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Last Update: 2 November 2017

Part I
M - 13
CANCER and CANCER GENETICS
Reference: Cooper, Russell, Bruce Alberts

Concept: (Oncogene, protooncogene, tumerogenic virus, provirsus and distinguish between v-src and
c-src. Structure and function of various sarcoma virus, structure and function of SV 40 and polyoma
virus, Philadelphia chromosome )

In multicellular organisms the proliferation, differentiation, and survival of individual cells are carefully
regulated to meet the needs of the organism as a whole. This regulation is lost in cancer cells, which grow
and divide in an uncontrolled manner, ultimately spreading throughout the body and interfering with the
function of normal tissues and organs. It results from a breakdown of the regulatory mechanisms that
govern normal cell behavior. In fact, many of the proteins that play key roles in cell signaling, regulation
of the cell cycle, and control of programmed cell death were first identified because abnormalities in their
activities led to the uncontrolled proliferation of cancer cells.
Definition and Terminology : Cancer may be defined as the uncontrolled proliferation of undifferentiated
cells at an exponential rate at any part of the body, with the formation of a malignant tumor and
interfering with the function of the normal tissues and organs.
Some cancerous cells or malignant tumor cells may pass through blood and lymph from the primary focus
to one or more secondary foci to form secondary malignant tumors. This property of the cancerous cells is
called metastasis.
The term cancer is derived from Latin Cancrum meaning crab probably because of the way a cancer
adheres to any part that it seizes upon in an obstinate manner like the crab. It is a popular, generic term
because the actual medical term for cancer is neoplasia which, from the Greek, means new formation.
Cancers are new growths of the cells in our bodies. Cancer is variously known as malignant tumor,
neoplasm (new growth) or neoplastic tumor.
The initiation of tumor in an organism is called oncogenesis (oncos, mass or bulk ; genesis birth )
and therefore the study of cancer is called oncology.
Types of cancer: Cancer can result from abnormal proliferation of any of the different kinds of cells in the
body, so there are more than a hundred distinct types of cancer, which can vary substantially in their
behavior and response to treatment.
The most important issue in cancer pathology is the distinction between benign and malignant tumors. A
benign tumor, such as a common skin wart, remains confined to its original location, invading neither
surrounding normal tissue nor spreading to distant body sites. Benign tumors are usually not life
threatening, and their surgical removal generally results in a complete cure. Exception include many
brain tumors, which are life threatening because they impinge on essential cells.
A malignant tumor, however, is capable of both invading surrounding normal tissue and spreading
throughout the body via circulatory or lymphatic systems (metastasis), forming new tumors at other
locations in the body. Only malignant tumors are properly referred to as cancers and malignancy can
result in death because of damage to critical organs, starvation, secondary infection, metabolic problems,
second malignancies, and / or hemorrhage.
Both benign and malignant tumors are classified according to the type of cell from which they arise. Most
cancers fall into one of three main groups: carcinomas, sarcomas, and leukemias or lymphomas.
Carcinomas are malignancies of epithelial cells which include approximately 90% of human cancers
perhaps because most of the cell proliferation in the body occurs in epithelia, or because epithelial tissues
are most frequently exposed to the various forms of physical and chemical damage that favor the
development of cancer..
Sarcomas are solid tumors of connective tissues such as muscle, bone, cartilage, and fibrous tissues which
are rare in human.
Leukemias and lymphomas arise from blood forming cells and from cells of immune system respectively
which account for approximately 8% of human malignancies.

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Tumors are further classified according to tissue of origin( e.g. lung or breast carcinomas ) and type of
cell involved. For example, fibrosarcomas arise from fibroblasts and erythroid leukemias from precursors
of erythrocytes (RBCs).
A list of benign tumors and malignant tumors of different tissues are given below:

Tissues Benign tumor Malignant tumor


Cartilage: Chondroma Chondrosarcoma
Glandular epithelium: Adenoma Adenocarcinoma
Adipose tissue: Lipoma Liposarcoma
Epithelium: Papilloma Carcinoma
Bone: Osteoma Osteosarcoma

Each cancer has characteristics that reflect its origin. Thus, for example, the cells of an epidermal basal-
cell carcinoma, derived from a keratinocyte stem cell in the skin, will generally continue to synthesize
cytokeratin intermediate filaments, is only locally invasive and rarely forms metastases; whereas the cells
of a melanoma, derived from pigment cell in the skin, will often continue to make pigment granules and if
not removed promptly is much more malignant and rapidly gives rise to many metastases and
consequently fatal.

