Viruses, Cell Transformation and Cancer

516 Pages·2001·8.47 MB·English

by  A.J. Zuckerman, I.K. Mushahwar (Eds.)

Viruses, Cell
Transformation
and Cancer
Viruses, Cell
Transformation
and Cancer
Edi t o r
R.J.A. Grand
CRC Institutef or Cancer Studies
University of Birmingham
Edgbaston
Birmingham B15 2TT
UK
20 0 1
E L S E V I E R
A m s t e r d a m - L o n d o n - N e w Y o r k - O x f o r d - P a r i s - S h a n n o n - T o k y o
INTRODUCTION
Roger J.A. Grand
It is now firmly established that carcinogenesis is a multistage process that is the result
of several causative events. Most commonly, many of these events result from genetic
lesions attributable to environmental factors. The net result is a loss of growth control
leading to the unregulated proliferation characteristic of the cancer cell. The identity of
the majority of the environmental agents which cause the genetic mutations remains
unknown although a few, such as tobacco smoke and ultra-violet radiation are now very
familiar. Important cancer-causing environmental factors which do not induce mutation
are tumor viruses. Their relevance to cancer in the context of this book is two-fold -
firstly they induce tumors in the infected host and, secondly, they provide important
models for studying neoplastic transformation as they transform cells in tissue culture
and can also induce tumors in experimental animals.
Whilst viruses are not in general a major cause of cancer, it has recently been
suggested that 10.3% of human cancers world-wide are attributable to viral infection.
This was equivalent to about 835,000 cases in 1990 (Parkin et al., 1999). An additional
400,000 cancers are probably due to infection with non-viral agents such as
Helicobacter pylori. Not surprisingly, a greater proportion of cancers arise from
infection in developing countries (22.5% compared to 6.8% in developed countries
Parkin et al., 1999). The viruses responsible for most of these cases are human
papillomavirus (HPV), which is considered to be involved in about 90% of cervical
cancers, Epstein-Barr virus (EBV) which has been linked to Burkitt's lymphoma, non-
Hodgkin's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma and Hepatitis
B and C viruses (HBV and HCV) which are responsible for around half of the cases of
liver cancer world-wide. In addition, HIV-infected individuals become susceptible to
cancers due to further infection with other viruses such as human herpes virus 8
(HHV8) and EBV.
The realization that cancers can be linked to "infectious agents" is not recent.
Leukemias and sarcomas in chickens were demonstrated to be transmissable in the first
decade of this century (Ellerman and Bang, 1908; Rous, 1911). Similary, a
predisposition to tumors of the breast in C3H mice was shown to be passed from
mothers to offspring through milk (Bittner, 1936). A predisposition to lymphomas was
also found to be due to viral infection (Gross, 1957). Since that time these cancers in
mice have been attributed to infection with mouse mammary tumor virus (MMTV) and
mouse leukemia virus (MLV) respectively. In the 1930s cancers were also shown to be
caused by DNA viruses (although of course the difference was not realized at the time);
for example, skin cancers were induced in cotton tail rabbits by infection by pox virus
and papilloma virus (Shope, 1932; Shope and Hurst, 1933).
