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Mechanisms of acquisition of molecular genetic changes during tumor development and progression

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Mechanisms of acquisition of molecular genetic changes during tumor development and progression

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Content MECHANISMS OF ACQUISITION OF MOLECULAR GENETIC
CHANGES DURING TUMOR DEVELOPMENT AND
PROGRESSION
by
Jiamei Yu
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
(PATHOBIOLOGY)
December 2004
Copyright 2004 Jiamei Yu
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UMI Number: 3155500
Copyright 2004 by
Yu, Jiamei
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DEDICATION
This dissertation is dedicated to my husband, our daughter and my parents
with love.
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iii
ACKNOWLEDGEMENTS
I would like to express my thankfulness to my mentor Dr. Louis Dubeau for
his years of continuous guidance, support and understanding for me during
my Ph.D. study in his lab. His enthusiasm for science as a scientist and his
kindness as a mentor will continue to be what I greatly admire and learn from
in my future careers.
I am also very grateful to my other dissertation committee members Dr. Axel
H. Schonthal, Dr. Pradip Roy-Burman, Dr. Norbert Berndt for their valuable
and timely advice and help on my disseretation project.
I want to acknowledge Dr. Robert A. Weinberg from the whitehead institute
for providing us with the expression vector for human telomerase and Dr.
Jerry Shay from the University of Texas southwestern medical center for his
cooperation with us for the study of the determinants of crisis.
I greatly appreciate the help from a number of graduate student and
technicians in Dr. Louis Dubeau’s lab: Mihaela Velicescu, Andrew Rice,
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iv
Rajas Chodankar, Bongha Shin, Sepideh Karimi, Eileen Granada, Erika
delgadillo. I am very lucky to study in a friendly and well-communicated
atmosphere with them around.
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V
TABLE OF CONTENTS
Page
DEDICATION ii
ACKNOWLEDGEMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT x
PART I BACKGROUND 1
Chapter 1 Acquisition of molecular genetic changes by cancer
cells 2
I. Genetic instability in cancer cells 3
II. Current models of acquisition of molecular
genetic changes during tumor development
and progression 5
III. Aneuploidy versus mutation hypothesis
of cancer 9
IV. Ovarian tumors of low malignant potential 13
V. Rationale and hypothesis 18
PART II MECHANISMS OF ACQUISITION OF MOLECULAR
GENETIC CHANGES DURING TUMOR DEVELOPMENT
AND PROGRESSION 26
Chapter 2 Dynamics of acquisition of genomic instability during cancer
development 27
Abstract 28
Introduction 30
Materials and Methods 33
Results 35
Discussion 49
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Chapter 3 Role of numerical chromosomal instability in tumor
progression 58
Abstract 59
Introduction 62
Materials and Methods 66
Results 69
Discussion 101
Chapter 4 Differences in ploidy stability between ovarian tumors of
low malignant potential and ovarian cystadenomas 108
Abstract 109
Introduction 111
Materials and Methods 114
Results 119
Discussion 134
Chapter 5 HHR6A overexpression in aneuploid versus diploid
clone 139
Abstract 140
Introduction 142
Materials and Methods 144
Results 149
Discussion 160
PART III EPILOGUE 163
Chapter 6 Summary and future directions 164
Summary 165
Future directions 171
ALPHABETIZED BIBLIOGRAPHY 179
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vii
Table 1
Table 2
Table 3
Table 4
LIST OF TABLES
Page
Examination of loss of heterozygosity in low and high
passages of spontaneously immortalized ovarian
cystadenoma cells. 41
The stability of the cells after spontaneous
immortalization. 83
Telomerase-transfected diploid cells outgrow aneuploid
cells. 85
More diploid than aneuploid clones are obtained from
the aneuploid fraction after telomerase transfection. 152
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VIII
LIST OF FIGURES
Page
Figure 1.1 Classical model of tumor progression. 22
Figure 1.2 Alternative model of tumor progression. 24
Figure 2.1 Acquisition of alterations in cancer development. 39
Figure 2.2 Comparison of loss of heterozygosity before and
after crisis. 43
Figure 2.3 Comparison of LOH on 15q in clones of spontaneously
immortalized cells to clones before crisis. 45
Figure 2.4 An in vivo example potentially leading to cancer
development according to our proposed model. 47
Figure 3.1 The role of aneuploidy in immortalization of benign
ovarian epithelial tumor cells expressing
SV40 large T Ag. 77
Figure 3.2 Does aneuploidy facilitate transformation in the
presence of mutagen? - hypothesis and rationale. 79
Figure 3.3 Significance of aneuploidy compared to mutagenesis
on in vitro longevity of benign ovarian epithelial tumor
cells expressing SV40 T antigen. 81
Figure 3.4 Correlation of chromosome numbers with the growth
kinetics of MCV39 low passage clones. 88
Figure 3.5 Observation of numerical chromosomal instability in
MCV39 cell clones 25 population doublings later. 90
Figure 3.6 Correlation of chromosome numbers with population
doubling time of MCV39 cell clones 25 population
doublings later. 92
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ix
Figure 3.7 A model for tumor development.
Figure 3.8 The relationship between LOH on 15q and the growth
rate of clones before and after crisis.
Figure 3.9 The relationship between LOH on 15q and the
transformation ability of clones before and after
crisis.
Figure 4.1 Differences in ploidy stability between ovarian
cystadenomas and LMP tumors expressing
SV40 large T antigen.
Figure 4.2 Differences in ploidy stability between cystadenomas
and LMP tumors are not determined by differences
in levels of SV40 large T antigen expression.
Figure 4.3 Different roles for telomere attrition in initiating crisis of
cystadenomas versus LMP tumors.
Figure 5.1 DNA profiles of the diploid and aneuploid clone in our
experiment.
Figure 5.2 Overexpression of HHR6A protein in aneuploid clone
compared to diploid clone revealed by
western blot.
Figure 5.3 Overexpression of HHR6A protein in aneuploid clone
compared to diploid clone revealed by
immunohistochemistry.
94
96
98
125
127
132
154
156
158
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X
ABSTRACT
The mechanisms of acquisition of the molecular genetic changes by cancer
cells still remain poorly understood despite extensive studies. This thesis
takes advantage of two cell lines, called MCV39 and MCV50, spontaneously
immortalized after their parental cell strain, derived from human ovarian
cystadenoma cells transfected with SV40 large T antigen, had recovered
from crisis. I examined the role and dynamics of acquisition of aneuploidy
and other molecular genetic changes in this longitudinal model of tumor
progression. Not only were several losses of heterozygosity present
immediately after immortalization in both cell lines, but such losses were
even more numerous in subclones of the parental cells that had not yet
reached crisis. This suggests that at least some alterations in human cancers
come from clonal expansion of changes acquired before transformation,
Most of these abnormalities develop when the cells approach crisis, which is
characterized by severe changes in genomic DNA content. I propose a novel
mechanism for acquisition of chromosomal alterations during tumor
development that stipulates that the bulk of genetic alterations present in
mature cancer cells are not the result of gradual progression, but represent
clonal expansion of changes acquired before transformation.
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xi
MCV39 cells obtained at low passage were followed over as many as 60
population doublings in order to evaluate their chromosomal stability.
Although only a single clonal additional loss of heterozygosity was acquired
over this period of time, the ploidy status of the cells was constantly
changing, with a tendency for the cells to first double their chromosome
numbers, followed by development of aneuploidy. We conclude that although
the majority of molecular genetic abnormalities found in cancer cells may
have been acquired before transformation, these cells may continue to
acquire additional abnormalities after transformation, in a manner consistent
with the classical model of tumor progression.
Another part of my work was to examine the relative significance of
aneuploidy and mutagenesis on tumor progression. Cells were either treated
with a mutagen or separated based on their ploidy status and their growth
kinetics and in vitro longevity were observed. I present evidence that a
combination of aneuploidy and mutagenesis is more permissive to tumor
progression than either one alone.
Another section of this thesis deals with comparing the changes associated
with in vitro crisis in cell lines derived from ovarian cystadenomas versus
ovarian tumors of low malignant potential (LMP). The results suggest that
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while severe ploidy changes are the main determinant of crisis in
cystadenomas, such changes do not take place in LMP tumors, where crisis
is determined primarily by telomere attrtion.
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PARTI
b a c k g r o u n d
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2
Chapter 1
Acquisition of molecular genetic changes by cancer
cells
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I. Genetic instability in cancer cells
Cancer cells typically possess numerous molecular genetic changes and are
thought to be genetically unstable. Genetic instability has been proposed as
a mechanism for the acquisition of changes required for tumorigenesis. Thus,
genetic instability is believed to be essential and serve as driving force for
tumor development and progression (Coleman WB and others, 1999).
Genetic instability is proposed to cause the heterogeneity of genome, and
hence the heterogeneity of morphology and behavior of cells within the same
cancer (Bignold LP and others, 2003).
There are two main types of genetic instability in cancer cells: chromosomal
instability (CIN), caused by abnormalities in genes necessary for
chromosomal segregation (Lengauer C and others, 1998) and microsatellite
instability (MIN), caused by mutations in mismatch repair genes (Loeb KR
and others, 2000).
Chromosomal instability refers to the observation that cancer cells usually
gain or lose whole chromosomes or large fractions of chromosomes much
more frequently than normal cells, resulting in aneuploidy and losses of
heterozygosity (Nowak MA and others, 2002). Various changes causing
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4
defective mitosis, such as mutations of mitotic checkpoint genes, can lead to
chromosomal instability (Daniel PC and others, 1999). Chromosomal
instability was observed in very early stages of colorectal cancers and might
facilitate colorectal tumorigenesis (Rajagopalan H and others, 2003).
Microsatellite instability refers to change of length of microsatellite alleles due
to gain or loss of repeat units as a result of nucleotide insertions or deletions
(de la Chapelle A and others, 2003). Microsatellite instability has been
observed in various sporadic cancers, including colorectal, gastric, and
endometrial cancers (Cai KQ and others, 2004). Defective mismatch repair,
which causes microsatellite instability, can facilitate malignant transformation
by allowing the rapid accumulation of mutations that affect the function of
critical genes in the cells.
In colorectal cancers, 15% have MIN and are diploid, whereas 85% have CIN
and are aneuploid. It has been shown that although microsatellite instability
is recessive, chromosomal instability is dominant (Lengauer C and others,
1997).
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Despite the extensive demonstration of numerous genomic alterations in
cancer cells, little is known about the mechanisms responsible for the genetic
instability.
II. Current models of acquisition of molecular genetic changes during
tumor development and progression
1. Classical model versus alternative model
The classical model of tumor progression that is first advanced by Foulds
(Foulds L, 1954) and Nowell (Nowell PC and others, 1976) proposes that
cancer cells acquire molecular genetic changes in a gradual step-wise
manner after transformation of a single normal cell. After transformation, cells
that acquire new molecular genetic changes keep on showing up randomly
during the progression process, followed by elimination of cells which acquire
changes detrimental to cell survival, and selection of those cells which
acquire changes associated with a growth advantage (Figure 1.1).
Although the classical model of tumor progression are widely favored, the
observations in people with familial adenomatous polyposis (FAP) showed
that although these patients developed hundreds to thousands of
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adenomatous polyps during their second and third decades of life, 90% of
their polyps remained not to progress to colorectal cancer (Kinzler KW and
others, 1996). This is in strong contrast to the classical model of tumor
progression, which predicts for these patients that all of their polyps may
progress to colorectal cancer.
In addition, the risk of colorectal cancer remains stable over time, in contrast
to the exponentially increasing risk with age as predicted by the classical
model of tumor progression. The classical model of tumor progression can’t
explain some observations of Lynch syndrome as well. For example,
although the adenomatous polyps always take place at the left side, Lynch
syndrome associated colorectal cancers always happen at the right side.
All these seem to suggest that there may exist an alternative model of tumor
progression and things may happen very differently. In this thesis, an
alternative model of tumor progression proposes another hypothesis for the
mechanisms of acquisition of molecular genetic changes by cancer cells.
Instead of a gradual step-wise manner of acquisition of molecular genetic
changes, the alternative model proposes that the single normal cell acquires
many molecular genetic changes immediately when the transformation
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process occurs and chooses to become different stages of cancer. The
alternative model also hypothesizes there is not necessary progression
between different stages of cancer (Figure 1.2).
Up to now, most people favor the classical model of tumor progression
because they believe it is more likely for cancer cells to acquire the genomic
abnormalities by a gradual, step-wise manner. They don’t believe the
alternative model of tumor progression because they don’t think there exist
such a big catastrophic event that can generate so many genetic
abnormalities to be acquired by the cancer cells immediately when the
transformation process occurs.
2. Aneuploidy and crisis
But actually such a big catastrophic event that is associated with various
kinds of genetic abnormalities does exist. This big catastrophic event is
crisis.
In 1965, Hayflick observed that after approximately fifty active cell divisions,
primary human fetal cells in culture entered degeneration phase
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characterized by increased population doubling time, decreased mitotic
activity and aneuploidy. The finite lifetime of diploid cells in culture was
referred to as the Hayflick limit (Hayflick L, 1965).
It was observed that cultured human cells rarely bypassed the Hayflick limit
and underwent spontaneous immortalization (Levan A and others, 1958).
However, cultured primary rodent cells frequently bypassed the Hayflick limit
and underwent spontaneous immortalization (Todaro GT and others, 1963).
It was proposed that this was because of the chaotic progression of
aneuploidy experienced by mouse cells in contrast to a non-chaotic
progression of aneuploidy experienced by human cells, which enabled the
mouse cells to have more chance than human cells to acquire the right
combination of aneuploid chromosomes for immortalization (Rasnick D,
2000).
Aneuploid cells have a lower survival than normal diploid cells. However, all
diploid cells usually have a limited life span, in contrast, aneuploidy can lead
to immortalization on rare occasions (Levan A and others, 1958). Aneuploidy
has been linked with immortalization in modern textbooks (Lewin B, 1994).
On the one hand, aneuploidy results in non-viable cells and crisis. On the
other hand, rare aneuploid cells can arise and become spontaneously
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immortalized and transformed to cancer cells (Posadas EM and others,
1996). Aneuploidy provides a link between cancer and crisis.
Hi. Aneuploidy versus mutation hypothesis of cancer
1. Aneuploidy hypothesis of cancer
Nearly all of human solid cancers are aneuploid (Mitelman F and others,
1997). But it is still not clear whether aneuploidy, a common feature of cancer
cells, is the cause or consequence of cancer. This is an issue raised by an
article in science five years ago (Hieter P and others, 1999).
Over a century ago, Hansemann first discovered asymmetric mitoses in
epithelial cancer cells (Hansemann D and others, 1890). Soon after that,
Boveri found in his experiment that aneuploidy caused tumor-like phenotypes
in developing sea urchin embryos (Boveri T, 1902). Up to 1960s, aneuploidy
has been proposed to be the cause of cancer by Boveri and others (Levan A
and others, 1958; Hauschka TS and others, 1961; Atkin NB and others,
1966).
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1 0
However, ever since Morgan’s papers on Drosophilia genetics first appeared
in 1910, gene mutations, rather than aneuploidy, have been on everybody’s
mind as the mechanisms of generating abnormal phenotypes. The
aneuploidy hypothesis of cancer was abandoned when cancers were found
to be nonclonal for aneuploidy, but clonal for mutations (Cairns J and others,
1978).
The aneuploidy hypothesis of cancer that is currently supported by the
research groups of Dr. Peter Duesberg (Li R and others, 1997) believes that
aneuploidy is the cause of cancer. According to them, aneuploidy regroups
thousands of genes encoded on chromosomes as well as alters many
genetic programs, a process similar to regrouping assembly lines of a car
factory. Instead of producing cars with more or less than four wheels per
body, or two or more bodies per engine, aneuploidy makes a new biologic
entity, the cancer cells (Rasnick D and others, 1999). Aneuploidy itself has
been regarded as a specific random chromosome number mutation to cause
the unique and complex phenotypes of cancer cells (Duesberg P and others,
2000).
According to the aneuploidy hypothesis, genetic and karyotypic instability of
cancer cells was just an inherent consequence of aneuploidy (Duesberg P
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and others, 1998). The probability of acquiring a karyotype which growed
better than a normal cell, as predicted by Boveri, was as low as winning a
lottery, which accounted for the slow kinetics of carcinogenesis (Boveri T,
1914). In addition, aneuploidy was suggested to cause multidrug resistance
of cancer ceils by reassorting multiple drug-resistance genes on
chromosomes (Duesberg P and others, 2000).
The structurally altered or marker chromosomes of cancer cells were also
suggested to be caused by aneuploidy. It was proposed that aneuploidy
unbalanced either genes of enzymes maintaining nucleotide pools or genes
of histones. As a result, DNA breaks were created to initiate structural
abnormalities of chromosomes in cancer cells (Brinkley BR and others, 1998;
Duesberg P, 1999).
2. Mutation hypothesis of cancer
In 1976, Peter Nowell first postulated that genetic instability of cancer ceils
was generated by mutations (Nowell PC, 1976). Accumulation of gene
mutations in a single cell was believed to underlie the development and
progression of cancers. However, the known mutation rate in normal somatic
cells was not enough to accumulate enough mutations to allow cancers to
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occur during a human lifetime. Thus, Lawrence Loeb et al had predicted a
“mutator” phenotype. Mutations of the mutator genes, which had no direct
selective advantage or disadvantage themselves, increased the mutation
rates of those genes involved in DNA repair or chromosomal segregation
(Loeb LA, 1991). The cells generated by “mutator” suffered more and more
gene mutations and became cancer cells (Orr-Weaver TL and others, 1998).
However, there was still no experimental confirmation of the “mutator”
phenotype (Strauss BS, 1992). Thus, a “hit and run” mutator had been
postulated (Loeb LA, 1996).
The mutation hypothesis, which is currently supported by the research
groups of Dr. Robert A. Weinberg, believes mutation is the cause of cancer.
According to the mutation hypothesis, mutations convert protooncogenes to
oncogenes, in addition to inactivate tumor suppressor genes, thus disrupt
critical signaling pathways that cause cancer cells to arise (Alberts B and
others, 1994). They believe whether cancers are diploid or aneuploid is
secondary or nonessential.
However, the gene mutation theory can’t explain the growing lists of
nongenotoxic carcinogens (Lijinsky W, 1989). Moreover, there are no cancer-
specific gene mutations. Although the gene mutation hypothesis adopted
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13
dominant retroviral oncogenes as a support for their theory (Martin GS,
1970), it disregarded the fact that the promoters of the retroviral oncogenes
were one thousand times stronger than those of cellular oncogenes
(Duesberg P and others, 1992).