Properties of cancer cells: Cancer cells typically display abnormalities in the mechanisms that regulate
normal cell proliferation, differentiation, and survival. Taken together, these characteristic properties of
cancer cells provide a description of malignancy at the cellular level.
(1) Decreased Density-dependent inhibition of Growth: A primary distinction between cancer cells and
normal cells in culture is that normal cells display density dependent inhibition of cell
proliferation. Normal cells proliferate until they reach a finite cell density, which is determined in
part by the availability of growth factors added to the culture medium (usually in the form of
serum). They then cease proliferation and become quiescent, arrested in the Go stage of the cell
cycle. The proliferation of most cancer cells, however, is not sensitive to density-dependent
inhibition. Rather than responding to the signals that cause normal cells to cease proliferation and
enter Go, tumor cells generally continue growing to high cell density in culture, mimicking their
uncontrolled proliferation in vivo.
(2) Loss of adhesiveness: Cells in tissues are attached to one another and to the extracellular
matrix(ECM). Cancer cells are less stringently regulated than normal cells by cell-cell and cell-
extracellular matrix interactions. Disruption of these adhesion events leads to increased cell
motility and potential invasiveness of cells through the ECM. In addition, most cell types require
attachment to the ECM for normal growth, differentiation, and function. This attachment is
responsible for what was termed anchorage dependence. Most cancer cells are less adhesive than
normal cells, often as a result of reduced expression of cell surface adhesion molecules like E-
cadherin and integrins involved in binding to other cell and the ECM respectively. Consequently,
cancer cells are comparatively unrestrained by interactions with other cells and tissue components,
contributing to the ability of malignant cells to invade tissues and metastasize.
(3) Morphological and cytoskeletal differences: The reduced adhesiveness of cancer cells also results in
morphological and cytoloskeletal alterations. (a) Cancer cells are rounder than their normal
counterparts and (b) the most important thing is that their nucleus becomes much bigger than that
of a normal cells and at times, the nuclear outline is irregular. In other words, the nucleo-
cytoplasmic ratio is much higher than normal in cancer cells.(c) Cancer cells have very large
nucleoli also. (d) Many mitotic figures are shown by cancer cells. (e) chromatin shows unusual
clumping, and (f ) cancer cells almost always have an abnormal karyotype with abnormal number
of chromosomes (polypoid or aneuploid) or with abnormal chromosome structures.
(4) Loss of Anchorage Dependence: Most freshly isolated normal cells and cells from cultures of
normal diploid cells do not grow well when they are suspended in fluid or a semisolid agar gel. If
these cells make contact with a suitable surface, however, they attach, spread, and proliferate. This
type of growth is called anchorage-dependent growth. Many cell lines derived from tumors and
cells transformed by oncogenic agents are able to proliferate in suspension cultures or in a
semisolid medium( methylcellulose or agarose) without attachment to a surface. This is called
anchorage-independent growth. This property of transformed cells has been used to develop
clones of malignant cells. This technique has been widely used to compare the growth properties of
normal and malignant cells.
(5) Loss of Contact inhibition :A striking difference in the cell-cell interactions displayed by normal
cells and those of cancer cells is illustrated by the phenomenon of contact inhibition. Normal
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fibroblasts, placed 1mm apart in liquid medium on a glass surface of a culture dish, an outgrowth
toward each cell occurred or migrate across the surface of the culture dish until they make contact
with the neighboring cell. Further cell migration is then inhibited, and normal cells adhere to each
other, forming an orderly array of cells( monolayer) on the culture dish surface. It was concluded
that the cells inhibit one another by mutual contact of their cell surface, a phenomenon called
contact inhibition. A variety of malignant transformed cells, in contrast, continue moving after
contact with their neighbors, migrating over adjacent cells, and do not stop replicating when they
come in contact, growing in disordered, muitilayered patterns. Not only the movement but also the
proliferation of many normal cells is inhibited by cell-cell contact, and cancer cells are
characteristically insensitive to such contact inhibition of growth.
(6) Highly invasive: Transformed malignant cells in culture and human cells in vivo produce a variety
of lytic enzymes that degrade the ECM and allow cancer cells to invade tissues, lymphatic
channels, and the vasculature. These proteases include plasminogen activator, cathepsins, and a
number of matrix metalloproteases (MMPs).The MMPs are a large family of proteases that includes
collagenases (MMPs1,2, and 9) and stromelysins (MMPs 3 and 11). Secretion of collagenases, for
example, have been found at elevated levels in melanoma and in cancers of the colon, breast, lung,
prostate, and bladder. Usually, these elevated levels correlated with higher tumor grade and
invasiveness. In normal cells, the metalloproteinases are inhibited by the presence of a tissue
inhibitor of metalloproteinases( TIMP) . In tumor cells, TIMP inhibits metalloproteinases only when
the number of TIMP molecules are greater than the number of metalloproteinase molecules.
(7) Reduced requirement of extracellular growth factors/lower serum requirements: Tumor
cells are often able to survive in the absence of growth factors required by their normal
counterparts, contributing to the unregulated proliferation of tumor cells both in vitro and in vivo.
In some cases, cancer cells produce growth factors that stimulate their own proliferation. Such
abnormal production of a growth factor by a responsive cell leads to continuous autostimulation of
cell division (autocrine growth stimulation), and cancer cells are therefore less dependent on
growth factors or serum in the culture.
(8) Angiogenesis: Cancer cells secrete growth factors that promote the formation of new blood vessels,
called angiogenesis. Angiogenesis is needed to support the growth of a tumor beyond the size of
about a million cells, at which point new blood vessels are required to supply oxygen and nutrients
to the proliferating tumor cells. Such blood vessels are formed in response to growth factors,
secreted by the tumor cells, that stimulate proliferation of endothelial cells in the walls of
capillaries in surrounding tissues, resulting in the outgrowth of new capillaries into the tumor. The
formation of such new blood vessels are is important not only in supporting tumor growth, but
also in metastasis.
(9) Fails to differentiate normally: Another general characteristic of most cancer cells is that they fail
to differentiate normally. Such defective differentiation is closely coupled to abnormal
proliferation, since most fully differentiated cells either cease cell division or divide only rarely.
Rather than carrying out their normal differentiation program, cancer cells are usually blocked at
an early stage of differentiation, consistent with their continued active proliferation.
(10) Immortality of Transformed Cells in Culture: Most normal diploid mammalian cells have a limited
life expectancy in culture. For example, normal human fibroblast lines may live for 50 to 60
population doublings, but then viability begins to decrease rapidly unless they transform
spontaneously or are transformed by oncogenic agents. However, malignant cells once they
become established in culture, will generally live for an indefinite number of population doublings,
provided the right nutrients and growth factors are present. It is not clear what limits the life
expectancy of the normal diploid cells in culture, but it may be related to the continual shortening
of chromosomal telomeres each time cells divide. Transformed cells are known to have elevated
levels of telomerase that maintain telomere length.
(11) Resistance to Apoptosis: Normal cells respond to a variety of suboptimal growth conditions by
entering a quiescent phase in the cell division cycle, the Go state. If the cells are blocked at this
point for any length of time, they die. The events that regulate the cell cycle, called cell cycle check
points. This loss of cell cycle check point controlled by cancer cells may contribute to their
increased susceptibility to anticancer drugs. Many cancer cells fail to undergo apoptosis
(programmed cell death), and therefore exhibit increased life spans compared to their normal
counterparts. In this case, the failure to undergo apoptosis contributes to the resistance of cancer
cells to irradiation and many chemotherapeutic drugs, which act by damaging DNA.
(12) Other Biochemical properties: A number of changes in the biochemical characteristics of malignant
cells surface have been observed. These include appearance of new surface antigens,
proteoglycans, glycolipids, and mucins, and altered cell-cell and cell-extra cellular matrix
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communication. Some mucin antigens are shed from tumor cells and can be detected in the sera of
patients with pancreatic, ovarian, breast, and colon cancers ( e.g. CA19-9, CA125, CA15-3 are
used as tumor markers).