The study of DNA tumor viruses over the past fifty years has added greatly to our
knowledge of how viruses cause tumors in mammals and, perhaps more significantly,
has helped to identify some of the genes/proteins involved in non-virally induced
tumorigenesis. It should be noted, however, that many of the most commonly studied
2 R.J.A. GRAND
DNA tumor viruses such as Simian virus 40 (SV40), adenovirus, polyoma virus and BK
and JC viruses are all considered to be non-oncogenic in humans although they can
cause tumors in rodents. All of these, together with HPV, have small genomes, and this,
taken in conjunction with the fact that the viral DNA can generally be used to transform
cells in tissue culture has facilitated their study. Furthermore, it has become apparent
that expression of only a small number of the viral genes is necessary for transformation
or for oncogenesis. Studies using the small DNA tumor viruses have greatly enhanced
our understanding of cell transformation and over the past twenty years it has become
apparent that cellular proteins targeted by the DNA viruses are, in many cases, the
products of genes mutated in human cancers. The most obvious examples of this are
p53 and Rb. p53 was originally identified as a protein which formed a complex with
SV40 T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979) and was soon
demonstrated to be a ubiquitous cellular component, which was often over-expressed
and mutated in transformed cell lines and in tumor cells. It has now been confirmed as
the most commonly mutated gene in human cancers (Levine et al., 1991) with about 60-
65% of tumors expressing mutant p53 (Greenblatt et al., 1994). The retinoblastoma
gene product pRb is also a target for the DNA tumor viruses and like p53 is mutated in
an appreciable proportion of human cancers (Friend et al., 1986; Lee et al., 1988; Whyte
et al., 1988; De Caprio et al., 1988).
Whilst our knowledge of how the small DNA viruses transform cells has increased
greatly over the past two decades much less progress has been made with most of the
viruses responsible for the majority of virally-induced human cancers. Thus, we
understand reasonably well how the HPV E6 and E7 genes transform cells in culture
and it is presumed that the same biochemical events play role in the early stages of
cervical carcinoma. However, our understanding of the mechanism by which those
viruses, other than HPV, initiate tumor development is rather more sketchy. Hepatitis B
virus (HBV) is an important causative agent for an appreciable proportion of
hepatocellular carcinomas (HCC) in Africa and Asia. Interestingly, infected individuals
who are also exposed to aflatoxin have an appreciably increased incidence of the
disease. This highlights a role for a particular virus as a co-carcinogen in conjunction
with an environmental factor. Although the HBV genome is small (3kb) there is still
considerable controversy over the mechanism by which the cancers arise. It has been
suggested that chronic HBV infection can lead to inflammation and liver injury,
followed by recurrent cellular regeneration. If genetic damage has been caused (for
example, by chemical carcinogens) hepatocarcinomas could result. Perhaps more
relevantly from the point of view of the theme of this book there is considerable
evidence to suggest that the HBV HBx protein can play a role in hepatocarcinogenesis
possibly by binding to p53, activating kinase pathways and increasing transcription. The
activities of HBx are discussed in detail in Chapter 7 of this volume. In addition, it is
possible that HBV surface antigens can act as oncogenic proteins and that integration of
viral DNA into the host genome could play a role in transformation.
The situation with EBV is much more complex in that the viral genome is large
(around 180kb) and encodes a number of proteins which may be involved in
transformation and the production of tumors. In addition, EBV has been linked to
INTRODUCTION 3
several different cancers in both epithelial and lymphoid tissue - for example Burkitt's
and other lymphomas, nasopharyngeal carcinoma (NPC), and Hodgkin's disease. Two
viral proteins, EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1)
are essential for in vitro transformation of B cells but others also play a part.
Furthermore, LMP 1 is expressed in a number of EBV-associated tumors. The roles of
the EBV proteins are considered in detail in Chapter 8 of this volume.
Human herpes virus 8 (HHV8) is a recent addition to the list of viruses which can
cause tumors in humans and is now thought to be largely responsible for Karposi's
sarcoma (KS) and primary effusion lymphoma (PEL). Of course, these are primarily a
problem in immunosuppressed individuals but with increasing prevalence of HIV
infection, particularly in Africa and Asia, the incidence of cancers attributable to HHV8
infection is likely to increase greatly in the near future. HHV8 (also known as
Karposis's sarcoma-associated herpes virus KSHV) is a member of the y-herpes virus
subfamily and has a large genome (around 170kb) with in excess of 80 open reading
frames. The fact that the virus was only identified a few years ago and the complexity of
the genomic organisation has meant that our knowledge of its mode of action, both in
transformation and tumorigenesis, is relatively limited. However, a detai~led discussion
of evidence linking HHV8 and KS and lymphoproliferative disease is presented in
Chapter 9, together with a summary of the current knowledge of the properties of a
number of virally-encoded proteins. The role of putative transforming proteins such as
K1, K9, K12 and the K cyclin are presented in Chapters 9 and 10 as well as discussion
of viral proteins (vFLIP and Bcl-2 homologues) which may inhibit apoptosis in the
infected cell.