Identification of a dominant cellular oncogene or a combination of genes that
can transform normal human cells to cancer cells was not successful (Li R
and others, 2000). Although Weinberg and colleagues described the first
genetically defined human tumor cells by inactivation of p53 and Rb cell
cycle regulatory proteins with SV40 large T antigen followed by introduction
of the catalytic subunit of telomerase, hTERT, as well as upregulation of ras
growth promoting pathway, it was argued by Dr. Duesberg that it was the
aneuploidy instead of the introduced genes that caused cancer (Tracy W,
2001).
IV. Ovarian tumors of low malignant potential
Ovarian cancer ranks toppest among cancers of the female reproductive
organs with regard to death rate in women. The prognosis of ovarian cancer
is poor since most patients develop advanced stages of disease upon
diagnosis. Breast-feeding, pregnancy and oral contraceptive can greatly
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protect against ovarian cancer. In contrast, increasing age, early menarche,
late menopause, nulliparity and infertility increase the risks of developing
ovarian cancer. In addition, family histories of breast or ovarian cancer
account for greater risk (Holschneider CH and others, 2000). There is still
controversy regarding the origin of ovarian cancer, for example, whether
ovarian cancer arises from the ovarian surface epithelium or the secondary
Mullerian systems is still unknown (Dubeau L, 1999). Thus, both the
prevention and treatment of ovarian cancer are very difficult. The five-year
survival rate of ovarian cancer is about 30 percent (Wingo PA and others,
1995).
The common subtypes of ovarian epithelial cancer are serous, endometroid
and mucinous, identical to tumors arising from fallopian tubes, endometrium
and endocervix respectively, at the morphological level (Chodankar R and
others, 2002).
Recommended by both the International Federation of Gynecology and
Obstetrics (FIGO) (Santesson L and others, 1968) and the World Health
Organization (WHO) (Serov SF and others, 1973), ovarian epithelial cancer
is subdivided into benign tumors called cystadenomas, tumors of low
malignant potential (also called tumors of borderline malignancy), and
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carcinomas based on their differences in malignant potential. Evidence
suggests that ovarian cystadenomas, ovarian tumors of low malignant
potential and ovarian carcinomas are most likely to be distinct disease
entities instead of part of the sequential stages in the multistep model of
ovarian carcinogenesis.
1. Risk factors
Fertile nulliparous women have increased risk of developing ovarian tumors
of low malignant potential (LMP). In addition, familial tendency,
environmental factors, persistent ovulation, viral infection as well as fertility
medications are all risk factors of ovarian tumors of low malignant potential
(Elchalal R and others, 1995). There is correlation between ovarian tumors of
low malignant potential and infertility. Moreover, infertile women who used
fertility drugs had four times the risk to develop ovarian tumors of low
malignant potential (Harris R and others, 1992). The protective factors
against low malignant potential ovarian tumors include increasing parity,
breast-feeding and oral contraceptive.
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2. Diagnosis
The diagnosis of ovarian tumors of LMP is based on the following
histopathological criteria: epithelial stratification, cellular atypia, mitotic
activity and absence of ovarian stromal invasion. Morphometry and ploidy
status have also been used as diagnostic tests (Baak JPA and others, 1987).
Nuclear morphology can help make differentiation between benign ovarian
tumors, ovarian tumors of low malignant potential and ovarian carcinomas.
Ovarian tumors of low malignant potential usually possess smaller, elongated
nuclei with higher optical density (Komitowski D and others, 1989). They
have smaller and less variation in nuclear areas compared to invasive
ovarian cancers (Hytiroglou P and others, 1992). Flow cytometric analysis of
cellular DNA content has been suggested to complement histopathological
diagnosis to predict biological behavior of ovarian tumors of low malignant
potential (Friedlander ML and others, 1984).
3. Prognosis
Approximately 80% of ovarian tumors of low malignant potential are stage I
or stage II diseases (Trimble CL and others, 1994). Ovarian tumors of low
malignant potential usually have favorable prognosis. The five-year survival
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rate is 80-95%, much higher than that of ovarian carcinoma. Only 10 to 30
percent of ovarian tumors of low malignant potential recur long time after the
treatment, usually more than 10 years later (Tattersall MH and others, 1992).
Age, histology grade and type are not prognostic factors of LMPs (Russel P
and others, 1984). Instead, morphometric features and DNA ploidy status are
thought to be prognostic factors, both are important for helping to choose
appropriate treatment plans (Kaern J and others, 1993).
Although 65 percent of epithelial ovarian cancers are aneuploid, only 5
percent of LMPs are aneuploid (Lai CH and others, 1996). DNA aneuploidy
has been correlated with increased tumor recurrence and shortened patient
survival (Sykes PH and others, 1997). Morphologic features, such as the
mean nuclear area, volume percentage of epithelium, and mitotic activity
index have also been suggested to be strongly associated with prognosis
(Baak JPA and others, 1981).
4. Treatment
Primary conservative surgery consisting of unilateral salpingo-oophorectomy
is appropriate for young women with stage la ovarian tumors of LMP who
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wish to retain their fertility potential. Standard adjuvant chemotherapy or
radiotherapy are restricted to patients with borderline tumors of the ovary
with aneuploidy or unfavorable morphometry, their efficacy is still unknown
(Burger CW and others, 2000). Debulking surgical procedures supplemented
with chemotherapy and/or radiotherapy is the primary treatment for advanced
stage ovarian tumors of LMP (Chien RY and others, 1989).
V. Rationale and hypothesis
1. Working hypothesis
Previously, two cells lines, called MCV39 and MCV50, spontaneously
recovered from crisis of ML10, the ovarian cystadenoma cells expressing
SV40 large T antigen. They provide us with a good in vitro longitudinal model
for ovarian tumor development. We propose to study the mechanisms of
acquisition of molecular genetic changes during tumor development and
progression in this in vitro model. We start this work by doing subcloning of
the parental and derived immortalized cell lines of our in vitro model.
The hypothesis of my project is that cancer cells acquire genetic
abnormalities in a way proposed by the following model: A normal cell
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divides for a limited number of times and undergoes senescence. In the
presence of certain abnormalities, cells can bypass senescence and
continue to proliferate, but at the cost of developing severe ploidy changes.
Usually the cells with severe ploidy changes will undergo crisis. Our
hypothesis is that crisis is the big catastrophic event associated with various
kinds of genetic abnormalities that can be acquired by cancer cells. Under
very very rare occasion, a cell at crisis luckily acquires genomic
abnormalities compatible with cell survival and wins the genetic lottery to
recover from crisis and become cancer cells. Our hypothesis is that cancer
cells acquire genetic abnormalities from those present at the time of crisis.
The nature and severity of genetic abnormalities in the cell at crisis
determine whether the cancer will be a low-grade or a high-grade cancer,
and this is determined immediately when the transformation process occurs
(Figure 2.1).
We will verify our hypothesis and achieve the purpose of our study through
the following two specific aims: In aim 1, we want to test the hypothesis that
loss of heterozygosity (LOH) as a parameter of genetic abnormalities in
spontaneously immortalized cells are clonal expansion of changes acquired
before immortalization. To test this hypothesis, we did subcloning of cells in
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the in vitro model for ovarian tumor development developed in our laboratory
and performed allelotype analysis in the subcloned population of the in vitro
model. In aim 2, we want to test the hypothesis that aneuploidy and
mutagenesis both contribute to malignant transformation. To test this
hypothesis, we treated mortal human ovarian cystadenoma cells with a
mutagen and subsequently separated cells based on their DNA content. We
measured the effects of aneuploidy and mutagenesis on in vitro longevity,
alone and in combination.
In addition, we previously found that aneuploidy and telomere attrition were
independent determinants of crisis in SV40 transformed ovarian epithelial
cells, and the crisis of ovarian cystadenomas was determined by aneuploidy
instead of telomere attrition (Velicescu M and others, 2003). In this
dissertation, we study the ploidy stability and the determinant of crisis of
ovarian tumors of low malignant potential and compare them to ovarian
cystadenomas.
Moreover, we also investigate the relationship between HHR6A (the human
homologue of yeast Rad6) and aneuploidy. We examine the level of HHR6A
expression in an aneuploid clone compared to a diploid clone by both
western blot and immunohistochemistry analysis.
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Therefore, the following questions will be addressed in the following
chapters:
1. When do cancer cells acquire genetic abnormalities?
2. What is the significance of aneuploidy compared to that of mutation in
malignant transformation?
3. What determines the selective advantage of cells during tumor
progression?
4. What is the ploidy stability of ovarian tumors of LMP compared to that of
ovarian cystadenomas?
5. What is the determinant of crisis of ovarian tumors of LMP compared to
that of ovarian cystadenomas?
6. What is the difference of HHR6A expression between the aneuploid clone
and the diploid clone?
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22
Figure 1.1 Classical model of tumor progression. This cartoon shows the
hypothesis of the classical model of tumor progression first advanced by
Foulds and Nowell. Cancer cells acquire molecular genetic changes in a
gradual step-wise manner after transformation of a single normal cell (green
circle). After transformation, we can see cells that acquire different kinds of
molecular genetic changes represented by circles with different colors start to
show up, followed by elimination of cells (red and gray circles) which acquire
changes detrimental to cell survival, and selection of those cells (yellow
circles) which acquire changes associated with a growth advantage. Cells
that acquire new molecular genetic changes (blue, brown, black and purple
circles) keep on showing up randomly during the progression process.
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23
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24
Figure 1.2 Alternative model of tumor progression. Instead of a gradual
step-wise manner of acquisition of molecular genetic changes, this model
stipulates that the molecular genetic changes that characterize a mature
cancer cell are acquired simultaneously. The number of molecular
abnormalities present varies between various tumors and is related to
biological aggressiveness. The alternative model proposes that the single
normal cell (green circle) acquires many molecular genetic changes
immediately when the transformation process occurs and chooses to
become different stages of cancers. Clusters of yellow circles, red circles and
purple circles represent different stages of cancers, which have different
numbers of abnormalities. For example, cancer cells represented by clusters
of purple circles have more numbers of abnormalities than cancer cells
represented by clusters of red or yellow circles. Cancer cells represented by
clusters of red circles have more numbers of abnormalities than cancer cells
represented by clusters of yellow circles. The alternative model also
hypothesizes there is not necessary progression between different stages of
cancers.
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/
\
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26
PART II
MECHANISMS OF ACQUISITION OF MOLECULAR
GENETIC CHANGES DURING TUMOR
DEVELOPMENT AND PROGRESSION
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27
Chapter 2
Dynamics of acquisition of genomic instability during
cancer development
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2 8
Abstract
Ovarian cystadenomas, which are benign tumors of the same cell lineage as
ovarian carcinomas, were transfected with an expression vector carrying
SV40 large T antigen. The transfectants developed severe ploidy changes
typical of cells expressing this antigen and underwent crisis after about 60 in
vitro population doublings. Two cell clones, called MCV39 and MCV50,
spontaneously recovered from crisis and became immortal cell lines.
Allelotype analyses of both cell lines immediately after they recovered from
crisis showed multiple losses of heterozygosity. Only a single additional loss
occured in MCV39 cell line over 60 subsequent population doublings,
suggesting that most losses were already acquired immediately after the
cells become immortalized. We transfected the parental cystadenoma cell
strain, called ML10, with an expression vector for the catalytic subunit of
telomerase, which allowed the cells to maintain their logarithmic growth and
escape crisis. Allelotype analyses of subclones of the telomerase transfected
cells obtained as early as possible after the telomerase-transfection
procedures revealed greater losses in these cells than in subclones of
MCV39 cells. All chromosomes affected by losses of heterozygosity in the
subclones of MCV39 cells were also affected by such losses in at least some
subclones of the telomerase-transfected ML10 cells. LOH on 15q, which was
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29
the only loss that was associated with progression of MCV39, did not confer
survival advantages in the telomerase-transfected pre-crisis cells. We
conclude that the genetic abnormalities present in MCV39 ovarian carcinoma
cells are not acquired in a gradual, stepwise manner during the progression
process, but are acquired immediately after transformation and come from
clonal expansion of changes already acquired before malignant
transformation, when the parental ML10 cells undergo crisis. This scenario
may represent an important mechanism of acquisition of genomic
abnormalities in some human cancers, particularly those carrying
abnormalities with consequences similar to those of SV40 large T antigen
expression, such as p53 mutations.
Keywords: Loss of heterozygosity; Crisis; Genomic abnormalities; Cancer
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30
Introduction
Cancer cells harbor various kinds of genomic abnormalities such as changes
in DNA methylation, changes in DNA ploidy, losses of heterozygosity, point
mutations, insertions, deletions, translocations and amplifications (Barrett JC
and others, 1990; Windle B and others, 1991). However, very little is known
regarding how cancer cells acquire these changes.
The concept of multistep carcinogenesis suggests that five or six
independent events are required for tumorigenesis. Compatible with this,
almost every human cancer has multiple genetic alterations. Each of these
changes may represent a crucial step for a normal cell to become a
malignant tumor. For example, seven genetic events are believed to be
necessary for colon cancer development. Inactivation of APC gene followed
by activation of ras gene and ultimately, loss of p53 gene are critical for colon
epithelium to evolve from hyperplasia to adenoma and eventually become
invasive and metastatic (Kinzler KW and others, 1996).
An hypothesis first advanced by Foulds (Foulds L, 1954) and Nowell (Nowell
PC and others, 1976), referred to as the classical model of tumor
progression, proposes that cancer cells acquire various kinds of molecular
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31
genetic changes in a gradual step-wise manner, resulting in tumor
progression. This involves elimination of cells that acquire changes
detrimental to cell survival, and selection of cells that acquire changes
associated with a growth advantage.
The classical model of tumor progression is not able to explain all
observations about cancer development. Individuals with familial
adenomatous polyposis (FAP), who typically develop hundreds and even
thousands of polyps, show malignant transformation in less than 10% of
these lesions (Kinzler KW and others, 1996). Most of these polyps should
progress to cancer according to the classical model of tumor progression.
When we put ML10, the cystadenoma cells expressing SV40 large T antigen
in culture, these cells reach crisis eventually. A previous student in our
laboratory, Mihaela Velicescu, obtained two immortalized cell lines called
MCV39 and MCV500, both of which had spontaneously recovered from
crisis, MCV39 was truly immortal from the time it was first isolated. However,
MCV500 showed logarithmic growth for only 35 population doublings, at
which time underwent a second crisis. Recovery from the second crisis
resulted in an immortal cell line, called MCV50.1 have made 17 separate
attempts to obtain additional spontaneously immortalized clones from
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32
cultured ovarian cystadenomas. None of these attempts were successful. I
therefore used MCV39 and MCV50 and the parental ML10 cells as model for
tumor development and progression. We performed allelotype analyses at
various time points in these cell lines as well as in various subclones. We
also performed similar analyses on cells approaching crisis. The results
provided insights into the dynamics and timing of acquisition of LOH in this in
vitro model.
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33
Materials and Methods
Cell lines and cell culture
ML10 cells were established from primary cultures of human benign ovarian
cystadenomas infected with an adenovirus vector expressing SV40 large T
antigen (Luo MP and others, 1997). This vector has become stably
integrated into the host genome upon multiple reinfections. MCV39 and
MCV50 are spontaneously immortalized cell lines derived from ML10 cells.
MCV152 cell line was obtained by transfection of an expression vector for the
catalytic subunit of human telomerase, hTERT into ML10 cells to make them
immortal. All cells were grown in MEM (Invitrogen Corporation, Carlsbad,
CA) supplemented with 10% fetal bovine serum (FBS).
Subcloning of cultured cells
After counting cell numbers by coulter counter (Coulter electronics, INC.
hialeah, FL), suspensions of trypsinized cells were diluted with filtered
conditioned medium and cultured in 96 well plates at an average of 0.2 cell
per well.
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Allelotype analysis
High molecular weight cellular DNA was extracted from MCV152, MCV39
low and high passages as well as their subclones as described previously
(Ehlen T and others, 1990). By PCR in the presence of radioactive a-p32-
dCTP (ICN Pharmaceuticals, Irvine, CA), we amplified the polymorphic
microsatellite sequences at the selected informative loci located on each
chromosome arms. The radiolabeled PCR products were electrophoresed on
6% ployacrylamide gels under denaturing conditions and visualized by
phosphorimager (Model GS-525, Molecular Imager® System, Biorad
Laboratories, Richmond, CA) as described previously (Kim TM and others,
1994). The set of primers used for locus ANK1 is 5’-TCC CAG ATC GCT
CTA CAT GA-3’ and 5’-CAC AGC TTC AGA AGT CAC AG-3’. The set of
primers used for locus D1S116 is 5-TAC AAG GCA ACC ACATAATT-3’
and 5’-CTT TTC CTA ATT GTG TGT GT-3’, which are the representative
results of our allelotype analysis. The set of primers used for locus IGFR1
located on the chromosome arm of 15q are 5’-TCC CAG ATC GCT CTA
CAT GA-3’ and 5’-CAC AGC TTC AGA AGT CAC AG-3’.
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35
Results
Comparison of loss of heterozygosity before and after crisis
We were interested to determine the timing and dynamics of acquisition of
chromosomal instability in MCV39 and MCV50 cells. A previous student in
our laboratory, Mihaela Velicescu, performed allelotype analyses to examine
loss of heterozygosity (LOH) as a parameter of genetic abnormalities in both
MCV39 and MCV50 cell lines immediately after their isolation as well as 60
or 32 population doublings later. As shown in Table 1, for both MCV39 and
MCV50 cell lines, most losses of heterozygosity were already present early
after the cells had become spontaneously immortalized. For each cell line,
only one single additional loss was acquired during the progression process
from the low passage to the high passage. This suggested that most losses
of heterozygosity were already present when the cells recovered from crisis
and became immortalized.
We next tested the hypothesis that losses of heterozygosity were already
present before cell immortalization, when the parental ML10 cells
approached crisis. Because of the difficulties in obtaining clonal populations
of cells approaching crisis, we transfected the parental ML10 cells with an
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36
expression vector for the catalytic subunit of human telomerase, hTERT,
resulting in rapid immortalization. Several subclones of “pre-crisis” cells were
isolated using this approach. In addition, we also obtained six subclones of
the low passage MCV39 and seven subclones of the high passage MCV39
(60 population doublings later). To clarify how the spontaneously
immortalized cells acquired LOH, we performed allelotype analysis in all
subcloned populations and compared the changes present in pre-crisis cells
to those present in subclones of MCV39 at low and high passage numbers.
