Chronic myelogenous leukemia is an example of a proto-oncogene activation by

Oncogenes, Tumor Suppressor Genes, and the Genetic Basis of Carcinogenesis

Cellular Oncogenes and Tumor Suppressor Genes Are Implicated in Carcinogenesis

A number of cellular genes are now implicated in carcinogenesis. These genes are of two types; oncogenes and tumor suppressor genes. Oncogenes are activated form of cellular proto-oncogenes that normally encode proteins necessary for cellular functions. A proto-oncogene can be activated into an oncogene through structural or functional alterations. Broadly speaking, activation of oncogenes and inactivation of tumor suppressor genes may have similar consequences in terms of tumor development.

Activation of Oncogenes and Inactivation of Tumor Suppressor Genes are Intimately Associated With Tumor Development

Proto-oncogenes may be activated by mutation, chromosomal rearrangement (e.g., translocations and inversions), or gene amplification to become a cellular oncogene (c-onc). An example of cellular oncogenic activation through gene amplification is Myc, which codes for a transcription factor that plays a role in cell division. Generation of high amounts of Myc oncogene product can also be due to high levels of transcription without gene amplification. This has been reported in Burkitt’s lymphoma where translocation of the Myc proto-oncogene from its normal location in chromosome 8 to chromosome 14 brings it close to the immunoglobulin heavy chain gene promoter. As a result, c-Myc now finds itself in a region of vigorous transcriptional activity, with a consequent overproduction of its product.

Another example of chromosomal translocation and cellular oncogenic activation is found in chronic myelogenous leukemia (CML). In CML, a reciprocal translocation occurs between chromosomes 9 and 22 [t(9;22)]. A portion on the long arm of chromosome 9 (9q) containing the Abl gene is translocated next to the Bcr gene on the long arm of chromosome 22 (22q). The altered chromosome 22 is called the Philadelphia chromosome (Ph′). The Bcr-Abl fusion gene produces higher levels of a fusion protein Bcr-Abl. The Abl portion of the fusion protein has constitutive protein tyrosine kinase activity, whereas the Bcr portion of the fusion protein can bind to many proteins. Binding of the Bcr-Abl fusion proteins with proteins involved in the mitogenic signaling pathway can cause activation of mitogenic signaling and increased cell proliferation.

Tumor suppressor genes, which also participate in the regulation of normal cell growth, are usually inactivated by point mutations or truncation of their protein sequence coupled with the loss of the normal allele. The first mutation may be inherited or somatic. The second mutation will often be a gross event leading to loss of heterozygosity and tumor suppressor function. This mechanism provides support to the two-hit hypothesis of Alfred Knudson, discussed earlier in the chapter. Frequent loss of heterozygosity in the tumor cells provides support to Knudson’s hypothesis.

Oncogenes and Tumor Suppressor Genes Encode Protein Products That Are Involved in the Regulation of Growth and Survival of Cells as Well as Programmed Cell Death

Proto-oncogenes encode proteins that are involved in the regulation of cell growth as well as division and differentiation, such as growth factors, growth factor receptor-associated tyrosine kinases, membrane-associated nonreceptor tyrosine kinases, G-protein-coupled receptors, membrane-associated G-proteins, serine-threnine kinases, transcription factors, and regulators of programmed cell death. For example, Sis, Int-2 encode growth factors; Src, Abl, erbB encode protein tyrosine kinases; Ras is a GTP-binding GTPase; and Fos, Jun, Myc, and Myb encode transcription factors. There are many other such examples. These proteins, when encoded by oncogenes, are called oncoproteins, which are either mutated or with unregulated expressions. In most cases, the oncogenes encode mutant forms of the proteins so that they are not subject to the on-off regulation in response to mitogenic signals. In other words, the mitogenic signal is perpetually “on,” resulting in uncontrolled cell proliferation.

The cell growth and division suppressor effects are also lost in the mutant p53 gene; p53 is a tumor suppressor gene that encodes a transcription factor (p53 protein). The amino acids that are involved in DNA binding show the highest mutation rate in various cancers. This demonstrates how mutations in tumor suppressor genes can abrogate their tumor suppressor function by disrupting the transcriptional regulation of their target genes. Another role of p53 is to regulate apoptosis or programmed cell death by upregulating the proapoptotic gene Bax. Mutant p53 cannot mediate apoptosis; thus cells with unrepaired DNA damage are prevented from undergoing apoptosis. Survival of these cells and their subsequent division may lead to the development of tumor.