Retroviruses have been of immense assistance in helping us to understand the
mechanisms of cellular transformation and the functions of a large number of important
cellular proteins (the proto-oncogenes) such as Ras and Myc which function in the
regulation of various aspects of cell growth. Whilst retroviruses can be tumorigenic in
animals (for example, Rous sarcoma virus in chickens and Abelson leukaemia virus in
mice) they appear to be directly responsible for few cancers in humans. However,
human T-lymphotropic virus type I (HTLVI) is the causative agent of adult T cell
lenkaemia/lymphoma, whilst HTLVII probably causes hairy cell leukaemia. The
number of cancers caused by HTLVI infection, worldwide, is relatively small (about
2600 in 1990) but as they are clustered in particular areas, giving a high prevalence in,
for example, the southern tip of Japan, southern Gabon, northern Zaire and parts of the
Caribbean they can pose a serious but geographically limited, health problem. The
transforming protein of HTLVI is Tax which serves as a transcriptional activator. The
activities of Tax with particular emphasis placed on its relationship to the cell cycle are
described in Chapter 11.
Although few cancers in humans are directly caused by retroviruses it must be
remembered that at the end of the twentieth century HIV is the infectious agent
responsible for more deaths than any other. HIV does not appear to have a direct
oncogenic capability but, by incapacitating the immune system, it is able to facilitate the
development of cancers caused by other agents (e.g. KS and non-Hodgkin's
lymphoma). Of course, many of the deaths due to AIDS are not a result of cancers but
4 R.J.A. GRAND
of opportunistic infections by non-cancer causing organisms. Despite the importance of
HIV infection as a contributory cause of cancers it was considered that a detailed
description of the properties of the virus was beyond the scope of this book. Similarly,
the large number of animal retroviruses which have been so important in the elucidation
of the mode of action of retroviral oncogenes have been ignored. This has been a
somewhat arbitrary decision based more on limitation of space than lack of relevance.
Whilst retroviruses such as avian erythroblastosis virus, avian myclecytomatosis virus-
29 and Harvey sarcoma virus do not infect humans their study has highlighted the
importance of cellular proto-oncogenes (in the case of those viruses erbB, myc and H-
ras respectively). Furthermore, it is now well-known that either changes in protein
expression due to chromosomal translocation (e.g. Myc and ErbB) or expression of an
aberrant protein with an altered activity (e.g. H-ras) following mutation are of particular
importance in the aetiology of some human cancers. In the examples mentioned it has
been shown that erbB is amplified in tumors such as glioblastomas and squamous cell
carcinomas, myc is amplified and over-expressed in, amongst others, tumors of the
breast, lung and colon, as well as leukemias, whilst mutant ras is expressed in acute
myeloid leukemias, colon and lung carcinomas. The subject of proto-oncogenes is so
large and complex that if requires treatment in greater detail than could be justified in
this book. Reluctantly, therefore discussion of proto-oncogenes has been omitted.
Within this volume the themes which I have very briefly touched upon here have
been considered in detail. Although the DNA tumor viruses, with the notable exception
of HPV, pose virtually no threat to humans, they have been discussed in considerable
detail in Chapters 2, 3 and 4. I feel that this can be justified by their scientific
importance in that results from the study of SV40, Ad and HPV have provided much of
our basic knowledge of the mechanism of cell transformation. Those organisms which
are the major causes the virally-induced cancers - HPV, HBV, EBV and the relatively
recently discovered HHV8 - are dealt with in detail in the central portion of the book.