The allelotype analysis was performed by PCR to amplify polymorphic
microsatellite sequences on each chromosome arms in the presence of
radioactive a-p32-dCTP. The radiolabeled PCR products were
electrophoresed on denatured 6% polyacrylamide gels and visualized by
autoradiography. Figure 2.2 showed results for two representative loci. The
upper and lower bands in Figure 2.2a represented the two alleles on this
locus D1S116. Loss of heterozygosity at this locus was not detected in
MCV39 cells, either low or high passage, but was present in several “pre
crisis” cell clones. LOH at the ANK1 locus was seen in all MCV39 cell clones
in Figure 2.2b. This loss was also present in several “pre-crisis” cells.
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37
Comparison of LOH on 15q in clones of spontaneously immortalized
cells to clones before crisis
For both MCV39 and MCV50 cell lines, only one single additional loss was
acquired during the progression process from the low passage to the high
passage (Table 1). We wanted to clarify when our spontaneously
immortalized cell line, MCV39, acquired this loss and to determine whether
or not it conferred a growth advantage to the cells harboring it.
Several frozen vials of MCV39 were revived and cultured until the cells
reached 60 population doublings to determine whether LOH on 15q would be
acquired reproducibly. The results (not shown) confirmed that this was
indeed the case, suggesting that the loss was already present in a subset of
cells at low passage and that those cells gradually outgrew those lacking this
loss. To confirm this conclusion, we examined this locus in subclones of
MCV39 cells (Figure 2.3). As expected, all high passage MCV39 clones
showed this loss. Five of the six early passage clones also showed this loss,
demonstrating its presence at this early time point. The fact that only a single
low passage clone showed both alleles while there was no detectable loss at
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38
this locus in the uncloned cell population is compatible with the idea that cells
harboring such a loss had some kind of growth advantage, resulting in more
efficient cloning.
Figure 2.3 also showed the presence of LOH on chromosome 15q in several
“pre-crisis” cells. The consequence of this loss in these cells is examined
further in the next chapter.
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39
Figure 2.1 Acquisition of alterations in cancer development. A normal
cell divides for a limited number of times and undergoes senescence. In the
presence of certain abnormalities, cells can bypass senescence and
continue to proliferate, but at the cost of developing severe ploidy changes.
Usually the cells with severe ploidy changes will undergo crisis. Our
hypothesis is that crisis is the big catastrophic event associated with various
kinds of genomic abnormalities that can be acquired by cancer cells. Under
very very rare occasion, a cell at crisis luckily acquires alterations compatible
with cell survival and wins the genetic lottery to recover from crisis and
becomes cancer cells. We hypothesize that cancer cells acquire alterations
from clonal expansion of those present at the time of crisis. The nature and
severity of changes in the cell at crisis determine whether the cancer will be a
low-grade cancer or a high-grade cancer, and this is determined immediately
when the transformation process occurs.
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40
Proliferation
signal
Senescence
Crisis
Low grade
carcinoma
High grade
carcinoma
Genetic instability
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41
Table 1. Examination of loss of heterozygosity in low and high
passages of spontaneously immortalized ovarian cystadenoma cells.
Cultured ovarian cystadenoma cells undergoing in vitro crisis were kept for
approximately 5 weeks, at which time 2 cell clones, called MCV39 and
MCV50, recovered from crisis, resumed their logarithmic growth and became
spontaneously immortalized. A previous student in our laboratory, Mihaela
Velicescu, performed allelotype analyses for MCV39 and MCV50 cell lines
immediately after the lines were established as well as 60 or 32 population
doublings later.
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42
Chromosomes with Chromosomes with
losses of heterozygosity losses of heterozygosity
in MCV39 cells jn MCV50 cells
low passage 60 population
doublings
2p
2p
5p 5p
8p 8p
10p tOp
18q 18q
Xq Xq
15q
low passage 32 population
doublings
6q 6q
10p
10p
18p 18p
Xp Xp
Xq Xq
12p
(Refer to: The Ph.D. thesis of Campan, Mihaela Velicescu:
Ph.D. Pa '99 C1868.
The role of cellular senescence in tumor development)
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43
Figure 2.2 Comparison of loss of heterozygosity before and after crisis.
Ovarian cystadenoma cells transfected with SV40 large T antigen were
cultured in vitro until they approached crisis. Some cells were transfected
with an expression vector for the catalytic subunit of human telomerase,
hTERT (obtained from Dr. Robert Weinberg, Whitehead Institute). We
obtained 7 different subclones of the transfectants (before crisis).
Cystadenoma cells that were not transfected with hTERT were kept in culture
until they stoped proliferating completely due to crisis. After approximately 5
additional weeks, a cell line named MCV39 recovered from crisis and
resumed its logarithmic growth. A total of 6 subclones of MCV39 cells were
obtained early after the immortalization event (early passage) and 7
subclones were obtained 60 population doublings later (late passage). DNA
samples from all clones were amplified by PCR using primers for the
polymorphic microsatellite sequences on each chromosome arms in the
presence of radioactive a-p32-dCTP, the radiolabled PCR products were
electrophoresed on denatured polyacrylamide gels and visualized by
phosphorimager. The arrows indicate the position of the upper and lower
bands that represent the two alleles, (a) and (b) show the representative data
of our allelotype analysis, on locus D1S116 and ANK1 respectively.
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44
BEFORE
CRISIS
AFTER RECOVERY FROM CRISIS
Early passage Late passage
s v m m
b
BEFORE AFTER RECOVERY FROM CRISIS
ML10 CRISIS Early passage Late passage
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45
Figure 2.3 Comparison of LOH on 15q in clones of spontaneously
immortalized cells to clones before crisis. Allelotype analysis on IGFR1
locus on 15q was performed in the subclones of our in vitro model. Five out
of six MCV39 low passage clones already showed LOH on 15q, only one
clone didn’t have LOH on 15q, suggesting that LOH on 15q were acquired
very early after immortalization. In addition, one of the seven clones obtained
before crisis also showed LOH on 15q, suggesting LOH on 15q already
happened in cells before immortalization, at the time of crisis.
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BEFORE AFTER RECOVERY FROM CRISIS
ML10 CRISIS Early passage Late passage
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47
Figure 2.4 An in vivo example potentially leading to cancer development
according to our proposed model. This tissue section was taken from a
cervical biopsy of a human patient with human papilloma virus infection. The
cells are stimulated to proliferate due to the virus and the chronic
inflammation (long arrow). The presence of the human papilloma virus
infection allows the cells to bypass senescence and continue to proliferate
until they reach the equivalent of in vitro crisis. This comes at the cost of
severe ploidy changes, which are readily recognized from the resulting large
nuclear sizes of the cells (short arrow). We hypothesize the alterations which
are acquired in a cell at crisis as result of severe ploidy changes may
occasionally be compatible with cell survival, leading to cancer with clonal
expansion of cells harboring multiple genomic abnormalities, which are
acquired before malignant transformation, at the time of crisis.
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48
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49
Discussion
Our results suggest that losses of heterozygosity are acquired during
recovery from in vitro crisis. More frequent losses of heterozygosity were
observed in the subclones of the parental cells that were approaching crisis
than in cells that had recovered from crisis to become immortal. All
chromosomes affected by losses of heterozygosity in the subclones of
spontaneous immortalized cells were also affected by such losses in at least
some cells approaching crisis. LOH on 15q, which is the only loss that was
associated with progression of MCV39, did not confer survival advantage to
pre-crisis cells artificially immortalized by transfection of the hTERT subunit.
These results strongly suggest that most losses of heterozygosity present in
mature cancer cells were acquired early after malignant transformation.
Whether this also applies to other genetic or epigenetic abnormalities
associated with cancer remains to be further investigated. Hence, we
propose an alternative model of acquisition of molecular genetic changes by
cancer cells. In this model, we hypothesize that the molecular genetic
changes in cancer cells may primarily come from clonal expansion of
changes acquired in the single aneuploid cell before malignant
transformation, at the time of crisis. The single aneuploid cell that recovers
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50
from crisis and acquires many molecular genetic abnormalities can choose to
become different stages of cancers immediately when the transformation
process occurs.
Our previous student Mihaela Velicescue observed the presence of DNA
methylation changes in the MCV39 and MCV50 cell lines as early as three
population doublings after they escaped from crisis. In addition, methylation
changes present in MCV39 cell line were found to take place before the
immortalization process, as the parental ML10 cells underwent crisis (data
not shown). Moreover, DNA methylation changes were previously found in
our lab to be early events in ovarian tumorigenesis (Cheng P and others,
1997). All these seem to suggest that DNA methylation changes are also
acquired by cancer cells in the way as proposed by the alternative model of
tumor progression. Thus, several of the genetic and epigenetic changes
associated with cancer may be acquired simultaneously at an early stage of
malignant transformation.
Our findings are compatible with the observations in colorectal cancer by
Shih et al (Shih IM and others, 2001) who found that over 90% of early
colorectal adenomas (size 1-3 mm) show similar loss of heterozygosity as
observed in colorectal carcinomas. The global hypomethylation of benign
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51
colorectal polyps were observed to be similar to those that are present in the
colorectal carcinomas. Such changes were suggested to precede the
appearance of malignant phenotype and be involved in early steps of
neoplastic transformation (Fearon ER and others, 1990). The observation
that colonic polyps early in the tumor progression pathway showed similar
numbers of genomic abnormalities as colorectal carcinoma suggests that
acquisition of genomic abnormalities already happen preceding the initiation
of carcinogenesis of colorectal cancer and is an early event in colorectal
tumor progression (Daniel L and others, 1999).
Our findings suggest that crisis is associated with various kinds of genomic
abnormalities that may be acquired by cancer cells. At crisis, the genomes of
the cells are quite abnormal. Various kinds of genomic abnormalities can
arise, as a consequence of aneuploidy that primarily accumulates preceding
crisis.
On the one hand, crisis serves as a potent barrier to immortal cell growth in
culture, on the other hand, the massive genetic instability associated with
crisis may enable those rare cells which are able to survive crisis to acquire
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52
the multiple genomic alterations necessary for malignant transformation
(Counter CM and others, 1992; de Lange T and others, 1990; Hastie ND and
others, 1990; Harley CB and others, 1992).
A group of chronic high-turnover diseases in humans have been observed to
be associated with both end-organ failure and cancer predisposition. For
example, liver cirrhosis is associated with hepatocellular carcinoma, Barrett's
esophagus is associated with esophageal cancer, and ulcerative colitis is
associated with colorectal cancer (Kitada T and others, 1995; Rudolph KL
and others, 2000; DePinho RA and others, 2000). These observations
suggest that telomere attrition in dividing cells can sometimes promote the
development of cancer, although it paradoxically often induces cell death.
Previous research has proposed that telomere-induced crisis may be the
mechanism to generate the diverse genomic alterations required for cancer
initiation (Maser RS and others, 2002). Crisis induced by telomere attrition
can allow accumulation of chromosomal structural aberrations related to
cancer occurence. The fusion-bridge-breakage cycles may be the source to
generate various kinds of changes such as translocations, deletions and
amplifications (McClintock B, 1941).
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53
Crisis of our cell culture model is determined primarily by aneuploidy, instead
of telomere shortening (Velicescu M and others, 2003). The telomere length
of these cells at crisis is similar to the telomere length at their early
passages. Thus, in our cell culture model, aneuploidy-induced crisis instead
of telomere-induced crisis is the mechanism to generate the diverse
molecular genetic alterations that can be acquired by cancer cells. Cancer
can occur following recovery from aneuploidy-induced crisis. Our findings in
the in vitro model of ovarian tumor development suggest that recovery from
genetic abnormality related to aneuploidy developed at the time of crisis is
part of the transformation process. The fact that most cancers are aneuploid
and aneuploidy is a hallmark of cancer makes it very reasonable for us to
further study the role of aneuploidy in malignant transformation. The
clarification of the role of aneuploidy in tumor development and progression
will certainly help us know more of the cause of cancer and design new
efficient strategies for cancer therapy.
Recently, a “run-initiation” model was proposed by Dr. Nicolas Janin, which
provides an explanation for many features of Lynch syndrome that cann’t be
explained by the classical model of tumor progression (Janin N, 1999). For
example, the classical model of tumor progression predicts the risk of
colorectal cancer to increase exponentially with increasing age, since each
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54
change in the multistep model of carcinogenesis is proposed to confer a
selective advantage during the tumor progression process. However, the
stability of colorectal cancer risk is observed over time in patients with
hereditary non-polyposis colorectal carcinoma. In addition, the classical
model of tumor progression can’t explain why Lynch syndrome associated
colorectal cancers always happen at the right side, whereas the
adenomatous polyps always take place at the left side. It is also hard to be
explained by the classical model of tumor progression why advanced stage
colorectal cancer can be diagnosed in less than two years after a normal
colonoscopy, in contrast to the slow progression process as predicted by the
classical model. The proposed “run-initiation" model provides a good
explanation for these observations since it predicts that recovery from crisis
is all the rate-limiting steps of carcinogenesis. Once the cells recover from
crisis, the transformation process is switched on and an extremely rapid
progression will follow, that is, the brutal transition to cancer occurs. Although
the main focus of the “run-initiation” model is on colorectal carcinogenesis, it
provides some similar findings as shown in our alternative model of
acquisition of molecular genetic changes by cancer cells in the in vitro model
of ovarian tumor development.
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55
Approximately 90% of human cervical carcinomas occur because of
infections with human papilloma virus (HPV), especially HPV-16 and HPV-
18. The HPV E6 protein is found to bind to p53 and promote its degradation
via the ubiquitin proteolytic pathway, the HPV E7 protein is reported to
inactivate pRB and cause genetic instability (Homayoun V and others, 1999).
Aneuploidy due to abnormal mitosis induced by the HPV E6 and E7
oncoproteins is proposed to be early event and essential for HPV-associated
carcinogenesis (Stefan D and others, 2000).
Our study in the in vitro longitudinal model for ovarian tumor development
suggests that cancer cells acquire genetic abnormalities in a way as
proposed by the model in Figure 2.1: A normal cell usually has limited life
span and undergoes senescence. In the presence of certain abnormalities,
cells bypass senescence but they proliferate with the development of
aneuploidy and will enter crisis eventually. Although the chance is as low as
winning a lottery, a lucky cell at crisis happens to acquire genomic
abnormalities compatible with cell survival that enable it to recover from crisis
and become immortal cancer cell. Crisis is associated with various kinds of
genetic abnormalities that can be acquired by cancer cells and cancer occurs
following recovery from crisis. Cancer cells acquire genetic abnormalities
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56
from clonal expansion of those present at the time of crisis. The nature and
severity of genetic abnormalities in the cell at crisis determine whether the
cancer will be low-grade or high-grade.
Figure 2.4 shows an in vivo example potentially leading to cancer
development according to our proposed model. The tissue section shown in
this figure is taken from a cervical biopsy of a human patient with human
papilloma virus infection. The cells are stimulated to proliferate due to the
presence of the virus and the accompanying chronic inflammation (long
arrow). The presence of the human papilloma virus infection allows the cells
to bypass senescence and continue to proliferate until they reach the
equivalent of in vitro crisis. This comes at the cost of severe ploidy changes,
which are readily recognized from the resulting large nuclear sizes of the
cells (short arrow). We hypothesize the alterations acquired in the cell at
crisis as a result of severe ploidy changes may occasionally be compatible
with cell survival, leading to cervical cancer with clonal expansion of cells
harboring multiple genohiic abnormalities acquired before malignant
transformation, at the tirhe of crisis.
Our study suggests a link between aneuploidy, crisis and the mechanisms of
acquisition of molecular genetic changes by cancer cells. Our findings
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57
provide some clarification of the mechanisms of acquisition of molecular
genetic changes during tumor development and progression.
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58
Chapter 3
Role of numerical chromosomal instability in tumor
progression
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59
Abstract
In the previous chapter I showed that the development of numerical
chromosomal instability preceded spontaneous immortalization of ovarian
cystadenomas expressing SV40 large T antigen. In the present chapter, we
want to explore whether numerical chromosomal instability continues to
occur after spontaneous immortalization and try to clarify the role of
numerical chromosomal instability in both tumor development and
progression.
I first sought to examine the role of numerical chromosomal instability leading
to aneuploidy in malignant transformation by comparing the frequency at
which diploid and aneuploid populations to recover from crisis of the ovarian
cystadenoma cells expressing SV40 large T antigen and give rise to
spontaneously immortalized cell lines. I also sought to compare the relative
importance of aneuploidy versus mutation by treating cultured mortal human
ovarian cystadenoma cells with a mutagen and subsequently separating the
cells based on their DNA content in order to determine which fraction is more
likely to show features of malignant transformation. Although aneuploidy
alone decreased in vitro longevity of benign ovarian epithelial tumor cells
expressing SV40 large T antigen, the combined effects of aneuploidy and
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60
mutagen treatment resulted in increased longevity. Mutagenesis alone didn’t
have obvious effect on in vitro longevity. These findings suggest that
cooperation between numerical chromosomal instability due to aneuploidy
and mutagenesis mediate malignant transformation of benign ovarian
epithelial tumor cells expressing SV40 large T antigen.
I examined the chromosomal stability of cells after they recovered from crisis
and spontaneously immortalized by comparing the number of chromosomes
in clones of MCV39 cells at different time points. The results showed a trend
for a gradual increase in chromosome numbers. Although clones of MCV39
cells showed chromosomal losses as well as increases during a period of
time spanhing 25 population doubings, there seemed to be a selective
advantage for chromosomal increases. Growth curve analyses showed a
correlation between the growth kinetics of the MCV39 clones and their
chromosome numbers. Different clones with different chromosome numbers
had different growth rate. All these findings suggest that numerical
chromosomal instability persists after spontaneous immortalization,
accounting for the continuous creation of progenitors of aneuploid cells with
variable chromosome numbers. Such numerical chromosomal instability may
result in selective advantage of individual cell clones, resulting in clonal
evolution during the tumor progression process.
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61
Although several clonal losses of heterozygosity were detected in MCV39
cells soon after their isolation, they acquired only a single additional loss over
a period of 60 population doublings. This loss involved chromosome 15q.
Given that substantial changes in chromosome numbers took place during
that time period, I conclude that is not a true reflection of the degree of
aneuploidy in cancer cells. I further investigated the influence of LOH on
chromosome 15q in this cell culture model. Although MCV39 clones
harboring this loss had a shorter doubling time and a higher cloning
efficiency than those that did show this loss, similar advantages were not
observed in pre-crisis parental cells with LOH on chromosome 15q. The
results suggest that LOH on chromosome 15q is either a biomarker with no
direct influence on tumor development or progression, or that another genetic
event, such as a mutation affecting a tumor suppressor gene on the retained
allele of this chromosome, takes place in MCV39 cells.
