadegan, M., Naghashfar, Z., Cowie, A., Carr, A., Grisoni, M.,
Kamen, R. and Cuzin, F. (1983): Expression of the large T protein of
polyoma virus promotes the establishment in culture of "normal"
rodent fibroblast cell lines. . Proc. Natl. Acad. Sci. USA 80, 4354-
4358.
Reznikoff.C.A., Brankow, D.W., Heidelberger, C. (1973): Establishment
of and Characterization of a cloned line of C3H mouse embryo cells
sensitive to postconfluence inhibition of cell division. Cancer Res
33: 3231-3238.
Rosner, B. (1990): Regression and Correlation Methods. In Rosner,
B.(eds) Fundamentals of Biostatistics. PW S-Kent Publishing
Company, Boston, pp 389-473
Rovinhski, B. and Benchimol, S.(1988): Immortalization of rat embryo
fibroblasts by the cellular p53 oncogene. Oncogene, 2, 445-452.
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 80: 7601-7605.
196
Sager, R. (1989): Tumor suppressor genes: The puzzle and the
promise. Science 246: 1406-1412.
Sambrook, J, Fritsch, E.F., Maniatis, T. (1989): Extraction and
purification of plasmid DNA in Sambrook, J, Fritsch, E.F., Maniatis, T.
(eds) Molecular Cloning 2nd ed, Cold Spring Harbor Laboratory Press,
pp 1.42-1.50.
Sambrook, J, Fritsch, E.F., Maniatis, T. (1989): Preparation and
transformation of competent E.Coli, in Sambrook, J, Fritsch, E.F.,
Maniatis, T. (eds) Molecular Cloning 2nd ed, Cold Spring Harbor
Laboratory Press, pp 1.82-1.84.
Sedman, S. A., Hubbert, N. L., Vass, W. C., Lowy, D. R., and Schiller, J.
T. (1992): Mutant p53 can substitute for human papillomavirus type
16 E6 in immortalization of human keratinocytes but does not have
E6-associated trans-activation or transforming activity. J. Virol.
6 6 , 4201-4208.
Shaulsky, G., Ben-Ze’ev A., Rotter, V. (1990): Subcellular distribution
of the p53 protein during the cell cycle of Balb/c 3T3 cells.
Oncogene 5: 1707-1711.
Shaulsky, G, Goldfinger, N, Peled, A., Rotter, V. (1991): Involvement
of wild-type p53 protein in the cycle requires nuclear location . Ceil
Growth Devel 2: 661-667.
Shay, J.W. and Wright, W.E. (1989): Quantitation of the frequency of
immortalization of normal human diploid fibroblasts by SV40 large
T antigen. Exp. Cell Res. 18, 109-118.
Shay, J.W., Wright, W.E., Werbin, H. (1991a): Defining the molecular
mechanisms of human cell immortalization. Biochim Biophys Acta.
1072:1-7.
197
Shay, J. W., Pereira-Smith, O.M., Wright, W.E. (1991b): A role for both
Rb and p53 in the regulation of human cellular senescence. Exp Cell
Res 196:33-39.
Shay, J.W., Wright, W.E. (1991c): Both Rb and p53 are potential
regulators of human cellular senescence. In Brandi M.L., Wright, R
(eds) Hereditary Tumors. Raven Press, New York, pp 173-182.
Shay, J.W., Wright, W.E., and Werbin, H. (1993a): Toward a molecular
understanding of human breast cancer: A hypothesis. Breast Cancer
Res. Treatment, 25: 83-94.
Shay, J.W., Wright, W.E.,Brasiskyte, Dana, Haegen, A.V.D. (1993b): E6
of human papillomavirus type 16 can overcome the M1 stage of
immortalization in human mammary epithelial cells but not in human
fibroblasts. Oncogene, 8, 1407-1413.
Shay, J.W., Hagen, B.V.D., Ying, Y., and Wright, W.E. (1993c): The
frequency of Immortalization of Human Fibroblasts and Mammary
Epithelial Cells Transfected with SV40 Large T antigen. Exp Cell.
Res. 209, 45-52.
Shin, S.I., Freedman, V.H., Risser, R., Pollack, R (1975):
Tumorigenicity of virus transformed cells in nude mice is correlated
specifically with anchorage-independence growth in vitro. Proc Natl
Acad Sci. USA 72, 4435-4439.
Silinskas, K.C-, Kateley, S.A., Tower, J.E., Maher, V.M. and McCormick,
J.J. (1981): Induction of anchorage independent grow in human
fibroblasts by propane sultone, Cancre Res., 41, 1620-1627.
Smith, B., Sager, R. (1982): Multistep origin of tumor-forming ability
in Chinese hamster embryo fibroblast cells. Cancer Res 41: 389-396.
Smith, J. R. and Pereira-Smith, O. M. (1990): Genetic and molecular
studies of cellular immortalization. Adv. Cancer Res. 54: 63-71.
198
Southern, P.J. and Berg, P. (1982): Transformation of mammalian
cells to antibiotic resistance with a bacterial gene under control of
the SV40 early region promoter. J Mol. Appl. Genet. 1:327-341.
Spandidos, D.A. and Wilkie, N. M. (1984): Malignant transformation of
early passage rodent cells by a single mutated human oncogene.