Retroviruses and their use in the search for novel oncogenes are discussed in
Chapters 11 and 12 with emphasis placed on HTLV1 in the first of these. Towards the
end of the volume more general themes are considered - in particular how viruses evade
the host's immune system and how they either cause or limit an apoptotic response by
the infected host. Finally, the possibilities of using viruses as therapeutic agents against
human cancers have been discussed.
I believe that the broad-range of subjects covered will give a relatively up-to-date
• and fairly concise description of a very large body of scientific research. As the authors
have concentrated on these aspects of the subject which they find of particular interest,
it is hoped that their enthusiasm and knowledge will make this an illuminating and
instructive account of a subject of relevance to scientists and clinicians, students and
experienced researchers alike.
Finally, I should like to express my gratitude to all of the contributors and, in
particular, to Nicola Waldron at the Institute for Cancer Studies, University of
Birmingham for tireless endeavour in preparing this volume for publication.
INTRODUCTION 5
References
Bittner, J.J. (1936) Some possible effects of nursing on the mammary gland tumor incidence in mice. Science
84, 162.
DeCaprio, J.A., Ludlow, J.W., Figge, J., Shew, J.Y., Huang, C.M., Lee, W.H., Marsilio, E., Paucha, E. and
Livingston, D.M. (1988) SV40 large tumor antigen forms a specific complex with the product of the
retinoblastoma susceptibility gene. Cell 54, 275-283.
Ellerman, V. and Bang, O. (1908) Experimentelle Leukemic bei Huhnem. Zentralbl Bakteriol Alet 46, 595-
597.
Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M and Dryja, T.P. (1986).
A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosareoma.
Nature 323,643-646.
Greenblatt, M.S., Bennett, W.P., Holstein, M. and Harris, C.C. (1994) Mutations in the p53 tumor suppressor
gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855-4878.
Gross, L. (1957) Development and serial cell-free passage of a highly potent strain of mouse leukaemia virus.
Proc. Soc. Exp. Biol. Med. 94, 767-771.
Lane, D.P. and Crawford, L.V. (1979) T antigen is bound to a host protein in SV40 transformed cells. Nature
278, 261-263.
Lee, E.Y., To, H., Shew, J.Y., Brookstein, R., Scully, P. and Lee, W.H. (1988) Inactivation of the
retinoblastoma susceptibility gene in human breast cancers. Science 241,218-221.
Levine, A.J., Momand, J. and Finlay, C.A. (I991) The p53 tumor suppressor gene. Nature 35 I, 453-456.
Linzer, D.I. and Levine, A.J. (1979) Characterization of a 54K dalton cellular antigen present in SV40-
transformed cells and uninfected embryonal carcinoma cells. Cell 17, 43-52.
Parkin, D.M., Pisani, P., Munoz, N. and Ferlay, J. (1999) The global health burden of infection associated
cancers. Cancer Surveys 33, 5-33.
Rous, P. (1911 ) A sarcoma of fowl transmissable by an agent separable frorn the tumor cells. J. Exp. Med. 13,
397-399.
Shope, R.E. (1932) A filtrable virus causing a tumor-like condition in rabbits and its relationship to virus
myxomatosum. J. Exp. Med. 56, 803-822.
Shope, R.E. and Hurst, E.W. (1933) Infectious papillomatosis of rabbits. J. Exp. Med. 58,607-623.
Whyte, P., Buchkovich, K.J., Horowitz, J.M., Friend, S.H., Raybuck, M., Weinberg, R.A. and Harlow, E.
(1988) Association between an oncogene and an antioncogene: the adenovirus E la proteins bind to the
retinoblastoma gene product. Nature 334, 124-129.
