Role of Viruses in the Causation of Human Cancer

To prove a causal relationship between a putative cancer-causing virus and human cancer is not a

simple task. Such proof relies on evidence that is to a fair extent circumstantial. This evidence includes

(1) epidemiological data showing a correlation between living in an area of endemic viral infection and a type of cancer; (2) serological evidence of antibody titers to viral antigens in patients with a given cancer type; (3) evidence for insertion of viral DNA into a cancer-bearing host’s cell genome; (4) evidence for a consistent chromosomal translocation, particularly those involving an oncogene, in virally infected patients; (5) data showing that viral infection of cells in culture or transfection of viral genes into cells

causes cell transformation and the ability of such cells to produce tumors in nude mice; and (6)

development of cancers of the suspected target organ in transgenic mice produced by embryonic

gene transfer of viral genes.

On the basis of this sort of evidence, some human cancers are considered to be caused by viral infection either directly or indirectly. By ‘‘directly,’’ I mean that the viral gene(s) can themselves cause cells to become malignant (sometimes also requiring the loss of a tumor suppressor gene). By ‘‘indirectly,’’ I mean that viral infection may simply cause the progression of malignant cell growth by producing an

immunodeficiency state (e.g., the occurrence of non-Hodgkin’s lymphoma in HIV-infected patients)

or by stimulating the proliferation of already transformed cells. Sometimes viral infection acts in concert with other infectious agents or chemical carcinogens. Such is the case for malarial infection of Epstein-Barr virus (EBV)– infected patients and for aflatoxin exposure of individuals bearing the hepatitis B viral genome in their liver cells (see below). The types of human cancer thought to be caused by viral

infection and the strength of epidemiological associations Epstein-Barr virus has been linked to four different types of human cancer: Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC),

B-cell lymphomas in immunosuppressed individuals such as HIV-infected patients, and some

cases of Hodgkin’s lymphoma.149 The evidence is strongest for an association with BL and NPC.

Infection with EBV does not by itself cause cancer. On average, across the world, about 90% of the population may be infected by the time they reach adulthood.

 In some endemic areas, the incidence rate approaches 100%. In developing countries, EBV infection often occurs in young childhood. In more affluent societies, EBV infection tends to occur as the ‘‘kissing age’’ of adolescence or young adulthood is reached, and manifests itself as infectious mononucleosis.

In developing countries, particularly in equatorial Africa, concomitant or subsequent infection with the malarial parasite induces B-cell proliferation and an immunodeficiency state that leads to malignant transformation and progression. There is a consistent chromosomal translocation involving immunoglobulin genes, usually on chromosome 14, and sequences within or adjacent to the c-myc gene locus on chromosome. The role of EBV in NPC is less well characterized, but the evidence for an association includes high serum antibody titers against EBV antigens and the presence of EBV DNA in NPC cells. Similar evidence suggests an association between EBV infection and induction of some

B-cell lymphomas and some Hodgkin’s disease cases in immunosuppressed individuals, although

the exact role of EBV remains to be elucidated.

Read more...

VIRAL CARCINOGENESIS

It has long been suspected that various forms of cancer, particularly certain lymphomas and

leukemias, are caused or at least ‘‘co-caused’’ by transmissible viruses. This theory has had its ups

and downs during the first half of this century, and it was not generally accepted until the 1950s

that viruses can cause malignant tumors in animals. The known carcinogenic effects of certain

chemicals, irradiation, chronic irritation, and hormones did not fit with the idea of an infectious

origin of cancer. In early experiments, the basic assay to determine whether cancer could

be induced by a transmissible agent involved transmititng malignant disease by inoculation of

filtered extracts prepared from diseased tissues. If the disease occurred in animals inoculated

with such filtrates, it was assumed to be caused by a virus. In 1908, Ellermann and Bang137

transmitted chicken leukemia by cell-free, filtered extracts and thus were among the first to

demonstrate the viral etiology of this disease. In 1911, Rous induced sarcomas in chickens by

filtrates obtained by passing tumor extracts through filters that were impermeable to cells

and bacteria. These findings remained dormant for two decades until Shope showed, in 1933,

that the common cutaneous papillomas of wild rabbits in Kansas and Iowa were caused by a

filterable agent.139 It was later found that when these tumors were transplanted subcutaneously

they became invasive squamous cell carcinomas.  