Keywords: Aneuploidy; Mutation; Clonal evolution; Maligndnt transformation
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62
Introduction
It has been suggested that during the multistep carcinogenesis process, it
takes months to decades for a single, normal diploid cell to become a clinical
visible entity of cancer as well as for the cells of a primary cancer to evolve to
more aggressive phenotypes after the transformation process (Duesberg P
and others, 2003). A miniscule selective advantage in a cell clone, such as
one percent increase in the ratio between cell birth and cell death, has been
reported to lead significant clonal dominance over prolonged periods of time
(Harith R and others, 2003).
What determines the selective advantage of cells and serves as the driving
force for clonal evolution during tumor progression is still not clear. In the
previous chapter, I showed the development of numerical chromosomal
instability preceding crisis in ovarian cystadenomas expressing SV40 large T
antigen. Two clones that spontaneously recovered from crisis and became
immortal cell lines, called MCV39 and MCV50, were recovered from
aneuploid, but not from diploid cell populations. In this chapter, I investigate
whether numerical chromosomal instability persists after the spontaneous
immortalization and whether numerical chromosomal instability plays a
significant role in tumor development and progression.
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63
Currently, there are two hypotheses for the cause of cancer: aneuploidy
versus mutation hypothesis of cancer. The aneuploidy hypothesis of cancer,
currently supported by the research groups of Dr. Peter Duesberg (Li R and
others, 1997), states that aneuploidy is the primary cause of cancer.
According to this hypothesis, aneuploidy regroups thousands of genes
encoded on chromosomes and alters many genetic programs. The process
whereby aneuploidy leads to a new biologic entity, the cancer cells, has been
likened to the regrouping assembly lines in a car factory (Rasnick D and
others, 1999). On the other hand, the mutation hypothesis, which is currently
supported by the research groups of Dr. Robert A. Weinberg, states that
mutations are the primary cause of cancer. Mutations activate oncogenes
and inactivate tumor suppressor genes, resulting in disruption of critical
signaling pathways that trigger the cancer phenotype to arise (Alberts B and
others, 1994). Proponents of this theory regard aneuploidy as secondary or
nonessential. I sought to clarify the role of numerical chromosomal instability
in malignant transformation by comparing the in vitro lifespan and ability to
recover from crisis in diploid and aneuploid cell populations.
According to Dr. Brinkley William from the Baylor College of Medicine,
cooperation between both aneuploidy and mutagenesis may be necessary
for the establishment of the cancer phenotype (Tracy W, 2001). In order to
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64
compare the role of numerical chromosomal instability to that of mutagenesis
in malignant transformation, I treated mortal human ovarian cystadenoma
cells with a mutagen, benzo[a]pyrene, and subsequently separated the cells
based on their DNA content. I compared the effects of numerical
chromosomal instability versus mutagenesis, alone and in combination, on in
vitro longevity.
To further explore the dynamics and role of the numerical chromosomal
instability due to aneuploidy in the progression process, I determined the
chromosome numbers of MCV39 cell clones at various time points after their
isolation and investigated the consequences of numerical chromosomal
changes on in vitro kinetics.
Although several clonal losses of heterozygosity were detected in MCV39
cells soon after their isolation, they acquired only a single additional loss over
a period of 60 population doublings. This loss involved chromosome 15q.
Our observation is compatible with the findings of Dodson MK et al who have
previously observed that chromosome arm 15q were lost significantly more
frequently in high-grade epithelial ovarian carcinomas compared to low-grade
epithelial ovarian carcinomas (Dodson MK and others, 1993). Given that
substantial changes in chromosome numbers took place over the 60
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65
population doublings of MCV39 cells, I conclude that is not a true reflection of
the degree of aneuploidy in cancer cells. I further investigated the influence
of LOH on chromosome 15q in this cell culture model. Although MCV39
clones harboring this loss had a shorter doubling time and a higher cloning
efficiency than those that did show this loss, similar advantages were not
observed in pre-crisis parental cells with LOH on chromosome 15q. The
results suggest that LOH on chromosome 15q is either a biomarker with no
direct influence on tumor development or progression, or that another genetic
event, such as a mutation affecting a tumor suppressor gene on the retained
allele of this chromosome, takes place in MCV39 cells.
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66
Materials and Methods
Cell proliferation assays
Cell proliferation assays were performed using the MTT cell proliferation
assay kit (ATCC, Manassas, VA), Cells were plated onto 8 mm wells of
microtiter plates at a density of 1 x 104 cells per well and incubated at 37°C.
Ten p ,l of the MTT reagent was added to each well, including controls. After
incubation at 37°C for 4 hours, 100 pi of the detergent reagent was added,
followed by incubation in a 37°C incubator in the dark overnight. Absorbance
at 570 nm was read in a microtiter plate reader (Spectra-Rainbow, Tecan,
Austria) in quadruplicate wells. A total of 8 time points over a 32 days period
for each cell line. Rate of proliferation was compared in diploid and aneuploid
cells treated with either vehicle (acetone) alone, or with 1 pg/ml of
benzo[a]pyrene. Culture medium was used as blank control for absorbance.
Metaphase spread preparation
Cultured cells were treated with 70 pi of colcemid (Gibco® laboratories, life
technologies, INC. Grand Island, New York) overnight at 37°C. Cells were
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67
then resuspended in 0.56% KCI in H2O and incubated in a 37°C water bath
for 8 minutes, followed by fixation in 45ml methanol plus 5ml acetic acid at
room temperature for 10 minutes. After centrifugation at position 5 for 3
minutes in the IEC clinical centrifuge (Model 115VAC, International
equipment co. Needham HTS, Mass), the cell pellets were resuspended in
fixative and incubated in 4 °C refrigerator overnight. The next day, the cell
pellets were washed in fixative 2 to 3 times, resuspended in 1 ml of fixative,
and dropped on precleaned glass slides (superfrost® plus 25 x 75 x 1 mm,
Approx. 1 4 Gross, VWR Scientific, West Chester, PA).
Growth curve analysis
Cells were plated onto fifteen 35 mm dishes at a density of 25,000 cells per
plate. Every other day, cell numbers were determined in triplicate dishes
using a model Zf Coulter counter (Coulter electronics, INC. hialeah, FL).
Colony formation assay
Ten thousand cells were plated in 0.3% top agar over 0.5% base agar in 60
mm petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ). After
incubation at 37°C in a humidified incubator for 10 to 14 days, each petri dish
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68
was stained with 0.5 ml of 0.005% crystal violet for over one hour. Numbers
of colonies in each petri dish were counted under a dissecting microscope
(LeiCA MZ125).
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Results
69
Role of aneuploidy on epithelial cell immortalization
In chapter 2 ,1 showed evidence that numerical chromosomal abnormalities
and losses of heterozygosity, as two hallmarks of malignancy, were present
in cancer cells, at least in part, due to clonal expansions of abnormalities that
had developed before acquisition of replicative immortality, when the parental
cells underwent crisis. I showed evidence that cancer cells acquired genetic
abnormalities from clonal expansion of alterations. To further investigate the
role of numerical chromosomal instability leading to aneuploidy in malignant
transformation, we separated benign ovarian cystadenomas expressing
SV40 T Ag into diploid and aneuploid cell populations by fluorescence
activated cell sorting and compared the rate of spontaneous immortalization
of these two fractions. Because DNA content was evaluated using Hoechst
33342, a dye compatible with cell survival, the diploid dnd aneuploid cell
populations could be put back in culture after the sorting procedures.
Out of 17 different attempts, only two immortal cell clones were recovered.
These are the MCV39 and MCV50 cell lines described in chapter 2. Both of
them were derived from aneuploid populations. No immortal cell line was
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70
recovered from diploid fractions (Figure 3.1). Although this difference in rate
of spontaneous immortalization between diploid and aneuploid fractions is
not statistically significant, the results nevertheless suggest that the presence
of aneuploidy may facilitate the establishment of the neoplastic phenotype.
Differential effect of aneuploidy and mutagenesis on in vitro longevity
According to Knudson’s two hit hypothesis, inactivation of tumor suppressor
genes is due to mutagenesis of one allele followed by loss of heterozygosity
of the other. Since I showed before that ovarian cystadenoma cells
expressing SV40 T Ag developed losses of heterozygosity at the time of
crisis, I sought to treat these cells at early passage with a chemical mutagen.
My hypothesis was that this would increase the chances that tumor
suppressor genes would become inactivated when the cells reached crisis,
which, as I already knew, was associated with frequent losses of
heterozygosity. I anticipated that this would enhance the rate of spontaneous
immortalization (Figure 3.2a).
The mutagen that I chose is benzo [a] pyrene (Bp), a polycyclic aromatic
hydrocarbon. The carcinogenic and mutagenic effects of benzo [a] pyrene
have been shown in several cell systems including human (Tokiwa H and
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71
others, 1993; Harvey RG, 1991). Active metabolites of benzo [a] pyrene have
been shown to bind covalently to DNA and form DNA adducts at the N2
position of guanine, causing mostly G to T transversions (Denissenko MF
and others, 1996).
Thus I treated cultured ovarian cystadenoma cells expressing SV40 large T
antigen with benzo [a] pyrene at early passage and separated the cells into
diploid and aneuploid cell populations 6 to 10 population doublings after
treatment with this mutagen. Cells treated with the vehicle only (acetone)
were used as control (Figure 3.2b).
No immortal cell line was obtained in 4 experiments using this approach. I
therefore performed cell proliferation assays in the various fractions at
different time points to examine the effects of mutagenesis and aneuploidy,
alone and in combination, on in vitro longevity. Aneuploid cells untreated with
benzo [a] pyrene invariably reached crisis 16-20 days (approximately 6
population doublings) earlier than untreated diploid cells (Figure 3.3). In
contrast, aneuploid cells which had been treated with the mutagen continued
to proliferate for an average of 24 days longer than treated diploid cells, at
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72
which time their total cell numbers started to show a gradual decrease due to
crisis. Mutagensis alone had no measurable effect on in vitro longevity
(Figure 3.3).
Chromosomal stability before and after spontaneous immortalization
Our allelotype analyses (see chapter 2) showed that MCV39 cells acquired a
single additional loss of heterozygosity when they were maintained in culture
for as long as 60 population doublings. This suggested that the cells were
significantly more stable genetically than before they acquired replicative
immortality. This conclusion was also supported by the DNA profiles obtained
from flow cytometry experiments, which were more stable and closer to
normal after acquisition of immortalization. To further examine the stabilty of
cells after spontaneous immortalization, we performed metaphase spreads to
count chromosome numbers in subclones of MCV39 cells obtained at low
and high passages. I found significant variations in chromosome numbers
among the various subclones, with a tendency to a chromosomal increase at
higher passages (Table 2). Thus, cycles of asymmetric division and mitotic
errors continued to occur after spontaneous immortalization.
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73
I next sought to investigate the chromosomal stability of cells that did not
spontaneously recover from crisis, but were immortalized artificially after
transfecting the catalytic subunit of human telomerase. The cells were
fractionated into diploid and aneuploid populations by FACS, were
subcloned, and chromosome numbers were examined at various time points
in one aneuploid clone. The results showed a gradual loss of chromosomes,
with a return to near diploidity (Table 3). These results are consistent with
those of an earlier student in our laboratory, Mihaela Velicescu, who showed
that cells recovered after telomerase transfection were primarily diploid. This
also contrasts with the trend toward an increase in chromosome numbers
that was observed overtime in spontaneously immortalized cells (Table 2).
Correlation between chromosome numbers and growth kinetics in vitro
We hypothesized that the numerical chromosomal instability that is present in
MCV39 cells may drive clonal evolution during the progression process in our
in vitro model. I performed growth curve analyses of the six MCV39 low
passage clones described earlier. The data (Figure 3.4) clearly showed that
the various changes in chromosome numbers that had taken place in the
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74
different clones were also associated with changes in cell doubling times.
These results support the idea that chromosomal instability drives tumor
evolution as suggested by the classical model of tumor progression.
To further examine the dynamics of chromosomal changes in MCV39 cells, 6
subclones of this cell line were obtained at low passage. Chromosome
numbers were examined at low passage. Chromosome numbers were
examined immediately after the clones were isolated as well as 25 population
doublings later. All clones showed extensive variability in their chromosome
numbers over that period of time, with a tendency to become near tetraploid
(Figure 3.5).
This suggests that aneuploid cells continue to accumulate, perhaps due to
G2/M abnormalities leading to tetraploidy, followed by subsequent
chromosomal losses and gains resulting in aneuploidy. As was observed
earlier with random subclones of MCV39 (Figure 3.4), the clones that were
followed over this period of 25 population doublings showed marked changes
in their doubling times (Figure 3.6).
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75
A model for tumor development
In chapter 2 ,1 have presented a model for acquisition of alterations in cancer
development, which suggested that several alterations occured before the
malignant phenotype was fully developed. Based on observations in this
chapter, numerical chromosomal instability seems to continue to occur after
spontaneous immortalization and drive clonal evolution further. Thus I
propose to modify the model presented in chapter 2 by adding an arrow
between low-grade and high-grade tumors (Figure 3.7). This implies that
although grade of malignancy may be determined early after tumor
development, progression from low to high-grade lesions can also occur at
later points.
Consequences of LOH on 15q on tumor development and progression
In chapter 2 ,1 reported that LOH on 15q was the only additional loss
acquired by MCV39 cells after their spontaneous immortalization. I sought to
investigate the significance of this loss by performing growth curve analyses
of clones with and without this abnormality before and after recovery from
crisis. After recovery from crisis, clones carrying this loss had shorter
doubling times. However, clones obtained before crisis had longer doubling
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76
times if they harbored this loss (Figure 3.8). Likewise, clones obtained after
recovery from crisis that carried the loss on chromosome 15q had increased
cloning efficiency, while clones obtained before crisis that carried the same
loss had decreased cloning efficiency (Figure 3.9). I conclude that LOH on
15q is a mere marker with no reference to the tumor phenotype in pre-crisis
cells. It is possible that this loss actively drove tumor progression in cells that
recovered from crisis. If so, a mutation must have occurred on a gene in the
retained allele because LOH on 15q by itself, without an accompanying
mutation, does not confer any selective advantage in vitro under our culture
conditions.
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77
Figure 3.1 The role of aneuploidy in immortalization of benign ovarian
epithelial tumor cells expressing SV40 large T Ag. To examine the role of
aneuploidy in malignant transformation, we separated benign ovarian
cystadenomas expressing SV40 T Ag, based on their DNA content, to diploid
and aneuploid cell populations by fluorescent activated cell sorting and
examined which cell populations were easier to be transformed. Both the
diploid and aneuploid cell populations underwent crisis eventually. Out of 17
separate experiments, we were ctble to obtain only two cell lines, called
MCV39 and MCV50, both spontaneously recovered from crisis of the
aneuploid cell populations and became immortalized. No cell line recovered
from the crisis of the diploid cell populations. This suggests aneuploid cell
populations are easier to be transformed than diploid cell populations and
aneuploidy may facilitate malignant transformation of benign ovarian
epithelial tumor cells expressing SV40 large T Ag.
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Diploid
1
Crisis
ML10
FACS
Aneuploid
I
Crisis
X
MCV39 MCV50
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79
Figure 3.2 Does aneuploidy facilitate transformation in the presence of
mutagen? - hypothesis and rationale, (a) One kind of the “two hit”
mechanism to inactivate tumor suppressor gene is hit of the first allele by
mutation followed by hit of the second allele by loss of heterozygosity. Since
we observed that ovarian cystadenoma cells expressing SV40 T Ag
developed loss of heterozygosity as a result of severe aneuploidy changes at
the time of crisis, if we treat these cells at their early passages with some
chemical mutagen to introduce mutations into these cells, the chemical
mutagen treatment will increase the chance to inactivate tumor suppressor
genes in these cells when they later develop many losses of heterozygosity
and reach crisis. We hypothesize this will greatly enhance the opportunity to
obtain spontaneously immortalized cell lines to recover from crisis, (b) We
treated cultured cystadenoma cells with benzo[a]pyrene (Bp), a chemical
mutagen, at their early passages, then we used FACS to separate the cells
based on their DNA content to diploid and aneuploid cell populations 6 to 10
population doublings after treatment with this mutagen. Cells treated with
vehicle acetone were used as control. We compared the significance of
aneuploidy to that of mutagenesis in malignant transformation by comparing
the frequency to acquire immortal cell lines from the diploid versus aneuploid
cell populations obtained after Bp or acetone treatment.
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80
«
mutation |_OH
-fr
Mutagen Crisis
FACS based on DNA content FACS based on DNA content
Compare
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81
Figure 3.3 Significance of aneuploidy compared to mutagenesis on in
vitro longevity of benign ovarian epithelial tumor cells expressing SV40
T antigen. Since no immortalized cell line was obtained from any group of
cells after one-year trial, we performed cell proliferation assay to compare
their in vitro life span. Aneuploid cell populations without Bp treatment
invariably reached crisis 16-20 days (approximately 6 population doublings)
earlier than untreated diploid cells, which recovered from the same sorting
experiment. In contrast, aneuploid cells treated with Bp continued to
proliferate for an average of 24 days more than treated diploid cells, at which
time their total cell numbers started to show a gradual decrease due to crisis.
Bp treatment had no measurable effect on in vitro longevity in diploid cells.
Thus, although aneuploidy alone decreased in vitro longevity, the combined
effects of aneuploidy and mutagenesis resulted in increased longevity. We
conclude aneuploidy and mutagenesis cooperate to mediate malignant
transformation.
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82
o 0.15
■ ML10+Bp(G2)1
■ ML10+Bp(G1)1
□ ML10+Ace(G1)1
ML10+Ace(G2)1
8 12 16 20 24 28 32
Tim e (D ays)
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83
Table 2. The stability of the cells after spontaneous immortalization.
Allelotype analysis and flow cytometry all suggested the stability of cells
during spontaneous immortalization. To determine the stability of cells after
spontaneous immortalization, we performed metaphase spread to count
chromosome numbers of MCV39 cell clones. Both the MCV39 low passage
and high passage cell clones were not constant for their chromosome
numbers. Even in the same clone, there existed variable chromosome
numbers between the metaphase counts. There was a trend for incfease in
chromosome numbers when MCV39 cell clones progressed to high
passages.