Viral Carcinogenesis

Viruses are implicated in approximately 15% of all cancers, such as nasopharyngeal carcinoma, T-cell leukemias, hepatocellular carcinoma, and Kaposi’s sarcoma. Only a small proportion of people infected with any of the human tumor viruses develop tumors, and those that are infected rarely (if ever) serve as sources for ongoing transmission. Instead, most human tumor virus transmissions are asymptomatic or mildly symptomatic but do not lead to neoplasia (Butel, 2000; Moore and Chang, 2010). Oncogenes are found in both cancer-causing DNA viruses and RNA viruses. DNA viruses with oncogenic potential are from six distinct viral families: hepatitis B viruses, simian virus 40 (SV40) and polyomavirus, papillomaviruses, adenoviruses, herpesviruses, and poxviruses. In contrast, members of only one family of RNA viruses, the retroviruses, are capable of inducing oncogenic potential (Cooper, 1995). Papillomavirus, hepatitis B virus and Kaposi’s sarcoma-associated herpes virus are the main DNA tumor viruses relevant in human cancer development. Like the tumorigenic DNA viruses, there are tumorigenic retroviruses. Whereas DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation, transforming retroviruses carry oncogenes derived from cellular proto-oncogenes that are involved in mitogenic signaling and growth control (Butel, 2000).

The model for the acquisition of oncogenes by retroviruses from cellular proto-oncogenes was first provided by Takeya and Hanafusa (1983) from their work on the c-Src proto-oncogene. Cellular proto-oncogenes contain introns while the corresponding viral oncogenes lack introns. The retroviral oncogene capture model postulates that the c-onc sequence was captured by virus through recombination that occurred at the level of proviral DNA. Retroviruses replicate inside the cell through a DNA intermediate, called provirus, which is integrated into the chromosomal DNA of the infected cell. Chance integration of provirus next to the cellular proto-oncogene creates a viral-cellular fusion gene. Read-through transcription of this fusion gene creates a hybrid (viral+cellular) RNA. Processing of this read-through transcript removes the introns. When this hybrid RNA sequence undergoes recombination with the viral RNA during reverse-transcription, the cellular oncogene (without the introns) is captured by the viral genome. Fig. 20.7 shows how a cellular proto-oncogene (c-onc) could be acquired by the viral genome. The function of the v-onc products is similar or identical to that of the c-onc products, and the expression is generally unregulated.

Figure 20.7. Mechanism by which a cellular proto-oncogene (c-onc) is captured by retrovirus to give rise to a viral oncogene (v-onc). It is triggered by proviral integration next to the cellular proto-oncogene creating a viral-cellular fusion gene. RNA processing following read-through transcription removes the introns from this hybrid (viral+cellular) RNA sequence. When this hybrid RNA sequence undergoes recombination with the viral RNA during reverse-transcription, the cellular oncogene is captured by the viral genome without the introns.

Oncogenes

Proto-oncogenes are normal cellular genes that regulate cell growth and differentiation. They often encode products such as growth factors and their receptors, cell cycle regulators, DNA-binding proteins, transcription factors, protein kinases involved in signal transduction, and others. When “activated” by overexpression or mutation, proto-oncogenes are termed oncogenes. Oncogenes drive proliferation and render the cell unresponsive to normal growth inhibitory signals, ultimately resulting in tumor formation.

There are a number of ways in which proto-oncogenes can be activated. The gene can be amplified, so that a signal to transcribe the gene results in the production of many more copies of mRNA than usual. Oncogenes can undergo mutations that cause constitutive activation of the encoded protein. In these cases the protein product is always “turned on” and is unresponsive to inhibitory signals. This scenario is common for tyrosine kinase receptors, such as the epidermal growth factor receptor (EGFR). Activating mutations in genes encoding these receptors result in constitutive kinase activity even in the absence of appropriate triggers or ligands. Tumor cells may also synthesize large amounts of both tyrosine kinase receptors and their activating ligands, forming a growth-promoting autocrine loop.