In 1934, Lucke´ observed that kidney carcinomas commonly found in frogs in New

England lakes could be transmitted by lyophilized cell-free extracts.141Twoyears later, Bittner

demonstrated the transmission of mouse mammary carcinoma through the milk of mothers to

offspring.142 This was the first documented example of transmission of a tumor-inducing virus

from one generation to another. Drawing on the experiments of Bittner, Gross postulated that

mouse leukemia was also caused by a virus and that occurrence of the disease in successive generations of mice was due to transmission of virus from parents to offspring. The proof of this hypothesis eluded Gross for a number of years until he was prompted, by evidence based on transmission of Coxsackie viruses to newborn mice, to attempt inoculation of mice less than

48 hours old. Using this approach, he successfully transmitted mouse leukemia by injecting

filtered extracts preparedfrom borgans of inbred AK or C58 mice, which have a high incidence

of ‘‘spontaneous’’ leukemia, or from embryos of these mice, into newborn C3H mice, which have

a very low incidence of leukemia. These experiments demonstrated for the first time that mouse

 leukemia is caused by a virus and that the virus is transmitted in its latent form through embryos.

This led to the isolation of a mouse leukemia virus.The isolated virus was also found to induce

Leukemias and lymphomas in inbred strains of mice. Electron-microscopic studies145 showed that

the mouse leukemia virus is spheroid, has a diameter of about 100 nm, and contains a dense,

centrally located ‘‘nucleus’’ separated from the external envelope by a clear circular zone. The

Gross mouse leukemia virus was classified as a type C virus, a term now used to describe a wide

variety of RNA-containing oncogenic viruses of similar morphology.

The RNA oncoviruses have been classified by morphological criteria. Intracytoplasmic type A

particles were initially observed in early embryos of mice and in certain murine tumors. These A

particles are noninfectious, bud into intracellular membranes rather than through the plasma

membrane, and thus stay within the cell. They have an active reverse transcriptase and exist as

a proviral form in chromosomal DNA. Type B viruses have spikes on their outer envelope, bud

from cells, and have been identified primarily in murine species, mouse mammary tumor virus

(MMTV) being an example. Type C viruses have been found widely distributed among birds and

mammals, can induce leukemias, sarcomas, and other tumors in various species, and have certain

gene sequences that are homologous to ‘‘transforming’’ sequences isolated from various

human tumors (see below). Another subgroup, type D RNA oncoviruses, has been isolated

from primate species but their oncogenic potential is not well established. The subtypes of

RNA tumor viruses, known as Retroviridiae, share a genetically related genome containing a

gag-pol-env gene sequence coding for virus internal structural proteins, the special type of

RNA-directed DNA polymerase called reverse transcriptase and viral envelope proteins, respectively.

Thus, they most likely share a common evolutionary heritage.146 However, distinct subclasses of retrovirus evolution, based on pol gene sequence homologies, have been found; one major pathway gives rise to mammalian type C viruses and a second to A, B, D, and avian type C oncoviruses.146 A more recent addition to the retrovirus classification is the human Tcell leukemia virus (HTLV), isolated from patients with certain forms of adult T-cell leukemias (discussed later). The pol gene of HTLV

appears to have evolved from a progenitor common to the types A, B, D, and avian C oncoviruses

rather than from the mammalian C type.146 If true, this would be unusual because most mammalian type C viruses share antigenic determinants among several gag, pol, and env gene products, suggesting a common progenitor for this subclass of retroviruses.

Read more...

DNA REPAIR MECHANISMS

Not all interactions of chemicals and irradiation with DNA produce mutations. In fact, all cells

have efficient repair mechanisms that repair such lesions. DNA repair mechanisms include sets of

enzymes that survey DNA for specific kinds of damage, remove the altered portion ofDNA, and

then restore the correct nucleotide sequence. The important role of DNA repair in human

cancer has been established by the finding that a number of inherited defects in DNA repair systems

predispose individuals to getting cancer. These diseases include xeroderma pigmentosum,

ataxia telangiectasia, Fanconi’s anemia, Bloom’s syndrome, Cokayne’s syndrome, and hereditary

retinoblastoma. There are several types of DNA repair systems, a number of which have been preserved