The Development of Cancer: One of the fundamental features of cancer is tumor clonality, the
development of tumors from single cells that begin to proliferate abnormally. The clonal origin of tumors
does not, however, imply that the original progenitor cell that gives rise to a tumor has initially acquired
all of the characteristics of a cancer cell. On the contrary, the development of cancer is a multistep
process in which cells gradually become malignant through a progressive series of alterations. One
indication of the multistep development of cancer is that most cancers develop late in life.
At the cellular level, the development of cancer is viewed as a multistep process involving mutation and
selection for cells with progressively increasing capacity for proliferation, survival, invasion, and
metastasis.
(1) The first step is the process, tumor initiation, is thought to be the result of a genetic alteration
leading to abnormal proliferation of a single cell.
(2) Cell proliferation then leads to the outgrowth of a population of clonally derived tumor cells.
(3) Tumor progression continues as additional mutations occur within cells of the tumor population.
Some of these mutations confer a selective advantage to the cell, such as more rapid growth, and
the descendants of a cell bearing such a mutation will consequently become dominant within the
tumor population. The process is called clonal selection, since a new clone of tumor cells has
evolved on the basis of its increase growth rate or other properties (such as survival, invasion, or
metastasis) that confer a selective advantage. Clonal selection continues throughout tumor
development, so tumors continuously become more rapid-growing and increasingly malignant.