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84
MCV39
HP
Clone
7
8
MCV39
HP
Clone
6
8
MCV39
HP
Clone
5
70-123
MCV39
HP
Clone
4
70-100
MCV39
HP
Clone
3
65-100
MCV39
HP
Clone
2
8
MCV39
HP
Clone
1
8
MCV39
U P
Clone
6
65-82,
101-124
MCV39
LP
Clone
5
4 4 - 50,
92-100
MCV39
LP
Clone
4
60-70
MCV39
LP
Clone
3
§
MCV39
LP
Clone
2
8 -
MCV39
LP
Clone
1
*
Chromosome
number
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85
Table 3 Telomerase-transfected diploid ceils outgrow aneuploid cells.
MCV167 was an immortal cell line we obtained by transfection of ML10, the
ovarian cystadenoma cells expressing SV40 T Ag, with the catalytic subunit
of human telomerase, hTERT. After telomerase transfection, most
populations of cells were diploid, although few aneuploid populations still
remained. By fluorescence activated cell sorting, we were able to isolate the
few aneuploid populations and put them back to culture, called
MCV167(G2)3- By subcloning, we obtained seven subclones from the sorted
aneuploid cell populations, four of which were diploid, three of which were
aneuploid. We chose one aneuploid subclone, clone 3, and put it in culture
from 13 population doublings to 30 population doublings after subcloning. We
counted chromosome numbers of clone 3 every passage (3.3 population
doublings) by metaphase spread. Clone 3 had 71 chromosomes at 13
population doublings after subcloning. Then small numbers of near-diploid
cells with 47 or 41 chromosomes started to show up in clone 3 and gradually
outgrew aneuploid cells, eventually all the cells in clone 3 became near
diploid and remained to be near-diploid thereafter. There was a trend of
decrease of chromosome numbers in the cell clones with ectopic expression
of telomerase. In contrast, we observed a trend of increase of chromosome
numbers in spontaneously immortalized MCV39 cell clones as they
progressed to high passages. All these suggest numerical chromosomal
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86
instability manifested as either increase or decrease of chromosome
numbers continue to occur duHng the progression process.
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0 )
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88
Figure 3.4 Correlation of chromosome numbers with the growth kinetics
of MCV39 low passage clones. Growth curve analysis was performed for
the six MCV39 low passage clones. There was correlation between the
growth kinetics of the MCV39 low passage cell clones and their chromosome
numbers. Different clones with different chromosome numbers had different
growth rate.
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89
6,5
W
Q
2
Q )
n
E
3
C
©
a
o
O )
o
5.5
X '
4.5
i a 1:54 chromosomes
-m --~ 02:50 chromosomes
-±— 03:40 chromosomes
04:60-70
chromosomes
0 5 :44-50, 92-100
chromosomes
06:65-82,101-124
chromosomes
1 0
Culture Time (Days)
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90
Figure 3.5 Observation of numerical chromosomal instability in MCV39
cell clones 25 population doublings later. Previously, we revived the
frozen vials of MCV39 low passage and high passage cells and did
subcloning of them. We were able to obtain six subclones of MCV39 low
passage cells and seven subclones of MCV39 high passage cells, their
chromosme numbers were obtained by metaphae spread (Table 2). We next
sought to examine the stabilty of cells after spontaneous immortalization by
examining the numerical chromosomal stability in single cell clones over the
progression process. So we put all six MCV39 low passage clones in culture
and cultured them for an additional twenty five population doublings, at which
time we counted their chromosome numbers again. Twenty five population
doublings later, all the MCV39 cell clones showed a more extensive
variability of chromosome numbers within the same clone and the modal
chromosome number range showed that all the clones developed near-
tetraploidy (MN ranges in red were the major modal chromosome numbers).
This suggests that aneuploid cells indeed continue to divide with endless
mitotic errors to generate progenitors of cells with different chromosome
numbers. Hyper- and hypo- tetraploidy development seems to be a trend of
chromosome number changes and may give the clone some selective
advantage during the progression process.
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91
CHROMOSOME
NUMBERS
AT 13 PDs
CHROMOSOME
NUMBERS
AT 38 PDs
25 PDs
MN
RANGE
40-97
37-119
36-107
39-126
40-125
32-164
90-100
47-55, 90-100
32-164 ) 47-55, 90-100
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92
Figure 3.6 Correlation of chromosome numbers with population
doubling time of MCV39 cell clones 25 population doublings later. One
of the MCV39 low passage clones, clone 3, had 40 chromosomes at 13
population doublings, and grew very slowly with population doubling time of
7.96 ± 1.34 days. We initially thought it might not be able to grow up.
However, 25 population doublings later, it showed very good logarithmic
growth with greatly reduced population doubling time, which was 1.75 ± 0.02
days, corresponding to its changed chromosome numbers between 36-107,
mostly between 60-80 and 90-100. Another MCV39 low passage clone,
clone 4, had 60-70 chromosomes at 13 population doublings and showed a
middle fast growth rate with population doubling time of 3.01 ±0.15 days. 25
population doublings later, it showed greatly reduced population doubling
time, which was 2.35 ± 0.21 days, and very good logarithmic growth,
corresponding to its changed chromosome numbers between 39-126, mostly
between 90-100 chromosomes. The observation that the growth kinetics of
the cell clones vary in response to their altered chromosome numbers further
support that numerical chromosomal instability drives for clonal evolution
during tumor progression.
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93
CHROMOSOME PD TIME
NUMBER AT 13 PDs (DAYS)
CI1
7.96 ±1.34
3.01 ±0.15
CHROMOSOME PD TIME
NUMBER AT 38 PDs (DAYS)
40-97
37-119
36-107
39-126
40-125
32-164
36-107 ) 1-75 ±0.02
39-126 ) 2.35 ±0.21
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94
Figure 3.7 A model for tumor development In chapter 2, we have
presented a model for acquisition of alterations in cancer development.
Based on our observations in this chapter, numerical chromosomal instability
continues to occur after spontaneous immortalization and dirves clonal
evolutin during the progression process. Thus we propose a model for tumor
development by adding an arrow to the previous model we proposed in
chapter 2. The single aneuploid cell that recovers from crisis is unstable and
divides with mitotic errors to generate progenitors of aneuploid cells with
variable chromosome numbers. After the transformation process, this
numerical chromosomal instability determines the selective advantage of the
cells and may result in the outgrowth of some tumor masses. Numerical
chromosomal instability drives clonal evolution during the progression
process from the low-grade carcinoma to the high-grade carcinoma.
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Proliferation
signal
Senescence
Low grade
carcinoma
High grade
carcinoma
Genetic instability
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96
Figure 3.8 The relationship between LOH on 15q and the growth rate of
clones before and after crisis. Growth curve analyses were performed for
clones before crisis and after recovery from crisis with and without LOH on
15q. After recovery from crisis, clone with this loss growed faster and LOH on
15q gave selective advantage for its growth kinetics. However, before crisis,
clone with this loss growed slower and LOH on 15q didn’t give selective
advantage for its growth kinetics. Thus, LOH on 15q has differential effect on
the growth rate of clones before and after recovery from crisis.
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97
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98
Figure 3.9 The relationship between LOH on 15q and the transformation
ability of clones before and after crisis. Colony formation assays were
performed for clones before crisis and after recovery from crisis with and
without LOH on 15q. (a) The dots in the rectangular areas are colonies
formed within one square cm area in the middle of the petri dishes. After
crisis, the clone with loss formed more colonies and LOH on 15q gave the
clone selective advantage for its transformation ability. Before crisis, the
clone with loss formed fewer colonies and LOH on 15q didn’t give the clone
selective advantage for its transformation ability, (b) The histogram
summarizes the number of colonies we observed in each group. Thus LOH
on 15q has differential effect on the transformation ability of clones before
and after recovery from crisis. The results of the growth curve analysis and
the colony formation assay suggest that LOH on 15q is either a biomarker
with no direct influence on tumor development or progression, or that another
genetic event, such as a mutation affecting a tumor suppressor gene on the
retained allele of this chromosome, takes place in MCV39 cells.
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99
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101
Discussion
Our allelotype analyses showed that all the MCV39 low passage subclones
were quite clonal for loss of heterozygosity and suggested only one single
aneuploid cell recovered from crisis and became spontaneously
immortalized. Our flow cytometry data also suggested the stability of the cell
during spontaneous immortalization. Thus it is unexpected for us to find that
different clones after spontaneous immortalization are not constant for their
chromosomal numbers, and even in the same clone, there exist variable
chromosome numbers between the metaphase counts. When we put all six
MCV39 low passage clones in culture and culture them 25 more population
doublings, all the six MCV39 clones show a more extensive variability of
chromosome numbers within the same clone. All these suggest that the
single aneuploid cell that recovers from crisis is very unstable, it continues to
divide with mitotic errors to generate progenitors of aneuploid cells with
different chromosome numbers. Numerical chromosomal instability continues
to occur after spontaneous immortalization, during the progression process.
Impressively, two MCV39 low passage clones, clone 3 and clone 4, show
greatly reduced population doubling time and very good logarithmic growth
corresponding to their changed chromosome numbers 25 population
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102
doublings later. The growth curve analysis shows that the growth kinetics of
the MCV39 cell clones always correlate with their chromosome numbers.
Different cell clones with different chromosome numbers but derive from the
same parental cell have different growth rate. Our findings suggest that the
numerical chromosomal instability, which correlates with the growth kinetics
of the cells, may serve as driving force for clonal evolution during tumor
progression, for example, from low-grade cancer to high-grade cancer.
Accordingly, we have proposed a model for tumor development as shown in
Figure 3.7.
Compatible with our findings, numerical chromosomal instability has been
previously reported in chemically induced cancers of Chinese hamsters
(Mitelman F and others, 1972), rats and mice (Levan G and others, 1975).
Numerical chromosomal instability has also been observed in spontaneous
cancers of human beings (Heim S and others, 1987). The numerical
chromosomal instability we observe in our in vitro model for ovarian tumor
development may offer some explanation for the heterogeneous karyotypes
frequently observed in solid cancer cells. These heterogeneous karyotypes
may result from similar mitotic errors as a result of multipolar mitosis due to
imbalance of the spindle apparatus induced by aneuploidy (Brinkley BR and
others, 1998). The heterogeneous karyotypes of cancer cells may simply
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103
reflect the progenitors of aneuploid cells that are continuously created during
the progression process, due to persisted numerical chromosomal instability.
Dr. Peter Duesberg has suggested that the multistep carcinogenesis actually
correspond to the multiple steps of aneuploidization (Duesberg P and others,
2003).
Our data in the previous chapter have suggested that cancer cells acquire
genetic abnormalities from clonal expansion of changes in a single aneuploid
cell with numerical chromosomal instability at crisis. In this chapter, we show
that numerical chromosomal instability continues to occur in the spontaneous
immortalized cells and drives for clonal evolution during tumor progression.
Taken together, it seems to suggest that numerical chromosomal instability
leading to aneuploidy may play role in both tumor development and
progression.
In addition to aneuploidy, cancer cells also have other genomic
abnormalities, such as mutation. However, currently what is the role of
aneuploidy and the role of mutation in malignant transformation are still not
clear. Therefore, we sought to examine the relative significance of
aneuploidy compared to that of mutation in malignant transformation. Our
findings suggest that aneuploidy and mutation may cooperate to mediate
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104
malignant transformation. Although aneuploidy alone decreases in vitro
longevity of benign ovarian epithelial tumor cells expressing SV40 large T
antigen, the combined effects of aneuploidy and mutagenesis result in
increased longevity. These results are compatible with the idea that
aneuploidy contributes to malignant transformation by unmasking mutations
that will otherwise remain silent due to their recessive nature in diploid cells.
Our data are compatible with the findings of Wei Dai et al, who observed that
as a result of Bub1 haploinsufficiency, mouse embryonic fibroblasts
developed widespread aneuploidy due to significantly compromised spindle
checkpoint. In response to challenge with carcinogen azoxymethane, these
mice rapidly developed lung and intestinal adenocarcinomas (Dai W and
others, 2004), which suggested aneuploidy could greatly facilitate tumor
development in the presence of mutations. In addition, our findings are also
compatible with those of Richard A. Woo et al. who recently found that
aneuploidy due to abolishment of the p53 pathway could promote malignant
transformation in the presence of coexpression of two oncogenes, ras and
E1A (Richard AW and others, 2004).
During the process of multistep carcinogenesis, some genotoxic carcinogens
induce aneuploidy by mutating specific mitosis genes, then the resulting
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105
aneuploidy may evolve autocatalytically, corresponding to the evolvement of
a normal cell to a clinical entity of cancer as well as the evolvement of a
primary cancer to the more aggressive phenotypes. This cooperation
between mutation and aneuploidy in malignant transformation has recently
been proposed as a “two-stage” model of carcinogenesis. It also suggests
that sometimes it is not easy to separate mutations from aneuploidy, since
some mutations can induce aneuploidy (Duesberg P and others, 2003). Our
findings suggest that the type of mutations that cooperate with aneuploidy to
mediate malignant transformation is not necessarily mutations of mitosis
genes, since random mutations caused by mutagen, such as
benzo[a]pyrene, can also cooperate with aneuploidy to mediate malignant
transformation.
Further study, such as transfection of the catalytic subunit of human
telomerase, hTERT, into the diploid and aneuploid cell populations obtained
after benzo[a]pyrene or acetone treatment, followed by comparison of their in
vitro immortality may help us better compare the significance of aneuploidy to
that of mutation in malignant transformation.
We found almost all MCV39 high passage clones had near-tetraploid
chromosome numbers. Furthermore, when we took six MCV39 low passage
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106
clones and cultured them 25 more population doublings, we also observed
that they all developed near-tetraploid modal chromosomal numbers. Both
seem to suggest hypo or hyper-tetraploidy development as a trend of
changes in chromosome numbers during the progression process and they
may give the cells some selective advantage. Compatible with our findings,
many human solid cancers have been frequently found to have between 60
and 90 chromosomes, that is, near triploid or tetraploid (Sandberg AA, 1990).
For example, the DNA index of the colon cancer cells is 1.71 (Lengauer C
and others, 1997). It has also been reported that the DNA index of late stage
cancers is usually between 1.5 and 2 (Shackney SE and others, 1995).
It was proposed that these near tetraploid chromosome numbers might be
achieved either by doubling of hypodiploid cells directly (Giaretti W and
others, 1990) or by doubling of diploid or hyperdiploid cells followed by
chromosome loss (Heim S and others, 1995). During the progression
process, tetraploidization may promote the viability of the cancer cells. Near
doubling of the chromosome numbers of a normal diploid cell can easily
compensate any chromosome loss in cancer cells and improve their
adaptation to various tissue environment, thus facilitate their invasion and
metastasis (Duesberg P and others, 2000).
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107
Taken together, our study not only demonstrates the role of numerical
chromosomal instability in tumor development, but also provides comparison
of the relative significance of numerical chromosomal instability leading to
aneuploidy to that of mutation in mediation of malignant transformation. Our
study gives deeper insight into the complex dynamics of numerical
chromosomal instability in clonal evolution during tumor progression.
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108
Chapter 4
Differences in ploidy stability between ovarian
tumors of low malignant potential and ovarian
cystadenomas
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109
Abstract
We previously showed that ovarian cystadenomas developed aneuploidy and
the crisis of ovarian cystadenomas was determined by aneuploidy instead of
telomere shortening. The purpose of this study is to examine the ploidy
stability of ovarian tumors of low malignant potential (LMPs) and the
determinant of their crisis and compare to ovarian cystadenomas.
We used two cell lines of ovarian tumors of low malignant potential for our
study, ML38 and ML46. ML38 was cell line derived from patients with
mucinous LMP tumor. ML46 was cell line derived from patients with serous
LMP tumor with invasive implants. The DNA profiles of these two LMP cell
lines showed that they were diploid both at their early passages and at their
crisis. Western blot showed that both of them expressed lower level of SV40
T Ag compared to the ovarian cystadenoma cell line ML10. Impressively,
both ML38 and ML46 still remained diploid even after we increased their
SV40 T Ag expression to the level compatible with that of ovarian
cystadenomas, by infection with adenovirus vector carrying SV40 T Ag. The
SV40 T Ag in both LMP cell lines were shown to be active by
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1 1 0
immunoprecipitation with p53 Ab followed by western blot with SV40 T Ag
Ab. Southern blot showed that the telomere length of ML38 gradually
decreased from 19 PD to 28 PD till crisis.
Our results suggest that ovarian tumors of low malignant potential tend to
remain diploid and do not develop aneuploidy. This is not because of loss of
their SV40 T Ag expression or activity. The crisis of LMPs is determined by
short of telomere length, instead of aneuploidy. All these findings suggest
that ovarian tumors of low malignant potential is very different from ovarian
cystadenomas and is a distinct disease from ovarian cystadenomas.
Keywords: Ovarian tumors of low malignant potential; Ploidy stability;
Telomere length; Crisis
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111
Introduction
In 1929, Howard Taylor first described an intermediate "semimalignant"
group of ovarian serous tumors (Taylor JHC, 1929). The clinical existence of
such an ovarian disease with a favorable prognosis but histologic features
resembling ovarian carcinoma was soon established (Aure JC and others,
1971). Recommended by both the International Federation of Gynecology
and Obstetrics (FIGO) (Santesson L and others, 1968) and the World Health
Organization (WHO) (Serov SF and others, 1973), ovarian epithelial tumors
are currently classified into benign tumors called cystadenomas, tumors of
low malignant potential (also called tumors of borderline malignancy), and
carcinomas. More recently, a suggestion was made to abandon the category
of borderline tumors, based on the argument that these tumors can always
be divided into benign and malignant subtypes (Seidman JD and others,
2002). Regardless of the merit of this classification from a purely clinical point
of view, they represent a distinct biological entity with molecular biological
features that separate them from either cystadenomas or carcinomas.
Although the question of whether or not they are precursors of carcinomas
remains controversial, the finding of molecular biological alterations that are
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112
more frequent in LMP tumors than low-grade carcinomas, plus the fact
recurrent LMP tumors usually retain their LMP characteristics, argues against
the idea that such progression in a frequent event.
Although a number of molecular genetic alterations have been associated
with LMP tumors, very little is known about their underlying mechanism.
Progress in this area will have important implications in our understanding of
cancer biology because although these tumors can readily metastasize to
extra-ovarian sites, they show either little or no invasive ability. Thus, they
provide a model to dissect those two hallmarks of the malignant phenotype.