The prototype of signal transduction oncogenes are the ras genes, which encode the RAS family of guanosine triphosphate (GTP)-binding proteins (G proteins) (Fig. 6-39). In normal cells, RAS proteins transmit growth stimulatory signals from growth factor receptors to the nucleus, ultimately activating transcription of genes that regulate cell proliferation. RAS is normally located on the cytoplasmic side of the cell membrane and is closely associated with farnesyl transferase. Inactive RAS binds guanosine diphosphate (GDP). Upon receiving a stimulatory signal from an activated growth factor receptor, RAS exchanges GDP for GTP. RAS bound to GTP is the active form, which triggers the RAS–RAF–mitogen-activated protein kinase (MAPK) signaling cascade and results in transcription of genes that promote cell division. The activation of RAS is normally short lived, because RAS has an intrinsic guanosine triphosphatase (GTPase) activity that hydrolyzes GTP to GDP and converts RAS to its inactive state. In many cancers, RAS mutation renders RAS activation independent of upstream growth factor receptor activation or abrogates RAS GTPase activity. RAS family members, the farnesyl transferase membrane anchor, and other components of the downstream MAPK signal transduction pathway are all attractive molecular targets for therapeutic intervention in cancer patients.

Functions of oncogene products (oncoproteins)

Proto-oncogenes encode proteins that are mostly involved in the regulation of cell growth, division, and differentiation. Consequently, proto-oncogenes encode products that can act as growth factors, growth-factor-receptor-associated tyrosine kinases, membrane-associated non-receptor tyrosine kinases, G-protein-coupled receptors, membrane-associated G-proteins, serine–threonine kinases, transcription factors, and regulators of programmed cell death.

Growth factors play important roles in cell cycle control, and they signal a cell to either enter the G1 phase or bypass it. The right amount of growth factors should be produced at the right time, or else cell cycle control may be dysregulated. Examples of oncogene-encoded growth factors are sis oncogene-encoded platelet-derived growth factor (PDGF) B chain and int-2 oncogene-encoded fibroblast growth factor (FGF)-related growth factor. Receptor-associated and non-receptor tyrosine kinases as well as G-proteins, G-protein-coupled receptors, and serine–threonine kinases all encode products that are important in regulating the transduction of mitogenic signals in the cell, thereby regulating normal cell division. In most cases, these oncogenes encode mutant forms of the proteins so that they are not subject to the on–off regulation in response to mitogenic signals. In other words, the mitogenic signal is perpetually “on”, resulting in uncontrolled cell proliferation.

Oncogenes, such as src and abl, encode tyrosine kinases that are membrane associated, while erbB, neu, and fms encode transmembrane kinases. An example of an oncogene encoding a GTP-binding GTPase is ras. Normal GTP-binding proteins (G-proteins), including ras, are important signal transducers. The active form binds GTP and transduces the mitogenic signal; hydrolysis of GTP to GDP inactivates the protein and terminates the signal. Further activation involves exchange of GDP for GTP. Mutant forms of ras exist in an inactive GDP-bound form, resulting in a perpetual “on” mode of the mitogenic signal. Oncogenes encoding transcription factors also act by the same principle. The proto-oncogene products of fos (Fos) and jun (Jun) form a heterodimer to produce the transcription factor activator protein-1 (AP-1). Mutant AP-1 may act in a constitutive manner without activation by agents, such as phorbol esters. Other oncogenes, such as myc and myb, also code for transcription factors that, when mutated, can cause aberrant transcriptional dysregulation.

The cell growth and division suppressor effects are also lost in mutant p53 which is a tumor suppressor gene and whose product (p53 protein) is a transcription factor. The three dimensional structure of p53 protein with its target DNA molecule defined the core domain and the amino acids involved in DNA binding (Cho et al., 1994). These amino acids that are involved in DNA binding show the highest mutation rate in various cancers. This demonstrates how mutations in tumor suppressor genes can abrogate their tumor suppressor function by disrupting the transcriptional regulation of their target genes. Another role of p53 is to regulate apoptosis or programmed cell death by upregulating the pro-apoptotic gene Bax. Mutant p53 cannot mediate apoptosis; thus cells with unrepaired DNA damage are prevented from undergoing apoptosis. Survival of these cells and their subsequent division may lead to the development of cancer cells.

Mutagenic chemicals that can disrupt the structure and/or expression of these proto-oncogenes can cause their oncogenic activation. For example, phorbol ester is a known activator of AP-1-mediated transcription. The procarcinogen 1,2-dimethylhydrazine has been reported to induce mutation in ras which was detectable in pre-neoplastic and neoplastic rat colonic mucosa (Jacoby et al., 1991).