Studies of colon carcinomas (colorectal cancer) have provided a clear example of tumor progression
during the development of a common human malignancy.
(a) The earliest stage of in tumor development is increased proliferation of colon epithelial cells. One
of the cells within the proliferative cell population is then thought to give rise to a small benign
neoplasm (an adenoma or polyp).
(b) Further rounds of clonal selection leads to the growth of a adenomas of increasing size and
proliferative potential.
(c) Malignant carcinomas then arise from the benign adenomas, indicated by invasion of tumor cells
through basal lamina into underlying connective tissue.
(d) The cancer cells then continue to proliferate and spread through the connective tissues of the
colon wall.
(e) Eventually the cancer cells penetrate the wall of the colon and invade other abnormal organs,
such as the bladder or small intestine.
(f) In addition, the cancer cells invade blood and lymphatic vessels, allowing them to metastasize
throughout the body.
Causes of cancer: There are many different causes of cancer, such as spontaneous genetic changes (e.g.
spontaneous gene mutations or chromosome mutations) and exposure to chemical mutagens, radiation, or
cancer inducing viruses (tumor viruses). Chemicals or radiations that can induce cancer are called
carcinogens. There is also hereditary predisposition to cancer. Cancers that run in families are known as
familial (hereditary ) cancers; cancers that do not appear to be inherited are known as sporadic (or
nonhereditary) cancers. But cancer always starts from a single cell due to genetic defects. Thus cancer is a
genetic disorder at the cellular level.
Chemical carcinogens: Many quite disparate chemicals have been shown to be carcinogenic when they
are fed to experimental animals or painted repeatedly on their skin. Examples include a range of aromatic
hydrocarbons and derivatives of them such as aromatic amines; nitrosamines; and alkylating agents such
as mustard gas. Although these chemical carcinogens are diverse in structure, they have at least one
property in common- they cause mutations.
A few of these carcinogens act directly on the target DNA, but generally the more potent ones are
relatively inert chemically and become damaging only after they have been changed to more reactive form
by metabolic processes- notably by a set of intracellular enzymes known as the cytochrome P-450
oxidases. These enzymes normally help to convert ingested toxins into harmless and easily excreted
compounds. Unfortunately, their activity on certain chemicals generates products that are highly
mutagenic. Examples of carcinogens activated in this way include the fungal toxin aflatoxin B1 ( a potent

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liver carcinogen produced by some molds ) and benzo(a)-pyrene, a cancer causing chemical present in
coal tar and tobacco smoke.
Although some chemicals contribute cancer development by stimulating cell proliferation, not causing
DNA mutation. Such components are referred to as tumor promoters. The most widely studied tumor
promoters are phorbol esters, which behave as artificial activators of protein kinase C and hence activate
part of the phosphatidylinositol intracellular signaling pathway. Hormones, particularly estrogens, are
important as tumor promoters in the development of some human cancer. The proliferation of cells of the
uterine endometrium, for example, is stimulated by estrogen, and exposure to excess oestrogen
significantly increases the likelihood that a women will develop endometrial cancer. The risk is minimized
by administration of progesterone to counteract the stimulatory effect of estrogen on endometrial cell
proliferation. These substances cause cancers at high frequency only if they are applied after a treatment
with a mutagenic initiator (a carcinogen that shows seeds of cancer to act as a tumor initiator).
Radiations: like UV-rays and Gamma rays may be mutagenic and carcinogenic. UV-rays cause
dimerization of thymine in the DNA strands having two consecutive thymines. Thymine dimer formation
seriously hampers DNA replication and may produce apurine sites, breaks and cross-linking of DNA
strands, that may have a carcinogenic consequences, especially for skin. Again, X-ray and gamma rays are
called ionizing radiations. These rays induce production of perhydroxyl or HO2 and superoxide or O2-
radicals. Such radicals act on cellular DNA and produce breaks and cross-linking of DNA strands. Thus,
the damaging action of both non-ionizing and ionizing radiations on DNA may have a carcinogenic effect.
These carcinogens are generally referred to as initiating agents, since the induction of mutations in key
target genes is thought to be the initial event leading to cancer development.
Tumor viruses: Members of six distinct families of animal viruses, called tumor viruses are capable of
causing cancer in either experimental animals or humans. Viruses belonging to five of these families have
DNA genomes and are referred to as DNA tumor viruses. Members of the sixth family of tumor viruses,
the retroviruses, have RNA genomes in virus particles but replicate via synthesis of a DNA provirus in
infected cells. The RNA tumor viruses transform a cell because of the property of a gene( or genes) in the
viral genome called an oncogene. By definition, an oncogene is a gene whose action promotes cell
proliferation.
The viruses that cause human cancer include hepatitis B virus (liver cancer), papillomaviruses (cervical
and other anogenital cancers), Epstein-Barr virus (Burkitts lymphoma and nasopharyngeal carcinoma),
and human T-cell lymphotropic virus (adult T-cell leukemia). In addition, HIV is indirectly responsible for
the cancers that develop in AIDS patients as a result of immunodeficiency.