IMMORTALIZATION OF PRIMARY RODENT CELLS BY SV40
Alison J. Darmon and Parmjit S. Jat
Ludwig Institutef or Cancer Research, London
ABSTRACT
This review is predominantly concerned with the mechanisms by which simian virus 40
(SV40) induces immortalization of primary ceils. Primary cells in culture have a finite
mitotic life span, after which they undergo replicative senescence. Expression of SV40
in these cells, in particular the large tumor antigen (T antigen) of SV40, before they
have senesced, allows them to overcome their finite mitotic life span and results in the
establishment of immortal cell lines. The immortal state is dependent on the continued
presence of T antigen, although whether all of the functions initially required to induce
immortalization are required to maintain it is currently unclear. Here, we first discuss
the growth restrictions of primary cells in culture, and define immortalization and
transformation. We then briefly review cell cycle regulation and negative growth control
in normal cells, with particular reference to the retinoblastoma family of proteins and
p53, before discussing mechanisms used by SV40 to overcome these growth
restrictions. Finally, we briefly mention trans versus cis complementation between T
antigen mutants, initiation versus maintenance of immortalization, the putative role of
the SV40 small t antigen, and the biological counting mechanism that measures the
finite proliferative life span.
Immortalization Versus Transformation
When mammalian cells isolated from an embryo or an animal are cultured in vitro, they
initially proliferate but stop dividing after a finite number of division (Hayflick and
Moorhead, 1961). At this point the cultures undergo crisis and the cells senesce. Such
senescent cells cannot be induced to enter mitosis, even if supplemented with fresh
growth medium. However, the cells do not die but remain metabolically active (they
continue to synthesize RNA and protein) and responsive to mitogens (some immediate
early genes are expressed (Tavassoli and Shall, 1988). Analysis of senescent fibroblasts
suggests that the ceils arrest in the G1, and possibly G 2, phases of the cell cycle. This is
in contrast to the G O arrest for cells which enter quiescence in response to either serum
deprivation or contact inhibition (Gelfant, 1977; Grove and Cristofalo, 1977).
It has been observed that cells from progressively older animals undergo
progressively fewer divisions in culture before undergoing senescence, suggesting that
there is an inverse correlation between the age of the animal and the in vitro life span of
cells derived from that animal (Hayflick and Moorhead, 1961; Bierman, 1978; Rohme,
1981). Additionally, cells from the same animal species undergo a relatively constant
number of divisions (approximately 30 population doublings for rodent embryo
fibroblasts compared to 50-70 doublings for human fibroblasts), and the number of
8 A.J. DARMON, P.S. JAT
population doublings for a given cell type is highly reproducible. Thus, it has been
suggested that cellular senescence may be a programmed event and that entry into
senescence is a manifestation of aging at the cellular level (Kirkwood, 1996; Smith and
Pereira-Smith, 1996).
The molecular basis for programmed entry into senescence is poorly-defned,
although it has been suggested that it may be linked to the random accumulation of
cellular damage (Orgel, 1973). This hypothesis suggests that as cells divide in vitro they
accumulate mutations, karyotypic changes and other forms of DNA damage (such as
loss of DNA methylation) and this leads to changes in the expression of positive and
negative regulators of cell growth or to a predisposition to karyotypic instability,
resulting in loss of proliferative potential (Sherwood et al., 1988).
Another hypothesis proposes that the progressive loss of telomeric DNA and other
essential sequences from the ends of chromosomes determines the finite proliferative
potential (Harley et al., 1990; Allsop et al., 1992). In this hypothesis, once the telomeres
have shortened to a critical length, the cell stops dividing and becomes senescent.
Although this mechanism probably operates in human cells, it is doubtful that it plays a
role in regulating proliferative potential in murine cells (Zakian, 1995; Autexier and
Greider, 1996; Lansdorp, 1997; Zakian, 1997; de Lange, 1998; Sedivy, 1998). Primary
cells derived from telomerase-deficient mice enter senescence at the same time as
primary cells from normal mice (Blasco et al., 1997) and are able to escape senescence
at the same rate, suggesting no autonomous role for telomerase in regulating senescence
in the murine system. In contrast, other workers (Bodnar et al., 1998; Vaziri and
Benchimol, 1998) have found that ectopic expression of telomerase in normal human
cells results in prolonged life spans in these cells.