Proto-oncogenes

Proto-oncogenes such as Ras and Myc were first discovered in the 1970s and 1980s, and it was only in the 1980s and 1990s that these genes were integrated into signaling pathways and that mutations in these genes were linked to human cancers. The Ras genes (Hras, Kras, and Nras) encode GTPase proteins that help transduce survival and growth-promoting signals. When oncogenic mutations occur, the normal abrogation of RAS signaling by hydrolysis of bound GTP to GDP is impaired, resulting in persistent signaling (reviewed by Singhal et al., 2005). Point mutations (found most frequently in codon 12, 13, and 61) are detected in 20–30% of lung adenocarcinomas (Slebos et al., 1990) and 90% of these mutations are found in Kras (see Table 34.1). Mutations in Kras are markers for poor prognosis in NSCLCs (Graziano et al., 1999) as mutations rarely occur in SCLC. Myc genes (MYCL, MYCN, and CMYC), which encode transcription factors that regulate genes involved in cell cycle regulation, DNA synthesis, and RNA metabolism, become activated by loss of transcriptional control or by gene amplification, resulting in MYC protein overexpression. C-Myc amplification occurs in 5–10% of NSCLCs (Richardson and Johnson, 1993).

Table 34.1. Common molecular alterations in lung cancer

Type of mutationFrequencyProto-oncogenes k-RasPoint mutationNSCLC: 20–30% (mostly adeno) MYCTranslocation amplificationSCLC: 30–40% NSCLC: 5–10% Bcl-2TranslocationNSCLC: (30%) Squamous: 25%; Adeno: 10%
Growth factors EGFR (ERBB-1)Deletion/Mutation amplificationSCLC: 0%NSCLC: 10%(BAC: 25%) HER2 (erbB2)Translocation amplificationNSCLC 30–40%
Tumor suppressors p53Deletion/LOHSCLC: 75%NSCLC: 50% Rb genePoint mutationSCLC: 90%NSCLC: 15–30% P16 (CDKN2)Deletion/LOHSCLC: ∼80% NSCLC: 30–50% FHITDeletion/LOHSCLC: 100% NSCLC: 60%

Proto-oncogene Amplification

Proto-oncogenes importantly participate in regulating cellular growth and proliferation. Different categories of oncogenes significantly contribute in the development of precursors and SCC UADT. Important members of the cell cycle regulators are cyclin D1 and epidermal growth factor receptor (EGFR). Cyclin D1, located in the 11q13 chromosome region, has a central role in the cell cycle. It regulates the G1/S transition by phosphorylation and inactivation of the retinoblastoma gene, which is considered a key event in cell cycle control. Gene amplification and overexpression have been frequently described in SCC UADT and also in its precursor lesions.221–223 Cyclin D1 inhibitor, p16 gene, contributes to cell cycle control through decrease of Rb gene phosphorylation. Despite related but opposite functions of genes, gain of cyclin D1 and loss of p16 are considered to be independent mechanisms in G1/S phase dysregulation.224,225 It is believed that constitutive activation of the cyclin D1 pathway can reduce or overcome certain mitogen requirements for cell proliferation and thus contribute to oncogenic transformation.47

Amplification of the chromosome 11q13 is a frequently detected event in SCC UADT development, observed in 30% to 50% of cases.226 In addition to cyclin D1, several genes that have potential functional importance for head and neck tumorigenesis are frequently coamplified in this region, including the INT2 gene (a member of the fibroblast growth factor family), EMS1, FGF4, vascular endothelial growth factor-βɛτα, phosphatase-1a, and glutathione S-transferase p. INT2 gene amplification, in particular, has been detected as an early event in head and neck carcinogenesis, already present at the stage of dysplasia. These data provide evidence that gene amplification can also occur early in the UADT tumorigenesis process.227

The EGFR affects cell division, migration, adhesion, differentiation, and apoptosis through a tyrosine kinase pathway.228 Overexpression of EGFR was found to correlate with the severity of epithelial abnormalities, suggesting that its alteration is an early genetic event in head and neck cancer development.229,230