We recently studied molecular mechanisms associated with acquisition of in
vitro immortality in cultured ovarian cystadenoma cells transfected with an
expression vector for SV40 large T antigen. As those cells aged in culture,
they underwent severe changes in their DNA ploidy. We showed that such
ploidy changes, which triggered apoptosis, were an important determinant of
in vitro crisis, which occured without substantial telomere attrition in this cell
culture model. We now report that cell cultures derived from LMP tumors and
transfected with the same vector for SV40 large T antigen used in our studies
with cystadenomas fail to show any significant change in their DNA ploidy as
they approach crisis. In contrast to cystadenomas, crisis is determined
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113
primarily by telomere attrition in LMP tumor cells. These results point to a
fundamental difference in the mechanism of acquisition of replicative
immortality between ovarian cystadenomas and LMP tumors.
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114
Materials and Methods
Cell lines and culture
ML38 was a cell line derived from patients with mucinous LMP tumor. ML46
was a cell line derived from patients with serous LMP tumor with invasive
implants. Both cell strains were isolated and transfected with SV40 large T
antigen using published protocols (Luo MP and others, 1997). HOC-7
ovarian carcinoma cell line was obtained from Dr. Ronald N. Buick,
University of Toronto (Buick RN and others, 1985). The source of ML10 was
described earlier (Luo MP and others, 1997). All cells were grown in MEM
supplemented with 10% FBS. HOC-7 cell line was grown in RPMI
supplemented with 10% FBS.
Analysis of DNA ploidy by flow cytometry
One million cells were resuspended in phosphate-buffered saline (PBS) and
fixed in 70% ethanol. After centrifugation, the cell pellet was resuspended in
1 ml of PBS, 10 pg /ml propidium iodide and 100 pg/ml RNase. The cells
were analyzed for distribution of fluorescence intensity using a Coulter Profile
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115
II flowcytometer (Beckman Coulter, Hialeah, FL) and analyzed using the
MultiCycle software (Phoenix Flow Systems Inc., San Diego, CA).
Infection of cells with vector carrying SV40 large T antigen
The source of the adenoviral vector for SV40 large T antigen was described
earlier. We added 7.5 x 10 7 PFU to 90% confluent cultures of LMP cell lines
in 35 mm tissue culture dishes. The cells were re-infected 48 hours later.
Western blot analysis
Cell monolayers were rinsed with 10 ml of ice-cold phosphate-buffered saline
(PBS) and treated with ice-cold RIPA lysis buffer (20 mM Tris-HCI pH 8.0,
125 mM NaCI, 0.5% NP-40, 20 mM NaF, 0.2 mM Na3 P04 l 2 mM EDTA, 35
pg/ml PMSF, 0.7 pg/ml pepstatatin A, and 0.5 pg/ml leupeptin) at 4°C for 30
minutes. They were detached from the tissue culture dishes (Becton
Dickinson Labware, Franklin Lakes, NJ) by rubbing with a cell lifter (Fisher
Scientific, Pittsburgh, PA) and centrifuged at 12000 rpm for 30 minutes. The
supernatants were stored at -80°C. Protein concentrations were determined
using the BCA protein assay reagent kit (Pierce, Rockford, IL). Samples
containing 10 pg of protein were electrophoresed on 10% polyacrylamide gel
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1 1 6
and transferred onto nitrocellulose membranes (Biorad Laboratories,
Richmond, CA). The membranes were incubated overnight in 10% non-fat
milk (Biorad Laboratories, Richmond, CA), in 0.1% of PBS/Tween-20, and
exposed to primary antibody for 1 hour at room temperature. Following three
10 minutes washing in 0.1% of PBS/Tween-20, the membranes were
exposed to the secondary antibody coupled to horseradish peroxidase. The
signal was detected by the ECL western blotting detection reagents
(Amersham Biosciences, Buckinghamshire, England). For loading control,
the membrane was stripped in 100 mM p - mercaptoethanol, 2% SDS, 62.5
mM Tris-HCI, pH 6.7 at 65 °C for 30 minutes, followed by extensive washing
in 0.1% of PBS/Tween 20, and reprobed with monoclonal antibody against p-
actin (Sigma, Saint Louis, MO). Monoclonal antibody for SV40 large T
antigen was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, cat #
sc-147).
Immunoprecipitation with p53 antibody and western blot analysis of
SV40 large T Ag
Two pg of primary monoclonal antibody against p53 (Santa Cruz
Biotechnology, Santa Cruz, CA) was added to 500 pg of total cellular protein
extract in a 1.5 ml microcentrifuge tube and incubated at 4 °C for 2 hours.
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117
Fifty pi of resuspended protein A-agarose was added and incubated at 4 °C
on a rocker overnight. Immunoprecipitates were collected by centrifugation at
2500 rpm for 5 minutes at 4 °C. The supernatant was aspirated and the pellet
was washed 4 times with 1.0 ml RIPA buffer, resuspended in 20 pi of 2 x
electrophoresis sample buffer, and heated in boiling water for 2 to 3 minutes.
After removing the agarose beads by centrifugation, 20 pi aliquots of
supernatant were electrophoresed on SDS-PAGE followed by western
blotting with monoclonal antibody for SV40 large T Ag.
Southern blotting
Ten pg genomic DNA from ML38, obtained after 19, 28 and 38 population
doublings, were digested with Rsa l/Hinf I restriction endonucleases,
electrophoresed in 1 % agarose gels, and transferred onto Zeta Probe GT
membranes (BioRad Laboratories, Hercules, CA) in 0.4 M NaOH following
acid depurination as described previously (Dubeau L and others, 1988). The
probe, which consisted in a synthetic fragment containing 3 repeats of the
human telomeric sequence (TTAGGG)3 , was end-labelled with Y-3 2 P-dCTP.
The membrane was hybridized in 0.5 M NaHP04 , pH 7.2, 5 % SDS, 1 mM
EDTA, pH 8.0, 1 % BSA, 50 % Formamide at 42 °C overnight following pre
hybridization for 1 hour under the same conditions. The membrane was then
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118
washed for 15 minutes at 42 °C with 2 x SSC, 1 % SDS, followed by
washing with 0.2 x SSC, 1 % SDS for 15 minutes at room temperature. The
hybridization signals were visualized by exposure to a phosphorimager
(Model GS-525, Molecular Imager® System, Bio-Rad Laboratories, Hercules,
CA).
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119
Results
Differences in ploidy stability between ovarian cystadenomas and LMP
tumors expressing SV40 large T antigen
Previously, we observed that ovarian cystadenoma cells expressing SV40
large T antigen, called ML10, developed polyploidy / aneuploidy in the period
immediately preceding the initiation of crisis (Velicescu M and others, 2003).
In this study, we cultured the two LMP cell lines expressing SV40 large T
antigen, called ML38 and ML46, from their early passages until they reached
crisis to study the ploidy stability of ovarian tumors of low malignant potential
(LMPs) and compared to ovarian cystadenomas. By flow cytometry, we
observed that the DNA profiles of the two LMP cell lines ML38 and ML46
were diploid at their early passages and they remained diploid and didn’t
show any aneuploidy changes at the time of their crisis (Figure 4.1a). The
flow cytometry did show apoptosis in both LMP cell lines at crisis (Figure
4.1a) and western blot showed the LMP cell lines at crisis expressed
elevated level of p21 compared to their early passages (Figure 4.1b), which
supported the LMP cells we analyzed at crisis were indeed in crisis. Our data
show that ovarian tumors of low malignant potential are very different in their
ploidy stability compared to ovarian cystadenomas.
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120
Differences in ploidy stability between cystadenomas and LMP tumors
are not determined by differences in levels of SV40 large T antigen
expression
Since aneuploidy characteristically developed in cells expressing SV40 T Ag,
we examined whether the two LMP cell lines expressed different levels of
SV40 T Ag compared to ovarian cystadenomas by western blot, followed by
exploring whether this was the reason why LMPs were different in their ploidy
stability from cystadenomas. Western blot showed that the two LMP cell lines
ML38 and ML46 expressed lower level of SV40 T Ag compared to the
cystadenoma cell line ML10 (Figure 4.2a). Although the difference was not
that huge, we still wanted to rule out the possibility this lower level of SV40 T
Ag expression was the reason why LMPs didn’t develop severe aneuploidy.
Thus, we tried to increase SV40 T Ag expression in the LMP cell lines by
infecting them with adenovirus vector expressing SV40 T Ag in order to
examine whether the LMPs developed aneuploidy with higher level of SV40
T Ag expression similar to cystadenomas. After infection of ML38 with
adenovirus vector expressing SV40 T Ag, we obtained the cell line called
ML38T. By western blot, we observed elevated level of SV40 T Ag
expression in ML38T at 33 population doublings after T Ag infection, almost
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121
compatible to the level expressed in ovarian cystadenomas ML10. By flow
cytometry, we found both ML38 and ML38T remained diploid at 33
population doublings after SV40 large T Ag infection, at which time these
cells reached crisis (Figure 4.2b). We performed similar experiments for
ML46 and showed us similar results (data not shown).
Our findings suggest that the differences in ploidy stability between LMPs
and cystadenomas are not because of differences in their levels of SV40 T
Ag expression.
Since lower level of SV40 T Ag expression is not the reason why the LMPs
are diploid, we try to examine whether the LMP cell lines do not develop
aneuploidy are because their SV40 T Ag are not active.
We did immunoprecipitation for the two LMP cell lines ML38 and ML46 with
p53 monoclonal antibody followed by western blot with SV40 T Ag
monoclonal antibody to find out whether their SV40 T Ag could bind to p53.
Immunoprecipitation with p53 antibody followed by western blot with antibody
for SV40 T Ag were performed in the following controls for our experiment:
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122
the ovarian cystadenoma cell line ML10 (positive control), the ovarian
carcinoma cell line Hoc-7 which is known not to have SV40 T Ag (negative
control), RIPA lysis buffer (negative control).
Both ML38 and ML46 showed positive SV40 T Ag expression by western blot
following immunoprecipitation with p53 antibody. So their SV40 T Ag can
bind to p53 and are active. ML38 seems to have lower SV40 T Ag activity
compared to ML46 (Figure 4.2c), since we have found that ML38 and ML46
have similar level of p53 expression by western blot (data not shown).
Our data suggest that the ploidy stability of the two LMP cell lines ML38 and
ML46 is not because their SV40 T Ag are not active.
Different roles for telomere attrition in initiating crisis of cystadenoma
versus LMP tumors
Previously we observed the telomere length of ovarian cystadenoma cell line
ML10 at crisis was similar to the telomere length at its early passage (Figure
4.3a), with the average telomere length of 8.60 ± 0.38 kb at 20 PDs, and 7.34
± 0.10 kb at 50 PDs, the time of crisis of ML10 cells. These data suggest that
telomere attrition does not play role in the initiation of crisis of ovarian
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123
cystadenomas. Our further more detailed work supports that aneuploidy
which accumulates mainly preceding the time of crisis of cystadenomas
determines the initiation of crisis in these cells (Velicescu M and others,
2003).
In this study, we put ML38 cells in culture from 19 population doublings until
38 population doublings, the time it reached crisis. By flow cytometry, we
observed ML38 remained diploid from 19 population doublings through 28
population doublings, until crisis (data not shown), suggesting the initiation of
crisis in these LMP cells was not determined by aneuploidy.
Since we had found that aneuploidy and telomere attrition were independent
determinants of crisis in SV40-transformed epithelial cells (Velicescu M and
others, 2003), we hypothesized the initiation of crisis of LMPs might be
determined by telomere attrition. To test this hypothesis, we compared the
telomere length of ML38 at 19 population doublings, 28 population doublings
and 38 population doublings, the time ML38 reached crisis, by southern blot
using Y-p32-dCTP radiolabelled probes for telomere repeats (TTAGGGh.
We found the telomere length of ML38 showed gradual decrease from 19
population doublings to 28 population doublings till crisis, with the average
telomere length of 8.69 ± 0.23 Kb at 19 PDs to 5.61 ± 0.18 Kb at 28 PDs and
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124
4.71 ± 0.21 Kb at 38 PDs, the time of crisis of ML38 (Figure 4.3b). All these
findings suggest that telomere attrition plays role in the initiation of crisis of
ovarian tumors of low malignant potential.
TRAP assay revealed negative telomerase expression in both ML38 and
ML46 LMP tumor cell strains (data not shown). This is compatible with our
observation that both ML38 and ML46 LMP cell strains are mortal and reach
in vitro crisis in culture at 38 population doublings and 54 population
doublings respectively.
Our findings suggest that telomere attrition plays different roles in initiating
the crisis of cystadenomas versus LMP tumors. Telomere attrition
determines the in vitro crisis of LMPs, but does not determine the in vitro
crisis of cystadenomas.
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125
Figure 4.1 Differences in ploidy stability between ovarian cystadenomas
and LMP tumors expressing SV40 large T antigen, (a) The DNA content
of one ovarian cystadenoma cell line ML10 and two LMP cell lines ML38 and
ML46 at their early passages and crisis. The ovarian cystadenoma cell line
ML10 and the two LMP cell lines ML38 and ML46, expressing SV40 T Ag,
were cultured from their early passages until they reached crisis. The DNA
profiles obtained by flow cytometry showed that the ovarian cystadenoma
cell line ML10 started to develop severe aneuploidy at 42 population
doublings, preceding the time of their crisis, which was at 50 population
doublings. In contrast, both LMP cell lines ML38 and ML46 were diploid at
their early passages and remained diploid until the time of their crisis, at 38
PDs and 54 PDs respectively. Flow cytometry showed apoptosis in both LMP
cell lines at crisis, (b) Western blot showed that ML46 at crisis expressed
elevated level of p21 compared to its early passage, suggesting the LMP
cells we analyzed at crisis were indeed in crisis.
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126
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ML38
1 54 PDs
L
2n 4n
ML46
b
ML46
PDs 25 54
P21 -►
A p * j | »
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127
Figure 4.2 Differences in ploidy stability between cystadenomas and
LMP tumors are not determined by differences in levels of SV40 large T
antigen expression, (a) Western blot were performed to compare the level
of SV40 T Ag expression in two LMP cell lines, ML38 and ML46, to ovarian
cystadenoma cell line ML10. LMP cell lines ML38 and ML46 expressed lower
level of SV40 T Ag than ovarian cystadenoma cell line ML10. (b) The DNA
content of ML38 LMP cell line after infection with adenovirus vector carrying
SV40 T Ag. We infected both ML38 and ML46 with adenovirus vector
carrying SV40 T Ag. The cell line we obtained after infection of ML38 with the
adenovirus vector was called ML38T. Western blot showed elevated level of
SV40 T Ag expression in ML38T at 33 population doublings after T Ag
infection, almost compatible to the level of SV40 T Ag expression in ovarian
cystadenomas ML10. Flow cytometry showed that both ML38 and ML38T
remained diploid at crisis, 33 population doublings after infection with
adenovirus vector carrying SV40 large T antigen. Apoptosis was observed by
flow cytometry in both ML38 and ML38T cells at crisis. Similar experiments
were performed for ML46 cell line and similar results were obtained (data not
shown), (c) Activity of SV40 large T antigen in two LMP cell lines ML38 and
ML46. Immunoprecipitation (IP) was performed for protein extract from ML38
and ML46 with p53 monoclonal antibody followed by western blot with
monoclonal antibody for SV40 T Ag to examine whether the SV40 T Ag in
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128
the ML38 and ML46 cell lines were active and could bind to p53. IP with p53
antibody followed by western blot with SV40 T Ag antibody were also
performed in the following as controls: the ovarian cystadenoma cell line
ML10 as positive control, the ovarian carcinoma cell line Hoc-7 which was
known not to have T Ag as negative control. The last lane was RIPA lysis
buffer alone as negative control. Both ML38 and ML46 showed positive SV40
T Ag expression by western blot following IP with p53 antibody. So their
SV40 T Ag can bind to p53 and are active. ML38 seems to have lower SV40
T Ag activity compared to ML46.
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129
a
ML10 ML46
SV40 T Ag
Actin — ►
ML38
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ML38
33 PDs
ML38T
33 PDs
SV40 T Ag -►
Actin -►
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Hoc-7
IP
ML10 ML38 ML46
IP IP IP
RIPA
IP
IP : p53
W T: SV40 T Ag
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132
Figure 4.3 Different roles for telomere attrition in initiating crisis of
cystadenomas versus LMP tumors, (a) Southern blot was performed using
Y-p32-dCTP radiolabelled probes for telomere repeats (TTAGGG)3 to
compare the telomere length of the ovarian cystadenomas ML10 at the time
of their crisis to their early passage. 20 PDs was the early passage and 50
PDs was the time the cells reached crisis. The telomere length of these cells
at crisis is similar to that of their early passage, suggesting that initiation of
crisis of the ovarian cystadenomas expressing SV40 T Ag is not because of
telomere attrition, (b) Southern blot using Y-p32-dCTP radiolabelled probes
for telomere repeats (TTAGGG) 3 was performed to compare the telomere
length of ML38 at 19 population doublings, 28 population doublings and 38
population doublings, the time of crisis of ML38. Flow cytometry showed that
ML38 at 19 PDs, 28 PDs and 38 PDs were all diploid (data not shown).
Telomere length of ML38 gradually decreases from 19 PDs to 28 PDs till
crisis, with the average telomere length of 8.69 ± 0.23 Kb at 19 PDs to 5.61 ±
0.18 Kb at 28 PDs and 4.71 ± 0.21 Kb at 38 PDs, the time of crisis of ML38.
These data suggest that telomere attrition determines the initiation of crisis of
ovarian tumors of low malignant potential. Thus, telomere attrition plays
different roles in initiating the crisis of ovarian cystadenomas versus LMP
tumors.
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133
ML10 ML38
Kb
23.0
9.4
5.5
4.3
2.3
2.0
20 50
PD PD
Kb
23.0
9.4
5.5
4.3
2.3
2.0
19 28 38
PD PD PD
mm
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134
Discussion
Previously, we had observed that the ovarian cystadenoma cell line
expressing SV40 T Ag, ML10, started to develop severe aneuploidy at 42
population doublings, preceding the time of their crisis, which was at 50
population doublings. The telomere length of ovarian cystadenomas
expressing SV40 T Ag at their crisis was similar to the telomere length of
their early passages, further more detailed work we did suggested that the
crisis of the cystadenomas expressing SV40 T Ag was not determined by
short of telomere length, but determined by aneuploidy (Velicescu M and
others, 2003).
Up to now, whether benign ovarian cystadenomas, ovarian tumors of low
malignant potential and ovarian carcinomas were sequential stages in the
multistep process of ovarian carcinogenesis was still of much debate
(Diebold J and others, 1996). Pathologists were still not clear about whether
or not they should subdivide non-invasive and non-metastatic subtypes of
ovarian tumors into cystadenomas and LMP tumors since some believed that
LMP tumors should not be regarded as a separate disease entity from
benign ovarian cystadenomas. Some LMPs with serous differentiation
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135
behaved very similar as benign serous cystadenomas and it was quite
debating whether or not to make distinction between these two ovarian
diseases (Kurman RJ and others, 1993).