DNA Tumor Viruses:


DNA tumor viruses normally progress through their life cycles without transforming a cell to a cancerous
state. Typically, the virus produces a viral protein that activates DNA replication in the host cell. Then,
through the use of host proteins, the virus genome is replicated and transcribed, ultimately producing a
large number of progeny viruses and killing the cell. The released viruses can then infect other cells.
Rarely, the viral DNA is not replicated and becomes integrated into the host genome. If the viral protein
that activates DNA replication of the host cell is now synthesized, this protein transforms the cell to the
cancerous state by stimulating the quiescent host cell to proliferate; that is, it causes the cell to move from
the Go phase to the S phase of the cell cycle.
Thus, the DNA tumor viruses are oncogenic- they induce cell proliferation- but they do not carry
oncogenes like those in RNA tumor viruses. The mechanism for transforming cells is completely different
from that of RNA tumor viruses. DNA tumor viruses transform cells to the cancerous state through the
action of a gene or genes that are essential parts of the viral genome. One example of tumor produced by
DNA tumor viruses is found in the papovavirus family. This family includes the many known
papillomavirus, some of which cause benign tumors such as skin and venereal warts in humans. Other
human papillomaviruses (HPV-16, HPV-18, or both) cause cervical cancer, which is a leading cause of
cancer deaths among women worldwide. The key viral genes involved in the transformation are E6 and
E7. The protein produced of these two genes cause a change in the levels of cellular proteins that are
important in regulating cell growth and cell division. In particular, E7 binds to Rb, and E6 stimulates the
degradation of p53 by ubiquitin-mediated proteolysis.
SV40 and Polyomavirus: The best studied DNA tumor viruses, from the standpoint of molecular biology,
are probably simian virus 40(SV40) and polyomavirus. Although neither of these viruses is associated
with human cancer, they have been critically important as models for understanding the molecular basis
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of cell transformation. The utility of these viruses in cancer research has stemmed from the availability of
good cell culture assays for both virus replication and transformation, as well as from the small size of
their genomes.
The natural host species of SV40 and polyomavirus are monkey and mice respectively. In cells of their
natural hosts (permissive cells), infection leads to virus replication, cell lysis, and release of progeny virus
particles. Since a permissive cell is killed as a consequence of virus replication, it cannot become
transformed. However, the transforming potential of these viruses is revealed by infection of
nonpermissive cells, in which virus replication is blocked.
The SV40 and polyomavirus genes that lead to transformation have been identified by detailed molecular
analyses. Transformation by these viruses has been found to result from expression of the same viral genes
that function in early stage of lytic infection. The genome of SV40 and polyomavirus are divided into early
and late regions. The early region is expressed immediately after infection and is required for synthesis for
viral DNA. The late region is not expressed until after viral DNA replication has begun, and includes genes
encoding structural components of the virus particle. The early region of SV40 encodes two proteins,
called small and large T antigens, of about 17kd and 94kd, respectively. Their mRNAs are generated by
alternative splicing of a single early-region primary transcript. Polyomavirus likewise encodes small and
large T antigens, as well as a third early region protein of about 55kd, designated middle T. The
transformation of cells by SV40 is as a result of large T, whereas it is middle T by polyomavirus.
During lytic infection, these early-region proteins fulfill multiple functions required for virus replication.
SV40 T antigen, for example, binds to the SV40 origin and initiates viral DNA replication. In addition, the
early-region proteins of SV40 and polyomavirus stimulate host cell gene expression and DNA synthesis.
This stimulation of cell proliferation by early gene products can lead to transformation if the viral DNA
becomes stably integrated and expressed in a nonpermissive cell. For example, SV40 T antigen binds to
and inactivates the host cell tumor suppressor proteins Rb and p53, which are key regulators of cell
proliferation and cell cycle progression.
Like SV40 and polyomavirus the adenoviruses are also a large family of DNA viruses, not associated with
naturally occurring cancers in either humans or other animals but can induce transformation in
nonpermissive hosts. However, they are widely studied and important models in experimental cancer
biology. Transformation by the adenoviruses results from expression of two early genes- E1A and E1B,
which are required for virus replication in permissive cells. They also act by the same way by inactivating
Rb and p53 tumor suppressor proteins like T antigens of SV40 and middle T of polyomavirus.
Retroviruses and oncogene: Members of one family of RNA viruses, the retroviruses, cause a variety of
diseases including tumors, wasting and auto-immune diseases, immunodeficiency syndromes and aplastic
and haemolytic anaemias in a variety of animal species, including humans. Four human retroviruses have
been identified HTLV-I, HTLV-II, HIV-I and HIV-II .

Retroviruses are infectious particles, a class of


enveloped viruses containing a single stranded
RNA molecule as the genome, packaged in a
protein capsid, surrounded by a lipid envelope.
This lipid envelope contains polypeptide chains
including receptor binding proteins which link
to the membrane receptors of the host cell,
initiating the process of infection. The name is
derived from the fact that the virus particle
contains an RNA-dependent DNA polymerase,
the reverse transcriptase( RTase).This enzyme
converts the RNA genome into DNA which
then integrates into the host chromosomal
DNA and is expressed as proteins.