Proto-oncogenes

Proto-oncogenes such as Ras and Myc were first discovered in the 1970s and 80s. It was only in the 1980s and 90s that these genes were integrated into signaling pathways and that mutations in them were linked to human cancers. The Ras genes (Hras, Kras, and Nras) encode GTPase proteins that help transduce survival- and growth-promoting signals. When oncogenic mutations occur, the normal abrogation of RAS signaling by hydrolysis of bound guanosine triphosphate (GTP) to guanosine diphosphate (GDP) is impaired, resulting in persistent signaling (reviewed by Singhal et al., 2005). Point mutations (found most frequently in codons 12, 13, and 61) are detected in 20–30% of lung adenocarcinomas (Slebos et al., 1990), and 90% of these mutations are found in Kras (see Table 59.1). Mutations in Kras are markers for poor prognosis in NSCLCs (Graziano et al., 1999), as mutations rarely occur in SCLC. Myc genes (MYCL, MYCN, and CMYC), which encode transcription factors that regulate genes involved in cell cycle regulation, DNA synthesis, and RNA metabolism, become activated by loss of transcriptional control or by gene amplification, resulting in MYC protein overexpression. C-Myc amplification occurs in 5–10% of NSCLCs (Richardson and Johnson, 1993).

Table 59.1. Common molecular alterations in lung cancer

Type of mutationFrequencyProto-oncogenesk-RasPoint mutationNSCLC 20–30% (mostly adeno)MYCTranslocationSCLC 30–40%AmplificationNSCLC 5–10%Bcl-2TranslocationNSCLC (30%)Squamous 25%; adeno 10%EML4-ALKTranslocationNSCLC 2–7% (mostly adeno)Growth factorsEGFR (ERBB-1)Deletion/mutationSCLC 0%AmplificationNSCLC 10% (BAC 25%)HER2 (erbB2)TranslocationNSCLC 30–40%AmplificationTumor-suppressorsp53Deletion/LOHSCLC 75%NSCLC 50%RbPoint mutationSCLC 90%NSCLC 15–30%P16 (CDKN2)Deletion /LOHSCLC >80%NSCLC 30–50%FHITDeletion/LOHSCLC 100%NSCLC 60%Lkb1MutationNSCLC 30%

BAC, Bronchioloalveolar carcinoma; LOH, loss of heterozygosity.

The anaplastic lymphoma kinase (ALK) fusion gene was first described in lymphoma in 1994. In 2007, Soda and colleagues described an EML4-ALK fusion oncogene that generates aberrant signaling in NSCLC (Soda et al., 2007). The fusion protein arises from an inversion on the short arm of chromosome 2 that joins exons 1–13 of EML4 to exons 20–29 of ALK. Lung tumors that contain the EML4-ALK fusion oncogene or its variants are associated with specific clinical features, including never- or light-smoking history, younger age, and adenocarcinoma. ALK gene arrangements are largely mutually exclusive with EGFR or KRAS mutations and occur in 2–7% of NSCLC patients (Takahashi et al., 2010). A recent trial demonstrated the antitumor efficacy of crizotinib, an Alk inhibitor, in lung tumors with the ALK rearrangement, resulting in tumor shrinkage or stable disease in most patients (Kwak et al., 2010). Two secondary mutations within the kinase domain of EML4-ALK in tumor cells have been demonstrated in patients who relapsed during Alk inhibitor treatment (Choi et al., 2010).

3.5 Molecular Epidemiology

Proto-oncogene and tumor suppressor gene mutation assays have become a popular tool for investigating tumor etiology in humans and rodents mainly because mutations tend to be chemical specific (see The Application of Toxicogenomics to the Interpretation of Toxicologic Pathology, Chapter 11). Because the inactivating mutations of the p53 gene are primarily missense mutations (90%) and there are many “hotspots,” it is “well suited for this form of molecular archaeology” in examining human and animal tumors. Study of the patterns of oncogene activation in spontaneous versus chemically-induced rodent neoplasms has provided data that suggest that the molecular lesions associated with chemically-induced cancer are sometimes different from those documented in spontaneous cancer. Furthermore, the patterns of oncogene activation in several rodent model systems appear to be carcinogen-specific, are consistent with known or expected DNA adduct formation, and in some cases are similar to patterns of oncogene activation documented in human neoplasms.

Mutations in the ras gene from skin tumors in sun-exposed areas appear to be induced by UV irradiation; K-ras mutations in human lung and colon tumors may be the result of adduct formation due to carcinogens from cigarette smoke; and aflatoxin B1-associated human liver tumors appear to have a specific mutation at codon 249 of the p53 gene. There is epidemiological evidence suggesting that for some solvent-associated human leukemias, the exposure was associated with an increased risk of developing a tumor with ras activation.