Nature 310: 469-475.
Stahl, H. and Knippers, R. (1987): The simian virus 40 large tumor
antigen. Biochim. Biophys. Acta. 910, 1-10.
Stamps, A.C., Gusterson, B.A., O'Hare, M J . (1992): Are tumor
immotal? Eur J of Cancer 28A,1495-1500.
Stein, G.H. (1985): SV40-transformed human fibroblasts: evidence
for cellular aging in precrisis cells. J. Cell Physiol. 125, 36-44.
Suarez, H.G., Nardeux, P.C. Andeol, Y., and Sarasin, A. (1987): Multiple
activated onciogenes in human tumors, Oncogene Res. 1, 201-207.
Sutherland, B.M., Delihas, N.C., Oliver, R.P., Sutherland, J.C. (1981):
Action spectra for the ultra-violet light-induced transformation of
human cells to anchorage independent growth, Cancer Res. 41, 2211-
2214.
Sutherland, B.M., Freeman, A.G., Bennett, P.V. (1988): Human cell
transformation in the study of sunlight-induced cancers in the skin
of man. Mutation Res., 199, 437-448.
Temin, H.M., Rubin, H (1958): Characteristics of an assay for Rous
sarcoma virus and Rous sarcoma cells in tissue culture. Virology 6:
6 6 9-6 8 8 .
Thomassen, D.G., DeMars, R. (1982): Clonal analysis of the stepwise
appearance of anchorage independence and tumorigenicity in CAK, a
permanent line of mouse cells. Cancer Res 42: 4054-4063.
199
Thompson, K.V.A., and Holliday, R. (1975): Chromosome changes
during the in vitro agin g of MRC-5 human fibroblatss, Exp Cell Res.
96, 1-6.
Tomatis, L. (1990): The occurence of cancer, in Tomatis, L (eds),
Cancer: Cause, Occurence and Control, IARC Scientific Publications,
Lyon, France, pp15-88.
Tooze, J., ed. (1981) DNA Tumor Viruses, Molecular Biology of Tumor
Viruses (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), 2nd
Ed., Part 2, Revised.
Treisman, R., Novak, U., Favaloro, J. and Kamen, R. (1981).
Transformation of rat cells by an altered polyoma virus genome
expressing only the middle T protein. Nature 292,595-600.
Tuck, S.P., and Crawford, L. (1989): Overexpression of normal human
p53 in established fibroblasts leads to their tumorigenic conversion.
Oncogene Res 4, 81-96.
Vaino, H. (1986): Occupational exposures and primary prevention of
cancer, in Eylenbosch, L., Depoorter, A-M, Larebeke, N.V. (eds),
Primary Prevention of Cancer,Raven Press, New York, pp113-128.
Vogt, M., and Dulbecco, R. (1963): Steps in the neoplastic
transformation of hamster embryo cells by polyoma virus. Proc. Natl.
Acad. Sci. 49, 171-179.
Vousden, K.H. and Marshall, C. J. (1984): Three different activated ras
genes in mouse tumors: evidence for oncogene activation during
progression of a mouse melanoma. EMBO J 3: 913-917.
Watson, J. D. (1972): Origin of concatameric T4 DNA. Nature New Biol
239: 197-201.
200
Wilson, D. M., Fry, D. G., Maher, V. M., McCormick, J. J. (1989):
Transformation of diploid human fibroblasts by transfection of N-
ras oncogenes. Carcinogenesis 10:635-640.
Wright, W.E.and Shay, J.W. (1992): The two stage mechanism
controlling cellular senescence and immortalization. Exp. Gerontol
27, 383-389.
Wu, X. and Levine, A. J. (1994): p53 and E2F-1 cooperate to mediate
apoptosis. Proc. Natl. Acad. Sci. USA 91, 3602-3606
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R., and Beach,
D. (1993): p21 is a universal inhibitor of cyclin kinase. Nature 366,
7 0 1 -7 0 4
Yang, D., Louden, C., Reinhold, D.S., Kohler, S. K., Maher, V. M.,
McCormick, J. J. (1992): Malignant transformation of human
fibroblast cell strain MSU-1.1 by (±)-7B,8a- dihydroxy-9a, 10a-
epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Proc. Natl. Acad. Sci. USA.
89: 2237-2241.
Yang, J-H, Thraves, P., Dritschilo, A., Rhim, J. S. (1992): Neoplastic
transformation of immortalized human keratinocytes by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Cancer Res. 52: 3478-3482.
Zimmerman, R.J., Little, J.B. (1981): Starvation for arg and glu
sensitizes human diploidcells to the transforming effects of N-
Acetoxy-2-acetylaminofluorene, Carcinogenesis 2, 1303-1310.
Zimmerman, R.J., Little, J.B (1983a): Characterization of a
quantitative assay for the in vitro transformation of normal human
201
diploid fibroblasts to anchorage independence by chemical
carcinogenesis, Cancer Res 43, 2176-2182.
Zimmerman, R.J., Little, J.B (1983b): Characteristics of human
diploid fibroblasts transformed in vitro by chemical carcinogens.
Cancer Res., 43, 2182-2189.
202

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