Stylized drawing of a retrovirus

All retrovirus genomes consist of two


molecules of RNA, which are s/s, (+) sense and
have 5' cap and 3' poly-(A) (equivalent to
mRNA). These two molecules are physically
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linked as a dimer by hydrogen bonds. In addition, there is a 3rd type of nucleic acid present in all
particles, a specific type of tRNA (usually trp, pro or lys) - required for replication. At the 5' end of the
RNA genome is the U5 region and at the 3' end is the U3 region. The U3 region contains all of the
promoter information that is necessary to start RNA transcription at the beginning of the R (repeat) region
while the U5 region contains all of the information necessary to terminate after the other R repeat. These
vary in size from ~8-11kb. Retrovirus genomes have 4 unique features:

1. They are the only viruses which are truly diploid.


2. They are the only RNA viruses whose genome is produced by cellular transcriptional machinery
(without any participation by a virus-encoded polymerase).
3. They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication.
4. They are the only (+)sense RNA viruses whose genome does not serve directly as mRNA
immediately after infection.
5. The viral genome containing at least three genes: gag (coding for core proteins), pol (coding for
reverse transcriptase), and env (coding for the viral envelope protein).
6. At each end of the genome are long terminal repeats (LTRs) which include promoter/enhancer
regions, 3-prime cleavage and polyadenylation of mRNAs, and sequences involved with
integration.

In addition there are sequences required for packaging the viral DNA(psi) and RNA splice sites in the env
gene. Some retroviruses contain proto-oncogenes, which when mutated can cause cancers, however, in
the production of vectors these are removed. Retroviruses can also transform cells by integrating near to a
cellular protogenes and driving inappropriate expression from the LTR, or by disrupting a tumor
suppressor gene. This event is termed insertional mutagenesis

Note: Most RNA viruses employ single-stranded RNA(ssRNA) as their genetic material. The RNA base
sequence may be identical with that of viral mRNA, in which case the RNA strand is called the plus strand
or positive strand( viral mRNA is defined as plus or positive). However, the viral RNA genome may instead
be complementary to viral mRNA, and then it is called a minus or negative strand. Polio, tobacco mosaic,
brome mosaic, and Rous sarcoma viruses are all positive strand RNA virus; rabies, mumps, measles, and
influenza viruses are example of negative strand RNA viruses.
Plus strand of viral RNA often resembles mRNA in more than the equivalence of its nucleotide sequence.
Just as eukaryotic mRNA usually has a 5-prime cap of 7-methylguanosine. In addition, most or all plus
strand RNA animal viruses also have a poly-A stretch at the 3-prime end of their genome, and thus closely
resemble eukaryotic mRNA with respect to the structure of both ends. In fact, most plus strand RNAs can
direct protein synthesis immediately after entering the cell.
A few viruses have double stranded RNA (dsRNA) genomes. All appear to be segmented; some, such as the
reoviruses, have 10-12 segments.

Rous sarcoma virus: Viral oncogenes were first defined in Rous sarcoma virus(RSV), which transforms
chicken embryo fibroblasts in culture and induces large sarcomas within 1 to 2 weeks after incubation
into chickens. In 1910 F. Peyton Rous showed that when pieces of a sarcoma were transplanted into other
chickens from the same stock, the second group of chickens also developed sarcomas. Rous showed
further that cell-free filtrates of the sarcoma inoculated into chickens resulted in tumor development.
More than 50 years later, studies of RSV led to identification of the first viral oncogene(src), which has
provided a model for understanding many aspects of tumor development at the molecular level.

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The transforming potential of RSV was due to the presence of a specific oncogene in the viral genome in
addition to life-cycle genes (gag, pol, and env). Because RSV causes sarcomas, its oncogene was called v-
src. Like other retroviral oncogenes, it is not involved in the viral life cycle. It encodes a 60kd protein that
was the first protein-tyrosine kinase to be identified.

Viral oncogene: In the case of RSV, tumor induction results from the activity of the src gene in the
retroviral genome. The discovery that the product of a single gene was both necessary and sufficient for
tumor induction and formation was significant breakthrough. Viral oncogenes, responsible for many
different cancers are called v-oncs. Only retroviruses that contain a v-onc gene are tumor virus. The v-
onc genes are named for the tumor that the virus cause, with the prefix v to indicate that the gene is of
viral origin. Thus, the v-onc gene of RSV is v-src.

Virus Oncogene
Rous sarcoma virus v-src
Simian sarcoma virus v-sis
Avian erythroblastosis virus v-erbA or v-erbB
Kirsten murine sarcoma virus v-kras
Moloney murine sarcoma virus v-mos
MC29 avian myelocytoma virus v-myc

The retroviruses that contain oncogene are called transducing retroviruses, because they have picked up
oncogene from the genome of the cell. Retroviruses, that do not carry oncogenes are called
nontransducing retroviruses. Thus, the v-src of the RSV, originally picked up from the host genome,
called cellular src ( c-src).
CELLULAR PROTO-ONCOGENE: In the mid-1970s J.M. Bishop and Harold Varmus, along with a number
of other researchers, demonstrated that normal animal cells contain genes with DNA sequences very
closely related to the viral oncogenes. (For this discovery they were awarded Nobel Prize in 1989). In the
early 1980s R.A.Weinberg and M. Wigler showed independently that a variety of human tumor cells
contain oncogenes. These genes, when introduced into other cells growing in culture, transformed those
cells into cancer cells. The human oncogenes were found to be very similar to viral oncogenes that had
been characterized earlier, even though virus did not induce the human cancers involved. Moreover, the
human oncogenes were shown also to be closely related to genes found in normally growing cells.

