Seminars in Cancer Biology 14 (2004) 441448

Genetic alterations and DNA repair in human carcinogenesis

Kathleen Dixon , Elizabeth Kopras
Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA

Abstract A causal association between genetic alterations and cancer is supported by extensive experimental and epidemiological data. Mutational inactivation of tumor suppressor genes and activation of oncogenes are associated with the development of a wide range of cancers. The link between mutagenesis and carcinogenesis is particularly evident for cancers induced by chemical exposures, which, in some cases, lead to characteristic patterns of mutations. These genotoxic, direct-acting carcinogens form covalent adducts with DNA, which cause mutations during DNA replication. The link between mutagenesis and carcinogenesis is also supported by the observation that DNA repair defects are associated with an increased cancer risk. Normally, DNA repair mechanisms serve to suppress mutagenesis by correcting DNA damage before it can lead to heritable mutations. It has been postulated that mutagenesis plays a role in both the initiation phase and the progression phase of carcinogenesis, and that an essential step in the carcinogenic process is the development of a mutator state in which the normal cellular processes that suppress mutagenesis become compromised. Given the link between mutations and cancer, attempts have been made to use the mutational prole of cancer cells as an indicator of the causative agent. While this may be a valid approach in some cases, it is complicated by the role of endogenous processes in promoting mutagenesis. In addition, many important carcinogenic agents may enhance mutagenesis indirectly through suppression of DNA repair functions or stimulation of inappropriate cell proliferation. Epigenetic phenomena may also suppress gene expression without causing overt changes in DNA sequence. 2004 Elsevier Ltd. All rights reserved.
Keywords: DNA repair; Mutagenesis; Tumor suppressors; Oncogenes

1. Genetic alterations in cancer The association between genetic alterations and human cancer was rst observed decades ago [1]. Cytogenetic studies revealed that specic chromosomal abnormalities were linked to the development of certain cancers. For example, a chromosomal translocation (the Philadelphia chromosome) was frequently found in white blood cells of leukemia patients. In addition, tumor cells often exhibited extensive genetic instability leading to chromosome aberrations, rearrangements, and aneuploidy. However, it was not clear whether this widespread genetic instability was a cause or a consequence of the cancer phenotype. An understanding of the role of genetic alterations in cancer development arose out of studies of oncogenic viruses and hereditary cancers. RNA tumor viruses were found to express certain oncogenes (e.g., c-ras and c-myc) that contributed to the transforming activity of the viruses and that had homologous counterparts (proto-oncogenes) in the human genome. Later, it was shown that RAS and MYC were over-expressed in cancer cells, often due to genetic translocations that placed
Corresponding

author. Tel.: +1 513 558 1728; fax: +1 513 558 3509. E-mail address: Kathleen.dixon@uc.edu (K. Dixon).

the genes under the control of strong heterologous promotors. The study of human retinoblastoma led to the discovery of the RB tumor suppressor gene; loss of function of this gene through inheritance of one mutant allele and the somatic loss of the other allele lead to the formation of retinal tumors in children. Another important tumor suppressor protein, p53, was rst identied as a target for the SV40 tumor virus, and was later found to be inactivated in a variety of tumor cells, and also in Li-Fraumeni syndrome, which is associated with a high cancer risk. Both point mutations and deletions are found among inherited and somatic mutations that inactivate RB and TP53; in addition, loss of the second allele in the inherited cancers can often occur though loss of part or all of one of two homologous chromosomes. A large number of tumor suppressor genes and oncogenes have now been identied and characterized through the analysis of tumor cell DNA [14]. It has been postulated that the minimum constellation of mutations required for oncogenic transformation in humans includes inactivation of TP53 and RB, activation of RAS (or other members of that pathway), and constitutive expression of hTERT [57]. These genes control cellular functions that prevent uncontrolled proliferation (Fig. 1). The most prevalent mutations in human cancers occur in the tumor suppressor genes, TP53 and RB

1044-579X/$ see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2004.06.007

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Fig. 1. Genetic alterations in cancer. Cancer develops over time as a consequence of successive mutation and expansion of mutant clones. Mutations that inactivate tumor suppressor genes, activate proto-oncogenes, and turn on telomerase stimulate cell proliferation and inhibit cell death, providing a growth advantage.