Our findings in the two in vitro LMP cell lines, ML38 and ML46, suggested
that ovarian tumors of LMP tended to remain diploid, metaphase spread
revealed that both cell lines had 46 chromosomes from their early passages
until they reached crisis (data not shown). Their telomere length at crisis was
much shorter than the telomere length of their early passages. We previously
found two independent determinants of crisis in SV40-transformed epithelial
cells: ploidy-dependent crisis determined by aneuploidy and telomere-
dependent crisis determined by shortening of telomere length. Our results in
this study suggested that ovarian tumors of low malignant potential might
either have recovered from ploidy-dependent crisis, or for some reason,
ploidy-dependent crisis didn’t occur at all in ovarian tumors of low malignant
potential. It seemed shortening of telomere length was the only determinant
of crisis in ovarian tumors of low malignant potential. Based on our
observation, ovarian tumors of low malignant potential were very different
from ovarian cystadenomas. Although ovarian cystadenomas tended to
develop aneuploidy and their crisis was determined by aneuploidy, ovarian
tumors of low malignant potential remained ploidy stable and their crisis was
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136
determined by telomere attrition. Our findings supported that it was very
necessary to maintain the distinction between ovarian cystadenomas and
ovarian tumors of low malignant potential.
SV40 T Ag was found to be active aneuploidogen in human cells. The
mechanisms for SV40 T Ag to induce aneuploidy might involve displacement
of histone proteins from chromosomal DNA and unwinding nucleosomally
organized DNA to cause blockage of normal chromosomal binding sites for
tubulin fibers (Ramsperger U and others, 1995; Ray FA and others, 1990).
Although our two LMP cell lines ML38 and ML46 expressed lower level of
SV40 T Ag compared to ovarian cystadenomas, our immunoprecipitation
data showed that their SV40 T Ag could bind to p53 and were active. In
addition, it was very impressive that both the two LMP cell strains ML38 and
ML46 still remained diploid even after we enabled overexpression of the
SV40 T Ag in them by infection with adenovirus vector carrying SV40 T Ag.
Thus, the ploidy stability of the ovarian tumors of LMP was not due to loss of
SV40 large T antigen expression or activity.
Numerical chromosomal changes were not only important for ovarian
carcinogenesis but also widely used for the differentiation and prediction of
prognosis in ovarian cancers (Pejovic T and others, 1992). Flow cytometry
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137
analysis of the ploidy status of the ovarian tumors of low malignant potential
was not only important for improving the accuracy of the diagnosis of these
tumors, but also important for predicting their prognosis and making decision
for the treatment plans (Kaern J and others, 1990). For example, DNA
profiles obtained by flow cytometry analysis could identify the patients at high
risk of progression or recurrence, hence appropriate relaparotomy might be
performed for these patients.
Although extensive cytogenetic analyses had been done for ovarian
carcinomas to demonstrate their karyotype changes, these kinds of research
were still very few for ovarian tumors of low malignant potential, mainly
because of the difficulty for cell culture of ovarian tumors of low malignant
potential. Since the tumor cells of LMPs proliferated relatively slowly, there
were very limited numbers of cells available for cytogenetic analysis of these
tumors (Persons DL and others, 1993). Our study in the two in vitro LMP cell
strains provide some in vitro analysis information for this unique ovarian
disease, both regarding their ploidy stability and the determinant of their
crisis. Our findings also provide clear evidence for the necessity of the
distinction between ovarian tumors of low malignant potential and ovarian
cystadenomas.
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138
As a tumor of interest to many people and challenging for pathologists,
ovarian tumors of low malignant potential certainly require further more
detailed research for their basic tumor biology, especially study for their in
vitro behaviors. Further cytogenetic analysis of chromosomal changes in this
disease and clarification of the related mechanisms followed by comparison
to other ovarian diseases, such as ovarian cystadenomas and carcinomas,
will certainly be very helpful for better understanding and clinical intervention
of this borderline tumor of the ovary.
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Chapter 5
HHR6A overexpression in aneuploid
versus diploid clone
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140
Abstract
The purpose of this study is to examine whether HHR6A, the human
homologue of yeast Rad6, plays a role in the development of aneuploidy.
We had obtained several diploid and aneuploid clones from the telomerase-
transfected ovarian cystadenoma cells expressing SV40 T Ag. We used one
diploid clone and one aneuploid clone and compared the level of HHR6A
expression in the aneuploid clone to the diploid clone by both western
blotting and immunohistochemistry analysis. The ploidy status of the diploid
clone and the aneuploid clone were confirmed by flow cytometry. We also
counted the chromosome numbers of the diploid clone and the aneuploid
clone by metaphase spread. Our results showed that HHR6A was
overexpressed in the aneuploid clone compared to the diploid clone,
revealed by both western blot and immunohistochemistry analysis.
Our preliminary findings suggest that HHR6A overexpression is correlated
with aneuploidy and may play an important role in the development of
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141
aneuploidy, as part of the mechanism of HHR6A to mediate malignant
transformation.
Keywords: HHR6A; Aneuploidy; Malignant transformation
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142
Introduction
Aneuploidy is one of the major genomic instability found in all kinds of solid
cancers and has been proposed to be the cause of cancer (Duesberg P and
others, 2004). Much research interest has arisen regarding the mechanism
for the development of aneuploidy. Recently, it was found that constitutive
overexpression of the human homologue of yeast Rad6, HHR6B, an
ubiquitin-conjugating enzyme (E2), in the human breast epithelial cell line,
MCF10A, enabled these diploid cells without the anchorage-independent
growth ability to develop aneuploidy and grow in soft agar. (Malathy PVS and
others, 2002), suggesting that the human homologue of Rad6, HHR6B, might
be involved in the development of aneuploidy and mediate malignant
transformation.
HHR6A and HHR6B are two closely related human homologues of yeast
Rad6. They are localized on human chromosome Xq24-q25 and 5q23-q31,
respectively (Koken MH and others, 1991) and share 95% identical amino
acid residues.
Previously, we had obtained several diploid and aneuploid clones from the
telomerase-transfected ovarian cystadenoma cells expressing SV40 T Ag. In
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143
this study, we try to clarify whether HHR6A, the human homologue of yeast
Rad6, plays a role in the development of aneuploidy in the ovarian epithelial
tumor cell line. We used one diploid clone and one aneuploid clone of the
telomerase-transfected ovarian cystadenoma cells expressing SV40 T Ag
and compared the level of HHR6A expression in the aneuploid clone to the
diploid clone by both western blotting and immunohistochemistry analysis. In
addition to DNA profiles obtained by flow cytometry, the ploidy statuses of
the diploid clone and the aneuploid clone were also confirmed by metaphase
spread to count their chromosome numbers.
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144
Materials and Methods
Subcloning of cultured cells
After counting cell numbers by a model Zf Coulter counter (Coulter
electronics, INC. hialeah, FL), suspension of trypsinized cells were diluted
with filtered conditioned medium and cultured in 96 well plates at an average
of 0.2 cell per well.
Analysis of DNA ploidy by flow cytometry
One million cells were resuspended in phosphate-buffered saline (PBS) and
fixed in 70% ethanol. After centrifugation, the cell pellet was resuspended in
I ml of PBS, 10 )L tg /ml propidium iodide and 100 pg/ml RNase. The cells
were analyzed for distribution of fluorescence intensity using a Coulter Profile
II flowcytometer (Beckman Coulter, Hialeah, FL) and analyzed using the
MultiCycle software (Phoenix Flow Systems Inc., San Diego, CA).
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145
Metaphase spread preparation
Cultured cells were treated with 70 pi of colcemid (Gibco® laboratories, life
technologies, INC. Grand Island, New York) overnight at 37°C. Cells were
then resuspended in 0.56% KCI in H2 0 and incubated in a 37°C water bath
for 8 minutes, followed by fixation in 45ml methanol plus 5ml acetic acid at
room temperature for 10 minutes. After centrifugation at position 5 for 3
minutes in the IEC clinical centrifuge (Model 115VAC, International
equipment co. Needham HTS, Mass), the cell pellets were resuspended in
fixative and incubated in 4 °C refrigerator overnight. The next day, the cell
pellets were washed in fixative 2 to 3 times, resuspended in 1 ml of fixative,
and dropped on precleaned glass slides (superfrost® plus 25 x 75 x 1 mm,
Approx. Vi Gross, VWR Scientific, West Chester, PA).
Western blot analysis
Cell monolayers were rinsed with 10 ml of ice-cold phosphate-buffered saline
(PBS) and treated with ice-cold RIPA lysis buffer (20 mM Tris-HCI pH 8.0,
125 mM NaCI, 0.5% NP-40, 20 mM NaF, 0.2 mM Na3 P04, 2 mM EDTA, 35
pg/ml PMSF, 0.7 pg/ml pepstatatin A, and 0.5 pg/nnl leupeptin) at 4°C for 30
minutes. They were detached from the tissue culture dishes (Becton
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146
Dickinson Labware, Franklin Lakes, NJ) by rubbing with a cell lifter (Fisher
Scientific, Pittsburgh, PA) and centrifuged at 12000 rpm for 30 minutes. The
supernatants were stored at -80°C. Protein concentrations were determined
using the BCA protein assay reagent kit (Pierce, Rockford, IL). Samples
containing 10 pg of protein were electrophoresed on 10% polyacrylamide gel
and transferred onto nitrocellulose membranes (Biorad Laboratories,
Richmond, CA). The membranes were incubated overnight in 10% non-fat
milk (Biorad Laboratories, Richmond, CA), in 0.1% of PBS/Tween-20, and
exposed to the primary rabbit polyclonal antibody for anti-HHR6A (obtained
from Dr. Boris Sarcevic, Garvan Institute of Medical Research, Sydney,
Australia) for 1 hour at room temperature. Following three 10 minutes
washing in 0.1% of PBS/Tween-20, the membranes were exposed to the
secondary antibody coupled to horseradish peroxidase. The signal was
detected by the ECL western blotting detection reagents (Amersham
Biosciences, Buckinghamshire, England). For loading control, the membrane
was stripped in 100 mM p - mercaptoethanol, 2% SDS, 62.5 mM Tris-HCI,
pH 6.7 at 65 °C for 30 minutes, followed by extensive washing in 0.1% of
PBS/Tween 20, and reprobed with monoclonal antibody against p-actin
(Sigma, Saint Louis, MO). Monoclonal antibody for SV40 large T antigen was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA, cat# sc-147).
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147
Immunohistochemistry analysis
The diploid clone and aneuploid clone were cultured on the chamber slides
and fixed with acetone for 10 minutes. After removal of acetone and air dry
for 10 minutes, the slides were washed in PBS for 10 minutes. Endogenous
peroxidase was then quenched with 3% H2O2 in methanol for 10 minutes.
After washing with PBS for 5 minutes, the slides were incubated with
blocking donkey serum in PBS (ABC kit, Santa Cruz Biotechnology, Santa
Cruz, CA) for 15 minutes at room temperature. The primary rabbit polyclonal
anti-HHR6A antibody (obtained from Dr. Boris Sarcevic, Garvan Institute of
Medical Research, Sydney, Australia) diluted with blocking serum in PBS
was added to the slides and incubated for 90 minutes at room temperature in
the humid box. Cells incubated with blocking serum alone were used as
negative control. After washing with PBS for 3 x 5 minutes, the slides were
incubated with secondary biotinylated donkey-anti-rabbit IgG diluted 1:200 in
blocking serum in PBS for 45 minutes. Following 3 x 5 minutes of PBS wash,
cells on chamber slides were incubated with AB enzyme reagent (20 ul
avidin plus 20 ul biotinylated horseradish peroxidase in 1 ml PBS) for 30
minutes at room temperature in the humid box. After PBS wash, cells on
chamber slides were incubated with peroxidase substrate (1.6 ml distilled
water, 5 drops 10 X substrate buffer, 1 drop 50 X DAB chromogen, 1 drop 50
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148
X peroxidase substrate) for 30 seconds to 10 minutes until color developed.
After washing with deionized water for 5 minutes, the slides were
counterstained with hematoxylin for 5 to 30 seconds and washed with
running tap water. The slides were then dehydrated with 90% ethanol for 5
seconds, 100% ethanol for 2 x 10 seconds, and xylene for 2 x 10 seconds.
Excess xylenes were removed by air dry for a few seconds. Slides were then
mounted using permanent mounting medium and covered with cover slip.
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149
Results
The ploidy status of the diploid and aneuploid clone in our experiment
Previously we transfected ML10, the ovarian cystadenoma cells expressing
SV40 T Ag, with the catalytic subunit of human telomerase, hTERT, and
obtained the cell line called MCV167. After telomerase transfection, most
cells were diploid, but there were still some aneuploid cells remaining in the
population. By fluoresecence activated cell sorting, we were able to isolate
the few aneuploid population and put them back to culture. By subcloning,
we were able to get several diploid and aneuploid clones derived from the
sorted aneuploid population. We were able to obtain seven subclones, four of
which were diploid, three of which were aneuploid (Table 3).
We chose to use one diploid clone, clone 4, and one aneuploid clone, clone
2, for our study for the relationship between HHR6A and aneuploidy. By flow
cytometry, we observed the DNA profiles of the diploid clone, clone 4, and
the aneuploid clone, clone 2 (Figure 5.1).
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150
By metaphase spread, we counted the chromosome numbers of the diploid
clone, clone 4, and the aneuploid clone, clone 2. We found clone 4 had 46
chromosomes and clone 2 had 61 chromosomes.
The DNA profiles obtained by flow cytometry as well as the chromosome
numbers obtained by metaphase spread all showed that clone 4 was indeed
a diploid clone and clone 2 was indeed an aneuploid clone.
The overexpression of HHR6A protein in aneuploid clone compared to
diploid clone
We did western blot using the rabbit polyclonal antibody for HHR6A
(obtained from Dr. Boris Sarcevic, Garvan Institute of Medical Research,
Sydney, Australia) to compare the level of HHR6A expression in the
aneuploid clone, clone 2, to the level of HHR6A expression in the diploid
clone, clone 4. For the control of loading, the membrane was stripped and
reprobed with mouse monoclonal antibody for p-actin. Western blot showed
that the aneuploid clone, clone 2, expressed higher level of HHR6A
compared to the diploid clone, clone 4 (Figure 5.2).
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151
We also performed immunohistochemistry analysis using the same rabbit
polyclonal antibody for HHR6A to compare both the localization and the level
of HHR6A expression in the aneuploid clone, clone 2, to the diploid clone,
clone 4. Immunohistochemistry analysis showed the aneuploid clone, clone
2, expressed higher level of HHR6A than the diploid clone, clone 4. There
was no difference in the nuclear versus cytoplasmic localization of HHR6A in
the aneuploid clone, clone 2, compared to the diploid clone, clone 4 (Figure
5.3).
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152
Table 4 More diploid than aneuploid clones are obtained from the
aneuploid fraction after telomerase transfection. We transfected ML10,
the ovarian cystadenoma cells expressing SV40 T Ag with the catalytic
subunit of human telomerase, hTERT, and obtained the cell line called
MCV167. After telomerase transfection, most cells were diploid, but there
were still some aneuploid cells remaining in the population. By fluoresecence
activated cell sorting, we were able to isolate the few aneuploid populations
and put them back to culture. By subcloning, we got the diploid and
aneuploid cell clones derived from the sorted aneuploid population. We were
able to obtain seven subclones, four were diploid, three were aneuploid. By
metaphase spread, we counted the chromosome numbers of the clones.
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153
Clone
7
Aneuploid
0 5
O
V ”
Clone
6
Diploid
C O
^ r
Clone
5
Diploid
C O
Clone
4
Diploid
S ?
Clone
3
Aneuploid
C O
Clone
2
Aneuploid
5
Clone
1
Diploid
C O
*3
3
E
3
C
i
5 2
o. 3 5
£
o
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154
Figure 5.1 DNA profiles of the diploid and aneuploid clone in our
experiment. By flow cytometry, we observed the DNA profiles of the diploid
clone, clone 4 (a), and the aneuploid clone, clone 2 (b). These were the two
clones we chose to use for our study for the relationship between HHR6A
and aneuploidy.
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155
JM MCV167(G2)3CL4-2 2003-04-24 001 B FL3.HST W Am
240: S LC V S 1-
C E LL CYCLE
D A TA
200
160
e :
H l2 0 '
80
128 192 64 256
FL3 LIN
512,
M ean G1 = 66.0
C V G 1 - 5.0
% G1 = 35.7
M ean G 2=125.6
C V G 2 - 7.9
% G 2 = 3 1 .7
(30.8-32.6)
% S = 3 2 .6
(30.9-34.4)
G2/G1 =1.904
% B.D. = 33.4
Chi Sq.= 1.0
C ell N o .- 8076
% DE BR IS =47.45
JM MCV167(G2)3CL2-2 2003-04-24 002 B FL3.HST
O ■
. a
£ I
Z 3 -
o i
2
i * a ,
64 192 256
FL3 LIN
320 384
04,24/03. D IP L O ID C Y C L E
s l c v 's i'iM e a n G1= 83.2
1CVG1 = 2.7
% G1 = 6 .9
■Mean G2=155.9
CV G2 = 2.9
1 ( .0 -82 .1)
: ? %S ? = 62.7
I ( .0-99.9)
IG2/G1 =1.875
\% Tot = 16.2
ANEUPL. CYCLE
Mean G1=139.5
C V G 1 = 3.0
. % G 1 =24.1
Mean G2=272.2
CV G2 = 3.0
: % G2 = 16 .2
: (11.9-20.5)
:% S = 5 9 .7
( .0-99.9)
:G2/G1 =1.952
: % Tot = 83.8
i D .l. - 1 . 6 7 7
: Ave. % S= 60.2
% B.D. = 4 2 .9
5 7 @hi Sq.= .4
Cell N o.= 988
% DEBR!S=68.22
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156
Figure 5.2 Overexpression of HHR6A protein in aneuploid clone
compared to diploid clone revealed by western blot. We did western blot
using the rabbit polyclonal antibody for HHR6A to compare the level of
HHR6A expression in the aneuploid clone, clone 2, to the level of HHR6A
expression in the diploid clone, clone 4. For the control of loading, the
membrane was stripped and reprobed with mouse monoclonal antibody for
p-actin. Western blots showed that the aneuploid clone, clone 2, expressed
higher level of HHR6A protein compared to the diploid clone, clone 4.