Others have suggested that senescence is regulated via a genetic program (Pereira-
Smith et al., 1989; Goldstein, 1990; Vojta and Barrett, 1995). A number of genes that
may be involved in regulating senescence have been identified from senescent cells
(Murano et al., 1991; Nuell et al., 1991; Noda et al., 1994).
When a cell overcomes senescence it is said to have become immortal, since it has
acquired an infinite life span. A number of viral and cellular oncogenes can overcome
senescence, including the large tumor antigen (T antigen) of simian virus 40 (SV40).
Alternatively, serial cultivation of rodent embryo fibroblasts occasionally results in
spontaneously immortal cell lines which have escaped senescence (Todaro and Green,
1963; Curatolo et al., 1984). The cellular lesions responsible for this escape from
senescence are poorly-defined, however, mutations in the negative growth regulator p53
and increased expression of c-myc have been observed in some immortal cell lines
(Tavasso!i and Shall, 1988; Harvey and Levine, 1991; Rittling and Denhardt, 1992).
Immortalization has been suggested to be one of two steps required to bring about
the complete malignant transformation of rodent cells in vitro (Weinberg, 1985). In
contrast to fully transformed cells, immortal cells remain dependent on the presence of
mitogens (although they have a reduced requirement for them), cannot overgrow a
confluent monolayer and cannot form tumors in nude mice. The continued expression of
the immortalizing oncogene is required to maintain the immortal state, however whether
all the functions which were initially required to overcome the fmite life span of the
IMMORTALIZATIONO F CELLS BY SV40 9
primary cells, or a subset of these functions, are required to maintain the immortal state
has not been determined.
Immortal cells can be transformed into fully malignant cells by either the
introduction of a second oncogene (Land et al., 1983; Ruley, 1983) or, at a low
frequency, through the occurrence of spontaneous second events such as chromosomal
mutations (Land et al., 1986).
Somatic cell fusions of normal diploid human fibroblasts with several immortal cell
lines, including HeLa and SV40-transformed cells, have suggested that senescence is
dominant over proliferation. The hybrids resulting from such fusions only proliferate for
a limited period of time prior to undergoing senescence (Bunn and Tarrant, 1980;
Pereira-Smith and Smith, 1981; 1bid., 1988; Pereira-Smith et al., 1990). This is
consistent with the idea that the inactivation of specific senescence-promoting genes
may be important for cells to escape from senescence, and that activation of specific
dominant oncogenes can overcome senescence.
Immortalization requires not only the ability to overcome the limited proliferation of
primary cells but also requires the inhibition of programmed cell death by apoptosis. It
is thought that apoptosis may be a cellular defense against deregulated growth in
inappropriate conditions. Thus, in order to successfully immortalize a cell, an oncogene
must not only deregulate cell growth but also overcome the apoptotic pathway(s) which
may be activated in response to this deregulated cell growth (King and Cidlowski,
1998).
It is unknown whether immortalization has a role to play in tumorigenesis in vivo, or
whether it is merely required for the in vitro establishment of transformed cell lines
(Strauss and Griffin, 1990; Stamps et al., 1992). While it is hard to envisage a situation
where a tumor could be derived without first overcoming the finite mitotic life span, it
remains to be demonstrated whether this is a critical step in tumorigenesis.
Before focusing on the mechanisms used by SV40 to overcome senescence, we will
first present a very brief review of cell cycle regulation in normal (that is, uninfected)
cells.
Cell Cycle Control
In order to understand how SV40 can induce cellular proliferation and immortalization,
it is first necessary to understand how cell cycling is regulated in the absence of SV40.
This section presents an overview of cell cycle control (summarized in Figure 1) so that
the affects of SV40 infection on cells can be more clearly understood. More in-depth
reviews of cell cycle regulation are presented elsewhere.
Cyclins, Cdks, and Cdk lnhibitors
Progression of eukaryotic cells through the cell cycle is regulated by the sequential
assembly and activation of key cyclin and cyclin-dependent kinase (cdk) complexes.
The cyclins constitute the regulatory subunit of the complex, while the cdk is the