from bacteria to humans. These include (1) abnormal precursor degradation, e.g., the hydrolysis of the oxidized nucleotide triphosphate 8-hydroxy-dGTP to its nucleotide 8-OHdGMP, preventing incorporation into DNA; (2) a visible light-activated photoreactivation repair mechanism for removal of UV-induced cyclobutane pyrimidine dimmers; (3) strand break repair via an action of DNA ligase, exonuclease,

and polymerase activities; (4) base excision repair that recognizes simple base alterations such

as cytosine deamination to uracil and requires the action of (a) a purine or pyrimidine glycosylase

that breaks the deoxyribose-base bond, (b) an endonuclease to cleave at the abasic site, (c) a phosphodiesterase to clip away the ‘‘naked’’ abasic site, (d) DNA polymerase, and (e) DNA

ligase to refill and reclose the site; (5) nucleotide excision repair that recognizes bulky DNA base

adducts, pyrimidine dimers, and base crosslinks and requires the concerted action of enzymes and recognition factors (see below); and (6) 06-alkyguanine-DNA alkyltransferase that recognizes and removes small alkyl adducts from DNA. In mammalian cells, key repair mechanisms are base excision repair, nucleotide excision repair, transcription-coupled repair, homologous recombination and end joining, and mismatch repair.

 Excision repair is the most generalDNArepair mechanism in higher organisms. Base excision repair removes damage such as deaminated bases, oxidized or ring-opened bases generated by hydroxyl or superoxide radicals, and abnormally methylated bases such as 3-methyladenine.126 Nucleotide excision repair requires sequential steps of (1) preincision recognition of damage; (2) incision of the damaged DNA strand at or near the damaged site; (3) excision of the damaged site and local removal of nucleotides in both directions from the defect in the affected DNA strand; (4) repair replication to replace

the excised region, using the undamaged strand as a template; and (5) ligation to join the repaired

sequence of nucleotides at its 30 end to the contiguous DNA strand.125 DNA repair is usually very accurate, but if repair cannot occur prior to or during DNA replication it may be error prone. This errorprone, post-replication repair seems to be brought into play by certain types of agents or

when a cell is overwhelmed by damage that it cannot handle by excision repair before the cell

enters S phase during the next round of cell division. In this case, the new DNA is synthesized

on templates that still contain damaged bases, leading to mispairing or recombinational events that transfer damaged bases to daughter strands. For example, in mammalian cells, 5% to 30% of UV-induced thymidine dimers are transferred from parental to daughter strands during postreplication repair.129

Nucleotide excision repair (NER) of DNA in eukaryotic cells requires several gene products. Some of these gene products appear to be identical or highly homologous in yeast, rodents, and humans.130,131 A number of defects in the NER system have been found by studying mutations in cells from patients with xeroderma pigmentosum, in whom at least nine different kinds of mutations (i.e., nine different  complementation groups) have been found.125 Some of these XP genes have been cloned and found to

be highly homologous to yeast RAD genes that are required for excision repair in Saccharomyces

cerevisiae.130–133 Some of the cloned human genes also correct repair defects in mutant rodent cells and are called excision repair crosscomplementing (ERCC) genes.

Read more...

MULTIPLE MUTATIONS IN CANCER

In most cases, it takes years for a full-blown invasive, metastatic cancer to develop from a

small clone of initiated cells. This process might take 20 years or more, during which time an

initiated clone of cells undergoes clonal expansion via multiple cell doublings. As these clones

expand, various cells in the population accumulate multiple genetic alterations, some of which

facilitate dysregulated cell proliferation and some of which lead to cell death. These genetic

alterations can include point mutations, chromosomal translocations, gene deletions, gene

amplifications, loss of genetic heterozygosity (LOH), and loss of genetic imprinting (LOI).

This accumulation of genetic defects that occurs during clonal expansion of transformed cells is

due to ‘‘genetic instability.’’ The cause of this genetic instability is not clearly understood, but

it includes defects in cell replication checkpoint controls and decreased ability to repair DNA

damage.