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157
Diploid
clone
Aneuploid
clone
HHR6A
I*
Diploid
clone
Aneuploid
clone
ACTIN
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158
Figure 5.3 Overexpression of HHR6A protein in aneuploid clone
compared to diploid clone revealed by immunohistochemistry. We
performed immunohistochemistry analyses using the rabbit polyclonal
antibody for HHR6A to compare both the localization and the level of HHR6A
expression in the aneuploid clone, clone 2(b), to the diploid clone, clone 4(a).
Immunohistochemistry analyses showed the aneuploid clone, clone 2(b),
expressed higher level of HHR6A compared to the diploid clone, clone 4(a).
There was no difference in the nuclear versus cytoplasmic localization of
HHR6A in the aneuploid clone, clone 2(b), compared to the diploid clone,
clone 4(a). Brown color indicates positive HHR6A expression.
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159
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160
Discussion
It had been found that human breast carcinomas showed overexpression of
HHR6B, the human homologue of yeast Rad 6, compared to normal
mammary cells. HHR6B showed predominant nuclear localization in the
human breast carcinomas, whereas normal mammary cells showed mainly
cytoplasmic distribution of HHR6B. Moreover, mammalian homologue of
yeast Rad6 was also overexpressed and showed predominant nuclear
localization in the mouse mammary metastatic cells, in contrast to the
prevalent cytoplasmic distribution in nonmetastatic or normal mouse
mammary cells (Malathy PVS and others, 2002).
In the ovarian epithelial tumor cell line we used in this study, the aneuploid
clone showed higher expression of HHR6A compared to the diploid clone,
revealed by western blot, suggesting that HHR6A overexpression might be
involved in the development of aneuploidy. In order to further explore the
underlying mechanism for HHR6A to induce aneuploidy, we were interested
to examine whether the subcellular localization of HHR6A in the diploid and
aneuploid clone were different by immunohistochemistry. However, our
findings suggested that there was no difference in the subcellular localization
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161
of HHR6A between the aneuploid clone and the diploid clone. Both the
diploid clone and the aneuploid clone showed mainly cytoplasmic localization
of HHR6A expression. Immunohistochemistry analyses also showed that
HHR6A was overexpressed in the aneuploid clone compared to the diploid
clone.
Rad6 had been previously reported to overexpress in both human breast
cancer cell lines as well as human breast carcinoma tissues. However, the
antibody used in that study failed to distinguish between HHR6A and HHR6B
protein, thus it was not clear whether the elevated levels of Rad6 protein
were derived from HHR6A or HHR6B (Malathy PVS and others, 2002).
Centrosome amplification and multipolar mitotic spindles were both observed
in the human breast epithelial cell lines, which developed aneuploidy after
ectopic expression of HHR6B (Malathy PVS and others, 2002). Rad6 had
been found to be associated with centrosomes throughout the interphase and
mitotic phases of the cell cycle, which might be important for its role to
maintain genomic integrity. Centrosome amplification and multipolar mitosis
resulting from HHR6B overexpression might lead to missegregation of
chromosomes and aneuploidy (Dowsy S, 1998). It will be interesting to
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1 6 2
examine whether there are centrosomal abnormalities in our aneuploid clone
that shows higher level of HHR6A expression than the diploid clone.
Since our study is performed only in one aneuploid clone and one diploid
clone, the data are very preliminary to draw the conclusion that HHR6A
overexpression is correlated with aneuploidy and plays role in aneuploidy
development. Further experiments to investigate HHR6A expression in more
diploid and aneuploid clones and further functional assay for the effect of
HHR6A expression on the in vitro transformation ability of the clones will be
very helpful to clarify the role of HHR6A in the development of aneuploidy
and mediation of malignant transformation.
It will be important to clarify whether ectopic expression of HHR6A in the
diploid clone of our ovarian epithelial tumor cells can induce aneuploidy in
the clone. It will also be interesting to examine whether inhibition of HHR6A
expression in the aneuploid clone of our ovdfian epithelial tumor cells will
make its ploidy status chdhge to diploid.
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PART III
EPlLOGllE
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164
Chapter 6
Summary and future directions
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165
Summary
Despite the observation that neoplastic cells possess numerous genomic
abnormalities, the mechanisms of acquisition of molecular genetic changes
by cancer cells remain poorly understood.
We previously observed the development of severe aneuploidy in ovarian
cystadenomas expressing SV40 large T antigen preceding the time of their
crisis. We reason that at the time of crisis, the genomes of the cells are quite
abnormal, various kinds of molecular genetic changes can arise. We
hypothesize that cancer can occur following recovery from crisis. In this
study, we propose an alternative model of acquisition of molecular genetic
changes by cancer cells.
Two cell lines, called MCV39 and MCV50, spontaneously recovered from
crisis of the ovarian cystadenoma cells expressing SV40 large T antigen and
became immortalized. They provided us with a good in vitro longitudinal
model of ovarian tumor development to study the mechanisms of acquisition
of molecular genetic changes during tumor development and progression. By
subcloning, we were able to obtain subclones of both the parental cell line
and the derived immortalized cell line. Our allelotype analyses showed that
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clones of cells obtained before crisis had more frequent losses of
heterogeneity than clones of spontaneously immortalized MCV39 cells. All
chromosomes affected by losses of heterozygosity in the spontaneously
immortalized MCV39 cells were also affected by such losses in at least some
clones of cells before crisis. These findings not only support the idea that
crisis is the big catastrophic event associated with various kinds of genetic
abnormalities that can be acquired by cancer cells, but also suggest that
genetic abnormalities in cancer cells are acquired immediately after
transformation and come from clonal expansion of those alterations present
at the time of crisis.
We next investigated what determined the selective advantage of cells after
spontaneous immortalization, during the progression process. We explored
whether numerical chromosomal instability continued to occur after
spontaneous immortalization and examined the role of numerical
chromosomal instability in tumor progression. Although our allelotype
analysis and flow cytometry data strongly suggested the stability of cell
during spontaneous immortalization, that is, only one single aneuploid cell
recovered from crisis and became spontaneously immortalized, we
unexpectedly observed that both the MCV39 low passage and high passage
clones were not constant for their chromosome numbers, and even in the
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167
same clone, there existed variable chromosome numbers between the
metaphase counts. There was a trend for increase in chromosome numbers
when MCV39 clones progressed to high passages. We sought to examine
the stabilty of cells after spontaneous immortalization by examining the
numerical chromosomal stability in single cell clones over the progression
process. We put all six MCV39 low passage clones in culture and cultured
them 25 more population doublings, then all the MCV39 clones showed a
more extensive variability of chromosome numbers within the same clone
and the modal chromosome range showed that all the clones developed
hypo- or hyper-tetraploidy. There was always correlation of the growth
kinetics of MCV39 cell clones with their chromosome numbers. Different
clones with different chromosome numbers had different growth kinetics. All
these findings suggest that numerical chromosomal instability continues to
occur after spontaneous immortalization and drives for the clonal evolution
during the progression process from the low-grade carcinoma to the high-
grade carcinoma.
To further study the role of numerical chromosomal instability leading to
aneuploidy in malignant transformation, we compared the frequency for the
diploid and aneuploid cell populations of ovarian cystadenoma cells
expressing SV40 large T antigen to recover from crisis and give rise to
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168
spontaneously immortalized cell lines. Two spontaneously immortalized cell
lines, called MCV39 and MCV50, both recovered from the crisis of the
aneuploid cell populations. It suggests that aneuploidy may facilitate
immortalization of benign ovarian epithelial tumor cells expressing SV40
large T antigen. To further compare the significance of aneuploidy to that of
mutation in malignant transformation, we treated mortal human ovarian
cystadenoma cells with mutagen benzo[a]pyrene and subsequently
separated the cells based on their DNA content to diploid and aneuploid cell
populations. We measured the effect of aneuploidy and mutagenesis on in
vitro longevity, alone and in combination. Although aneuploidy alone
decreased in vitro longevity, the combined effects of aneuploidy and
mutagenesis resulted in increased longevity. These findings suggest that
aneuploidy and mutagenesis cooperate to mediate malignant transformation
of benign ovarian epithelial tumor cells expressing SV40 large T antigen.
Allelotype analysis in the subclones of the in vitro model of ovarian tumor
development showed that LOH on 15q, as the single additional loss
associated with the progression of MCV39 cells, was acquired at the early
stage of spontaneous immortalization. Growth curve analysis and colony
formation assay in the subclones before and after recovery from crisis with
and without LOH on 15q showed that LOH on 15q had differential effect on
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169
the growth kinetics and the colony formation ability of clones before and after
crisis. We conclude that LOH on 15q is either a biomarker with no direct
influence on tumor development or progression, or that another genetic
event, such as a mutation affecting a tumor suppressor gene on the retained
allele of this chromosome, takes place in MCV39 cells.
We extended our study to investigate the ploidy stability and the determinant
of crisis of ovarian tumors of low malignant potential and compared to those
of ovarian cystadenomas. Although ovarian tumors of low malignant potential
were diploid and their crisis was determined by telomere attrition, ovarian
cystadenomas were aneuploid and aneuploidy determined their crisis. Our
data suggest that ovarian tumors of low malignant potential may be a distinct
disease entity from ovarian cystadenomas.
Finally, we also studied the relationship between HHR6A (the human
homologue of yeast Rad6) expression and aneuploidy. We found that an
aneuploid clone expressed higher level of HHR6A compared to a diploid
clone by both western blot and immunohistochemistry analysis. Our
preliminary findings seem to suggest that HHR6A overexpression may be
associated with the development of aneuploidy, as part of the mechanism of
HHR6A to mediate malignant transformation.
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170
These studies improve our understanding for the mechanisms of acquisition
of molecular genetic changes during tumor development and progression, by
proposing an alternative model of tumor progression. Our findings not only
implicate the role of aneuploidy in both tumor development and tumor
progression, but also clarify the significance of aneuploidy compared to that
of mutation in malignant transformation. Our study may provide better
information for both the prevention and therapy of cancers, especially those
with p53 mutations.
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171
Future directions
1. Examining changes in mitotic checkpoint genes such as Bub1 during
tumor progression.
The present study show hypo- and hyper- tetraploid development during the
progression process from MCV39 low passage clones to high passage
clones. We will be interested in examing the mechanism underlying the near-
tetraploidy development during the progression process. Dysregulation of the
mitotic spindle checkpoint by mutation of the spindle assembly checkpoint
gene Bub1 has been proposed to be one of the mechanisms leading to
aneuploidy development in cancer cells (Jablonski SA and others, 1998). It
was reported that Bub1 could sense the signal emitted by unattached
kinetochores (Taylor SS and others, 1997) and coordinated with other mitotic
checkpoint proteins such as Bub3, Mad1 and Mad2 to act as a potent
inhibitor of the anaphase-promoting complex (APC). Since degradation of
securin in an APC-dependent manner was required for the separation of
sister chromatids during metaphase and anaphase transition, the onset of
anaphase was delayed until all the chromosomes had attached to the spindle
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172
microtubules successfully. This mitotic checkpoint for metaphase and
anaphase transition ensured sister chromatids to divide equally upon
duplication of the cell (Hoyt MA 2001).
We speculate there may be some changes in the mitotic checkpoint gene
Bub1 during the progression process of the MCV39 clones, which accounts
for the development of hypo- or hyper- tetraploidy in high passages of
MCV39 clones. For this proposed study, we will compare changes, such as
mutations of Bub1 as MCV39 low passage clones progress to high passage
clones. Particularly, we will focus our study on changes of Bub1 in MCV39
low passage clone 3, which has 40 chromosomes at 13 population doublings
and grows very slowly, but has good logarithmic growth with modal
chromosome numbers between 90 and 100 after an additional 25 population
doublings. We will examine changes such as mutations of Bub1 during the
progression process of MCV39 clone 3.
Mutation of the mitotic spindle checkpoint gene Bub1, located at
chromosome 2q14, was found in colorectal tumor cell lines with aneuploidy.
It was also observed that expression of mutant hBUB1 gene had dominant
negative effect in euploid cells to cause chromosomal instability (Cahill D P
and others, 1998). It was proposed that development of aneuploidy due to
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173
defects in mitotic checkpoint genes contributed to tumor progression in
colorectal cancers (Lengauer C and others, 1997). More detailed
examination of changes of Bub1 in the MCV39 clones in our in vitro model of
ovarian tumor development may provide better understanding of the subtle
mechanisms involved in the numerical chromosome instability during the
progression process.
2. Clarify whether continuous creation of aneuploid cells with different
chromosome numbers during the progression process is due to
aneuploidy itself or SV40 T antigen.
We observed the continuous creation of cells with abnormal chromosome
numbers during the progression process of MCV39 clones. There are two
possible mechanisms underlying the creation of these cells: First possibility,
this happens because of the instability of aneuploidy itself, that is, the
aneuploid cell is trapped in endless cycles of asymmetric mitosis and divides
with mitotic errors to continue to generate progenitors of aneuploid cells with
abnormal chromosome numbers. Second possibility, this happens due to the
presence of SV40 T antigen. In future studies, we will try to clarify which one
of the two possibilities, the aneuploidy or the presence of SV40 T Ag, causes
continuous creation of aneuploid cells with variable chromosome numbers.
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174
The observations in mice carrying SV40 T antigen with a weak promoter in
the germ line showed that it took three to nine months for them to develop
tumors, which were aneuploid. Both SV40 T antigen and autocatalytic
aneuploidy had been proposed to drive the evolvement of preneoplastic
aneuploidy to neoplastic aneuploidy in these mice, after induction of
preneoplastic aneuploidy by SV40 T antigen alone (Keough R and others,
1995). It had also been reported that SV40 T antigen did not cause or
maintain transformation directly (Shein HM and others, 1962).
The underlying mechanism for SV40 T Ag to induce aneuploidy was
previously believed to be due to the ability of T antigen to inactivate cell cycle
regulatory proteins such as p53 or pRb. However, recently it was found that
fragment of T antigen unable to interact with p53 or pRb could still induce
aneuploidy (Woods C and others, 1994). This was explained by the findings
that interaction of SV40 large T antigen with the spindle assembly checkpoint
protein Bub1 was responsible for the ability of SV40 T antigen to cause
aneuploidy (Marina C and others, 2004).
Our study suggest that numerical chromosomal instability due to aneuploidy,
which continues to generate progenitors of cells with different chromosome
numbers, drives for clonal evolution during the progression process. To draw
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175
this conclusion, we need to rule out the possibility that continuous creation of
aneuploid cells with different chromosome numbers during the progression
process is due to the presence of SV40 T antigen itself. We will try to count
the chromosome numbers of the MCV39 clones by metaphase spread after
elimination of functional SV40 T antigen from them with antibody against
SV40 T antigen, using parallel cultures of MCV39 clones without antibody
treatment to eliminate functional SV40 T antigen as controls. To distinguish
whether the creation of cells with variable chromosome numbers is related to
the ability of SV40 T antigen to interact with p53, pRb or Bub1, we will treat
the MCV39 clones with different antibodies against different mutants of SV40
T antigen which lose their corresponding ability to interact with p53, pRb or
Bub1 respectively.
3. Finding whether transient G2/M arrest is responsible for hypo- and
hyper-tetrapioidy changes during tumor progression.
Normal cells begin to synthesize DNA only after completion of the previous
mitosis due to the presence of the cell cycle checkpoint (Levine DS and
others, 1991). It was reported that following transient mitotic arrest,
tetraploidy could develop in cells defective of the cell cycle regulatory
proteins such as p53 (Lanni JS and others, 1998), pRb (Khan SH and others,
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176
1998), p16 or p21 (Stewart ZA and others, 1999) due to two rounds of DNA
synthesis without an intervening mitosis, a process called endoreduplication.
Correspondingly, some cancer cells were found to overexpress G2/M phase
antiapoptotic protein survivin, which prevented apoptosis in the cells
undergoing abnormal mitosis (Li F and others, 1998), thus abnormal
aneuploid cells could still survive and progress through mitosis after transient
G2/M arrest.
Targeted deletion of exon 11 of the BRCA1 gene has been reported to cause
defective G2/M cell cycle checkpoint and aneuploidy development in mouse
embryonic fibroblasts (Xu X and others, 1999). In patients with germline
mutations of BRCA1 gene, the development of aneuploidy due to G2/M
arrest was proposed to be responsible for the tumorigenesis in these patients
(Subrata S, 2000).
In our lab, Sephideh Karami found that inhibition of BRCA1 expression either
by antisense or by retroviral vector infection all caused G2/M arrest and
development of aneuploidy in the ovarian epithelial tumor cells. Since we
observed near-tetraploidy development in our MCV39 clones as they
progressed to their high passages, we hypothesize there may be transient
G2/M arrest happening in these clones. In the future, we will try to examine
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177
whether there are alterations of BRCA1 expression in these clones as they
progress to high passages. We will also compare the level of survivin
expression in the MCV39 high passage clones to the MCV39 low passage
clones. We will treat the MCV39 low passage clones with caffeine, which is
proposed to release cells from G2/M arrest, and examine whether caffeine
treatment can prevent tetraploidy development in the MCV 39 clones as they
progress to higher passages, using MCV39 clones without treatment as
controls. These experiments will help us to determine whether transient
G2/M arrest is responsible for the near-tetraploidy development during the
progression process of MCV39 clones in our in vitro model for ovarian tumor
development.
4. Studying the effect of inhibition of HHR6A expression on ploidy
changes.
We will continue our investigation on the relationship between HHR6A and
aneuploidy. Our preliminary data presented in this study suggested that an
aneuploid clone expressed higher level of HHR6A protein compared to a
diploid clone by both western blot and immunohistochemistry analysis. These
findings will be reexamined in our future repeated experiments using more
diploid and aneuploid clones. We will also investigate whether inhibition of
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178
HHR6A expression in the aneuploid clone can change its ploidy status to
diploid. We will achieve this purpose by treating the aneuploid clone with
specific siRNA to inhibit HHR6A expression followed by examination of the
DNA profile of the clone by flow cytometry, using the aneuploid clone without
treatment to inhibit HHR6A expression as controls. It will also be interesting
to clarify whether HHR6A expression is related to the ploidy changes of the
MCV39 low passage clones as they progress to high passages. To achieve
this purpose, we will examine the level of HHR6A expression in the MCV39
low passage clones and compare them to the level of HHR6A expression as
these clones progress to higher passages.
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179
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Core Title Mechanisms of acquisition of molecular genetic changes during tumor development and progression

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