The analysis of subpopulations of humans at risk with unique genetic alterations in tumors may lead to the identification of more human carcinogens. Moreover, animal studies, such as in the mouse liver tumor models, may help further define these associations and elucidate the effects of dose.

It has been proposed that the carcinogen-specific patterns of ras gene mutation observed in tumors reflect the mechanisms of carcinogenesis, and some patterns of H-ras mutations in mouse liver tumors suggest that they occur from direct chemical–ras gene (genotoxic) interactions.

The B6C3Fl mouse liver is a model in which many genotoxic and non-genotoxic chemicals induce hepatocellular tumors. H-ras activation is a common event in tumorigenesis, and the frequency and pattern of activating mutations are compared between tumors that are chemically-induced and spontaneous to help in determining possible mechanisms by which chemicals induce tumors. Some genotoxic compounds cause mutations that can be explained by the type of DNA adducts formed and some non-genotoxic carcinogens induce tumors that have the same mutational spectra as spontaneous tumors, suggesting that these chemicals act by promoting the spontaneously initiated hepatocytes.

Molecular epidemiology of cancer is the study of molecular alterations, primarily mutations, in investigating the causative agents of cancer, as well as identifying individual cancer risk. The objective to identify cancer-causing agents based on the occurrence of predictable molecular alterations that are found in the neoplasm is intriguing. It is based on the hypothesis that there are carcinogen-specific patterns of mutations that reflect direct interactions of carcinogens with cancer genes. For example, lung and colon cancers from people who smoke tend to have a specific mutation in the ras oncogene or p53 tumor suppressor gene (i.e., mostly a G to T nucleotide base substitution) and that this mutation is likely due to direct interaction of the carcinogen in smoke benzo[a]pyrene with DNA.

Such chemical-specific mutational profiles (or “molecular signatures”) have been used to establish a causal nature between particular genetic events in tumors and carcinogen, such as neoplasms associated with exposure to radon, aflatoxin B1, vinyl chloride, and the nitrosamines. The strongest evidence for linkage between a cancer-causing agent and a specific type of neoplasm is that of the CC to TT double base changes observed in skin neoplasms of man and animals. This mutation is consistent with the predicted UV-induced damage of dipyrimidine dimers. In liver tumors from persons living in geographic areas with a high exposure to aflatoxin B1 there is a frequent mutation at the third nucleotide pair of codon 249 in the p53 gene, suggesting the mutation is chemical-specific and imparts a specific growth or survival advantage to the mutated liver cells.

Animal studies have confirmed that there are certainly chemical-specific mutational profiles in neoplasms; however, there are many examples where the mutational profile varies by strain (Table 5.15), species, dose, or dosing regimen. For example, diethylnitrosamine, a strong, cross-species hepatocarcinogen, will induce liver neoplasms in mice, rats, and rainbow trout, but the frequency and type of ras mutation in the neoplasm varies widely, and the mutations are not simply a reflection of direct DNA interaction.

TABLE 5.15. Species and Strain Comparison of the Variety of Frequencies and Spectra of ras Mutations in Hepatocellular Neoplasms Induced by Diethylnitrosamine (DEN)a

StrainH-ras codon 61 mutationsCodon 61 mutation (normal = CAA)Other rasAAACGACTACCAC3H54/114 (47%)2824200B6C3F163/239 (26%)16321500CD-19/25 (36%)7100N-ras (4/25; 16%)C57BL2/59 (3%)01010Rainbow trout–––––K-ras (6/7; 85%)F344 rat0/10––––0/10

Mouse data mainly from Maronpot et al. (1995). Mutations in the ras proto-oncogene: Clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101, 125–156.

In some studies, in vitro mutation assays are a poor predictor of liver tumor mutation profiles in the mouse. In this complex process, carcinogens might also be influencing events such as DNA repair, oxidative DNA damage, methylation, cell death, proliferation, and/or a hypermutable state.