There is evidence for the accumulation of thousands of mutations in cancer cells derived

from human tumors. For example, examination of the colon tumor–derivedDNA from patients with

hereditary non-polyposis colon cancer (HNPCC) reveals that as many as 100,000 repetitive DNA

sequences are altered from the mismatch DNA repair defects that these patients’ cells harbor

(reviewed in Reference 122). Mismatch repair defects have also been noted in ‘‘sporadic’’ (not

known to be hereditary) cancers. As noted earlier, one hypothesis explaining the genetic instability of transformed cells is the mutator phenotype hypothesis, championed by Loeb and colleagues.122 This hypothesis states that an ‘‘initial mutator [gene] mutation generates further mutations including mutations

in additional genetic stability genes, resulting in a cascade of mutations throughout the genome.’’ The molecular defect that could provide this phenotype could be a mutation in DNA polymerases that leads to error-prone DNA replication. The mutator phenotype would have to be generated early in tumorigenesis for this hypothesis to be valid. There are a number of arguments against this idea, such as observations

that there is not necessarily an increased mutation rate in cancer cells over that of normal cells123 and that a similar ‘‘evolution’’ of genetically altered cancer cells could arise by clonal selection followed by clonal expansion of cells with a genetic alteration that provides a proliferative advantage.

Read more...

GENETIC SUSCEPTIBILITY AND CANCER

As was noted above, there are a number of inherited cancer susceptibility gene mutations,

such as xeroderma pigmentosum, Fanconi’s anemia, and ataxia telangiectasia. These types of

inherited defects that lead to cancer are generally caused by a deficiency in DNA repair pathways.

Almost certainly we have only scratched the surface of inherited cancer susceptibility

genes that make an individual more prone to developing cancer. Other susceptibility genes

may include alterations in the metabolic enzymes that metabolize drugs and environmental

toxins, polymorphisms in genes that regulate utilization of certain essential nutrients such as

folic acid, or inherited mutations in tumor suppressor genes.

The completion of the Human Genome Project allows a systematic approach to discovering

the genetic alterations thatmakeindividuals prone to developing various diseases. The Environmental

Genome Project is producing a catalogue of variation in genes involved in catabolizing toxins,

nutrient metabolism, and DNA repair.121 These data, which will be largely generated by detection

of single nucleotide polymorphisms (SNPs), will enable toxicologists and cancer biologists to predict

individual susceptibility to diseases triggered or promoted by environmental pollutants, diet,

and other lifestyle factors. Some examples of this SNP analysis approach are the increased susceptibility

of individuals with altered folate metabolism genes to develop leukemia after benzene

exposure and the ethnic variation in the BRCA1 gene SNPs that affect susceptibility to

breast cancer.

Read more...

Ultraviolet Radiation

Ultraviolet radiation–induced lesions, generated by UV-B (280–320nm wavelength) or UV-A

(320–400nm wavelength), result from DNA damage, which is converted to mutations during

cellular repair processes. UB-B and UV-A generate different types of DNA damage and DNA

repair mechanisms (reviewed in Reference 113). Irradiation with UV-B produces cyclobutane

pyrimidine dimers that are repaired by nucleotide excision repair. If left unrepaired,

C?T and CC?TT base transitions occur. UVA- induced DNA damage produces mostly oxidative

lesions via photosensitization mechanisms and is repaired by base excision repair.

UV-B and UV-A also produce different effects on the immune system and elicit different transcriptional

and inflammatory responses. While the specific mechanisms by which UV radiation

induces basal cell or squamous cell carcinomas or melanoma are not clear, a number of signal

transduction pathways are affected that can either lead to apoptosis or to increased cell proliferation.

UV irradiation activates receptor tyrosine kinases and other cell surface receptors. It also enhances phosphorylation by ligand-independent mechanisms via inhibition of protein tyrosine phosphatase activity. Liganddependent cell surface receptor activation can also occur by activation of autocrine or paracrine

release of growth factors from keratinocytes, melanocytes, or neighboring fibroblasts. It is clear, however, that better animal models are needed to clearly define the mechanisms by which UV light causes human cancer. OXYGEN FREE RADICALS, AGING, AND CANCER The diseases of aging include  cardiovascular disease, decline in function of the immune system, brain dysfunction,

Read more...