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Definition of a proto-oncogene: A host gene is homologous to an oncogene that is found in a virus but
which can induce transformation only after being altered (such as mutation or a change of context such
as coming under the control of a highly active promotor).Thus, the cellular counterpart of the viral
oncogene is called proto-oncogene. It usually encodes a protein that functions in DNA replication or
growth control at some stage of the normal development of the organism.
CHARACTERISTICS OF CELLULAR PROTO-ONCOGENES
1) These are typical cellular genes with typical control sequences. As with most eucaryotic genes, most
have introns (retroviral oncogenes do not).
2) They show normal Mendelian inheritance.
3) As with all genes in the eucaryotic genome, they are always at same place in genome (cf. what would
be expected of endogenous retroviruses that had, over time, become incorporated into the cellular
genome).
4) There are no LTR sequences (v-oncs always are in an LTR context).
5) Viral oncogenes are most like the c-onc of the animal from which the virus is thought to have
acquired the gene. Thus, v-src of RSV is more like chicken src than human src. Note: v-onc was
picked up accidentally by the virus from the genome of a previous host cell.
6) Cellular oncogenes are expressed by the cell at some period in the life of the cell, often when the cell
is growing, replicating and differentiating normally. They are usually proteins that are involved in
growth control.
7) Cellular
oncogenes are (a) Chicken c-src proto-
highly oncogene
conserved.

In short, most
human and other
animal oncogenes
are mutant forms of
normal cellular
proto-oncogene
genes. They are
important cell
regulatory genes, in
many cases
encoding proteins
that function in the
signal transduction
pathways (b) RSV proviral DNA
controlling normal transcripts
cell proliferation. If
they are carried by
a virus are called v-
oncs. If they reside
in the host
chromosome,
oncogenes are
called cellular
oncogenes, or c-
oncs. ( For a
particular
oncogene, the onc is
replaced by the
three-letter
sequence of the
related viral
oncogene, e.g. v-src
9
and c-src.). A transducing retrovirus , then, carries a significantly altered form of a celluar proto-
oncogene( now a v-onc). When the transforming retrovirus infects a normal cell, the hitchhiking
oncogene transforms the cell into a cancer cell. A normal cell can also become transformed into cancer
cells even if a tumor virus does not infect them if the proto-oncogene is converted into a cellular
oncogene.
One significant difference between a cellular proto-oncogene and its viral oncogene counterpart is that
most proto-oncogene contain introns that are not present in the corresponding v-onc. For example, the
chicken src proto-oncogene is over 7kb long, with twelve exons separated by introns. In the RSV RNA
genome the v-src oncogene is 1.7kb long with no introns. Genomic RNA produced by the full-length
transcription of the proviral DNA is packaged into virus particles. Three types of mRNA are produced
from the RSV proviral DNA: a full length transcript and two mRNAs generated by differential splicing of
the full length transcript. mRNA transcription starts at a promoter in the left U3 sequence, and the
addition of the poly(A) tail is signaled by a sequence in the right R sequence.

Formation of Transducing Retrovirus:


Retroviral DNA( the provirus) integrates randomly into cellular DNA. Sometimes there occurs a genetic
rearrangements by which the transcriptional unit of the provirus connects to nearby cellular genes, often
by a deletion event involving the loss of some or all of the gag, pol, and env genes. In this way viral RNA
contains all or parts of a cellular gene. All viral progeny then carry the cellular gene and, under the
influence of viral promoters in the LTR, expressed the cellular protein in infected cells. If the cellular gene
picked up was a proto-oncogene, the modified retrovirus will be oncogenic.