Other possible mechanisms by which carcinogen treatment could affect ras mutation profile in tumor DNA include those caused by indirect oxidative DNA damage via oxygen or lipid radicals produced during carcinogen metabolism or cytotoxic injury; a selective growth promotion or death of tumor cells with a specific ras genotype; or a hypermutable state induced by the carcinogen (Table 5.11). Other factors that can affect the profile of ras mutations found in mouse liver tumors, and thus add an additional level of complexity in the evaluation of chemical genotoxicity in tumor DNA, include genetic background (strain) of mouse, dose, dosing regimen, and time of sampling. In some cases when humans and rodents are exposed to the same mutagenic carcinogen, such as vinyl chloride, the specific cells or tissue at risk and specific mutations differ.

Using tumor DNA from the National Toxicology Program (NTP) carcinogenesis testing program to detect dominant transforming genes, patterns of oncogene activation have been documented for spontaneous and chemically-induced neoplasms in Fischer 344 rats and in B6C3Fl mice. Results to date show a low frequency (about 3%) of activated oncogenes in spontaneous neoplasms of the Fischer 344 rats.

In contrast, a variety of epithelial neoplasms induced by benzidine congeners as well as lung tumors induced by tetranitromethane inhalation have shown a high frequency of oncogene activation. In the B6C3F1 mouse there is a high frequency (56%) of oncogene activation in spontaneous liver tumors and a lower frequency (8%) in spontaneous non-liver tumors. The oncogenes in spontaneous liver tumors in the B6C3F1 mouse most frequently involve activation of the H-ras proto-oncogene by point mutations in the sixty-first codon.

In chemically-induced hepatic neoplasia in this mouse hybrid, there is also a high frequency of oncogene activation, but the pattern of oncogene activation differs from that observed in spontaneous liver tumors. In addition to a “background” level of mutations in the sixty-first codon identical to those observed in spontaneous hepatocellular neoplasms, additional point mutations were documented in codons 12 or 117 of the H-ras proto-oncogene in addition to the activation of K-ras and N-ras oncogenes in chemically treated mice that had an unequivocal increase in hepatocellular tumors.

Molecular epidemiologic studies aimed at identifying an individual’s risk of developing cancer have found that persons with germ-line mutations in cancer genes (i.e. BRCA1 or BRCA2) or variations (polymorphisms) of carcinogen-metabolizing enzyme activities (i.e., cytochrome p450s or glutathione-S-transferases) or DNA repair capacities can be at increased risk of developing neoplasia in their lifetime. High-throughput analyses to examine single nucleotide polymorphisms (SNPs) are being used to search for biomarkers of cancer-risk individuals, and some of this information is being used to help people to take preventive measures to decrease their risk of developing cancer.

The most frequently identified activated oncogenes detected in chemically-induced neoplasms belong to the ras gene family. Ras oncogenes were originally detected and isolated by transfection of NIH3T3 cells (immortalized mouse fibroblasts) using DNA from tumors; this method of detection has been replaced by newer molecular techniques. Cloning of these genes from the tumor DNA revealed that many were H-ras, K-ras, or N-ras genes that differed from their proto-oncogene homologs by a single point mutation located in a specific codon. Thus, when adolescent rats are given a single exposure to nitrosomethylurea (NMU), mammary tumors result that have an activated H-ras oncogene with a G–A transition (mutation) in the twelfth codon.

G–A transitions are consistent with the Q6-methyl guanine adduct that is formed by methylating agents such as methyl-nitrosourea (MNU), and such mutations would be expected to lead to nucleotide mispairing during DNA replication. The altered DNA would be expected to give rise to an abnormal protein product that could theoretically alter cell growth or differentiation.

MNU is very labile, with an estimated biological half-life of a few minutes. Thus, it is likely that the effect produced by this chemical carcinogen occurred as an early event in the carcinogenic process. Because hormone-stimulated cell division in the mammary tissue is also necessary for the development of mammary neoplasia in this model, it is unlikely that activation of the H-ras gene is sufficient in and of itself for the production of mammary neoplasia. Another example of association of an activated H-ras gene with neoplasia is seen in the carcinomas produced in mouse skin by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with phorbol ester. In this instance there is an A–T transversion (mutation) in the sixty-first codon of the H-ras gene. Once again, the oncogene activation produced by the DMBA treatment was not sufficient to cause carcinoma development but required promotion by phorbol ester to obtain cancer. Rodent test systems used for the in vivo detection of chemical carcinogens often depend on demonstration of an increased incidence of common neoplasms or on the induction of novel neoplasms in chronically treated animals.