Ionizing Radiation

The history of radiation carcinogenesis goesback a long way.The harmful effects of X-rays were observed

soon after their discovery in 1895 by W. K. Ro¨ ntgen. The first observed effects were acute, such as reddening and blistering of the skin within hours or days after exposure. By 1902, it became apparent that cancer was one of the possible delayed effects of X-ray exposure. These cancers, which included  leukemia, skin cancers, lymphomas, and brain tumors, were usually seen in radiologists only after long-term exposure before adequate safety measures were adopted, thus it was thought that there was a

safe threshold for radiation exposure. The hypothesis that small doses of radiation might also cause cancer was not adopted until the 1950s, when data from atomic bomb survivors in Japan and certain groups of patients treated with Xrays for noncancerous conditions, such as enlarged thyroids, were analyzed. These and other data led to the concept that the incidence of radiation-induced cancers might increase as a linear, nonthreshold function of dose. Thus the debate about whether there is a safe threshold pertains to radiation carcinogenesis, just as it does to chemical carcinogenesis.

In radiation carcinogenesis, the damage to DNA, and hence its mutagenic and carcinogenic effect, is due to the generation of free radicals as the radiation passes through tissues. The amount of radical formation and ensuing DNA damage depend on the energy of the radiation. In general, X-rays and gamma rays have a low rate of linear energy transfer, generate ions sparsely along their tracks, and penetrate deeply into tissue. This profile contrasts with that of charged particles, such as protons and a particles, which

have a high linear energy transfer, generate many more radical ions locally, and have low penetration through tissues. Thedamage toDNA can include single- and double-strand breaks, point mutations due to misrepair deletions, and chromosomal translocations.107–109 The molecular genetic events that follow radiation damage to cells include (1) induction of early-response genes such as c-jun and Egr-1; (2) induction of later-response genes such as tumor necrosis factor-a (TNF-a), fibroblast growth factor

(FGF), and platelet-derived growth factor-a (PDGF-a); (3) activation of interleukin-1 (IL-1) PKC110; and (4) activation of oncogenes such as c-myc and K-ras.111 Induction of these genes may be involved in the cellular responses to irradiation and in the longer-range effects that lead to carcinogenesis. At any rate, the production of clinically detectable cancers in humans after known exposures generally occurs after long latent periods. Estimates of these latent periods are 7 to 10 years for leukemia, 10– 15 years for bone, 27 years for brain, 20 years for thyroid, 22 years for breast, 25 years for lung, 26 years for intestinal, and 24 years for skin cancers.

A more recent example of nuclear fallout leading to environmental exposure to radiation is the Chernobyl accident, which happened onApril 26, 1986. A steam explosion blew the lid off the reactor. The graphite core caught fire and over 1019 becquerels (Bqs) of radioisotopes were released, producing a fallout that covered much of Belarus, Northern Ukraine, and part of the Russian Federation. Estimates are that

10–20 million people were exposed to significant fallout. There were some deaths due to acute

radiation sickness from high levels of exposure. However, the long-term effects are still being

recorded. So far, the reliable reports of increases in cancer incidences are mostly limited to thyroid

cancer.112 This finding is in contrast to cancer incidence among atomic bomb survivors in Japan, some of whom developed cancers of various types, including cancers of the thyroid, breast, lung, stomach, esophagus, bladder, leukemia, and lymphoma (although the incidence of cancers in Japanese atomic bomb survivors was less than would have been predicted by radiation exposure). The reason for this discrepancy is most likely that those exposed to the Chernobyl fallout received primarily dosage from

b-emitters, mostly isotopes of iodine, which concentrates in the thyroid. Atomic bomb survivors,

by contrast, received whole-body irradiation from neutrons and gamma rays.

Another interesting point about the Chernobyl survivors is that the type of thyroid cancer they developed, mostly among those under 2 years of age if they were exposed, were 98% papillary, many with an unusual morphology, whereas in non-exposed populations, only 67% of childhood thyroid cancers are papillary.112 Expression of two families of oncogenes, the c-ret and ras families, has been shown to be

involved in papillary thyroid cancers. The oncogene c-ret is a receptor tyrosine kinase activated

by gene rearrangement, and two of these, ret-ptc 1 and ret-ptc 3, are activated in papillary carcinomas. Since c-ret is activated by rearrangement, the high proportion of doublestrand DNA breaks seen in radiation-induced papillary carcinomas of the thyroid may explain its activation. Since the thyroid is not the only tissue that concentrates iodine, malignancies of other tissues that also concentrate iodine, such as the breast, salivary gland, and stomach, may appear in higher incidence as time goes on. Moreover,

other isotopes including cesium were present inthe fallout, and inhabitants of parts of the Ukraine and Belarus are still exposed to low levels of radioactive cesium. The long-term effects, if any, of such exposure is not yet clear.

Read more...