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Protein products of Proto-oncogenes: About one hundred oncogenes have been identified through a
number of experiments. To understand how v-onc cause neoplasia, it is necessary to know the nature and
function of the proteins encoded by proto-oncogenes. Based on DNA sequence similarities and similarities
in amino acid sequences of the protein products, proto-oncogenes fall into several distinct classes, each
with a characteristic type of protein product.
In each case the proto-oncogene product is involved in the positive control of cell growth and division;
that is, the products are stimulatory to growth.
Class-I proto-oncogene: Growth factors are produced by this category of the proto-oncogene in normal
body cells. When modified by gene mutation or deletion they may produce some modified gene products
that cause uncontrolled cell division. For example, the product of the viral oncogene v-sis of simian
sarcoma virus was shown to identical to B chain of platelet-derived growth factor (PDGF, a factor found
in blood platelets in mammals),which is released after tissue damage. PDGF itself consists of two
polypeptides encoded by the proto-oncogene sis and affects only one type of cell, fibroblasts, causing them
to grow and divide. The mutated form of the sis gene in monkey ( c-sis) causes cancer of mesenchymal
cells(sarcoma).
The int-2 gene ( named for the fact that it is common site of integration of mouse mammary tumor virus)
encodes an FGF-related growth factor. The KGF (also called Hst) gene encodes an FGF-related growth
factor and was identified in gastric carcinoma and Kaposis sarcoma cells.
Class-II proto-oncogene: Growth factor receptors are produced by this category of genes, when mutated
produce modified growth factor receptor which generates constitutive stimulation to the cell in absence of
growth factors). The transmembrane receptor tyrosine kinases are the most important of the growth factor
receptors with respect to malignant transformation eg erbB, fms.
The erbB gene product is homologous to EGFR (epidermal growth factor receptor). Binding of EGF to the
receptor leads to internalisation of the EGFR complex, induction of the tyrosine kinase activity, stimulation
of cell DNA synthesis, & cell proliferation. v-erb encodes a truncated form of EGFR which does not have a
ligand binding domain -found in breast and ovarian tumours.
Class-III proto-oncogene: When active produce modified cytoplasmic protein kinases which cause
phosphorylation of various cellular proteins and initiates the signal transduction pathway constitutively
to induce cellular proliferation without any pause. e.g. abl oncogene concerned with chronic myeleoid
leukemia of man.
Class-IV proto-oncogene: Those encodes transcriptional regulators, when active, produces modified
transcription factors, which leads to production of different unusual proteins related to uncontrolled
proliferation of cells. e.g. myc oncogene concerned with Burkitts lymphoma of man, the gene was
originally identified in the avian myelocytomatosis virus.

FUNCTION OF PROTO-ONCOGENE- ENCODED EXAMPLE


PROTEINS
Control of DNA transcription (found in nucleus) myc
Signaling of hormone/growth factor binding such as a src is a membrane-bound tyr kinase.
tyrosine kinase
GTP-binding proteins involved in signal transduction ras
from a surface receptor to the nucleus
Growth factors sis is an altered form of platelet-derived growth factor B
chain.
Growth factor receptors erb-B is a homolog of the epidermal growth factor
receptor (it is also a tyrosine kinase). fms is a homolog of
the macrophage colony-stimulating factor (M-CSF)
receptor.

Activation of proto-oncogenes: In the normal cells expression of proto-oncogenes is tightly controlled so


that cell growth and division occur as appropriate for the cell type involved. However, when proto-
oncogenes are changed into oncogenes, the tight control can be lost, and unscheduled cell proliferation
can take place. The results of sequencing a number of known oncogenes indicate that many have
differences from the normal proto-oncogenes. The general types of changes that have been found are
point mutations, deletion, gene amplification, and chromosomal translocation.

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Point mutation: Point mutation in the coding region of a gene or in the controlling sequences (promoter,
regulatory elements, enhancers) can change a proto-oncogene into an oncogene by causing an increase in
either the activity of gene product or the expression of the gene, leading in turn to increase in the amount
in the gene product.
For example, the ras genes are a family of genes (N, H, and K) encoding membrane associated G proteins.
A single point mutation in the c-ras proto-oncogene, generally in codon 12,13, or 61, results in a mutant
protein that can transform normal cells into malignant cells by transmitting constitutive growth signals,
found in bladder cancer, lung cancer, colon cancer, and leukemia. The v-H-ras and v-K-ras differ from
corresponding c-ras genes by alterations of 3 and 7 amino acids respectively.

Deletion: Deletion of part of the coding region or a part of the controlling sequences of a proto-oncogene
have been found frequently in the oncogenes. The myc oncogene, for example, can arise from its proto-
oncogene by deletion. The normal gene consists of three exons and two introns; in some commonly found
myc oncogenes the first exon and most of the first intron are deleted.

Gene amplification: Some tumors have multi copies


of proto-oncogenes. These have probably occurred
by a random over replication of small segments of
the genomic DNA. For example, multiple copies of
ras are found in mouse adrenocortical tumors. In
general, extra copies of the proto-oncogene in the
cell result in an increased amount of gene product,
thereby inducing or contributing to unscheduled
proliferation.

Chromosomal translocation: Chromosomal


translocation in human tumor cells are rather
common, and some are specific for certain tumor
types. For example, Chronic Myelogenous Leukemia
(CML) and Philadelphia chromosome produced by a
reciprocal translocation involving chromosome 9
and 22, and Burkitts lymphoma resulting from a Fig The Philadelphia chromosome results when a
reciprocal translocation involving chromosome 8 piece of chromosome #9 switches places with a piece
and 14. In CML, the breakpoint on ch 9 is within an of chromosome #22.
intron of the ABL oncogene. The translocation joins
most of the ABL genomic sequence onto a limited region on ch..22 called the breakpoint cluster region
(BCR gene) on ch. 22. The novel gene product has enhanced tyrosine kinase activity resulting in
transforming properties

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