signaling. The MYC gene is often activated by DNA rearrangements that place the proto-oncogene under the control of a strong promoter, or gene amplication events that increase expression through an increase in gene copy number. The net effect of activation of these proto-oncogenes is the stimulation of cell proliferation, leading to the expansion of the transformed cell population and augmenting the effects of loss of tumor suppressor function. A related set of genes that operate in signal transduction are responsible for certain inherited conditions characterized by the development of scattered benign lesions that occasionally become malignant [3]. For example, the neurobromatosis (NF1) gene product regulates the RAS pathway, and the familial adenomatous polyposis (APC) gene regulates the WNT pathway. Affected individuals inherit mutations on one allele, and mutation of the second allele appears to be necessary for development of the benign lesions; further progression requires additional genetic hits. An additional requirement for cancer development is cell immortalization. Normal cells are able to undergo only a nite number of cell divisions before they reach crisis and die. This phenomenon has recently been attributed to a gradual shortening of the small repeat telomere sequences at the ends of chromosomes that protect them from both degradation and end-to-end fusions. The absence of telomeres leads to genetic instability and ultimately apoptotic cell death. Tumor cells overcome this process by switching on the gene (hTERT) telomerase, an enzyme that maintains telomere length [6].

2. Mutation spectra Extensive analysis of mutations in the TP53 gene has revealed that inactivating mutations are widely distributed throughout the gene, but certain types of mutations are more prevalent in some cancers than in others [8]. For example, in the case of sunlight-induced carcinoma, tandem double mutations at adjacent pyrimidines are observed at high frequency [11,12]; such mutations are rarely observed in other cancers. This observation is consistent with the fact that the UV portion of the suns spectrum dimerizes adjacent pyrimidines in skin, and such lesions have been shown to preferentially lead to tandem double mutations in in vitro mutagenesis assay systems [13,14]. Another example is the analysis of TP53 mutations in primary liver cancer derived from patients exposed to aatoxins, carcinogenic metabolites of certain spoilage molds [15]. In this case, a high frequency of G:C-to-T:A mutations at the third base in codon 249 were observed. A similar pattern of aatoxin-induced mutations was observed in mutagenesis test systems [16], and this correlation was used to strengthen the causal link between exposure to aatoxins and liver carcinogenesis in epidemiological studies [15]. An extensive database of tumor-associated TP53 mutations has been compiled (www.iarc.fr/p53) [17] and certain additional associations between environmental

[5]. Both base substitution mutations and gene deletions in these genes are found in a wide variety of cancer types [8]. The Rb protein is a key regulator of the cell cycle, and loss of this function can lead to increased cell proliferation and a failure in terminal differentiation, i.e., an increase in the birth rate of cells [7]. The p53 protein is important in cellular responses to stress, controlling DNA repair, cell cycle checkpoints, and apoptosis [9]. Perhaps, the most important of these pathways for cancer development is apoptosis; loss of p53 function can lead to decreased apoptosis, i.e., a decrease in the death rate of cells. Thus, loss of these two tumor suppressor genes leads to a net increase in cell numbers due to an increased birth rate and a decreased death rate. Cancer development can also be promoted through mutations that activate the expression of proto-oncogenes that regulate cell proliferation [5]. These are genes for secreted growth factors (e.g., PDGF), cell surface tyrosine kinase receptors (e.g., EGFR, HER), signal transduction G-proteins (e.g., RAS), and nuclear transcription factors (e.g., MYC) [10]. RAS mutations are found in about 20% of tumors, including colon, lung, breast, and bladder cancer. Activating mutations are often missense mutations that decrease the GTPase activity of the protein and prolong RAS-dependent

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Fig. 2. TP53 mutations in lung cancer. Diagram from the IARC TP53 mutation database (R8, June 2003) [17]. Analysis of mutations in TP53 from a variety of lung tumors reveals characteristic hotspots for mutagenesis.

exposures and mutational spectra have emerged. An example of one such association is shown in Fig. 2; there appears to be an association between G:C-to-T:A mutations at codons 157, 158, 245, 248, 249, and 273 in TP53 and cigarette smoking-associated lung cancer [18]. While these associations cannot conrm the link between a particular exposure and cancer development in a single individual, they are suggestive of a link on a population basis. In an attempt to determine whether general rules for the mutagenic specicity of carcinogens could be derived from the analysis of mutational patterns (mutation spectra) of individual carcinogens, several different mutation assay systems have been used [19]. One such system, the pZ189 shuttle vector system, has been used to analyze the specicity of almost 40 different carcinogens [20]. Cluster analysis of these mutation spectra revealed patterns of mutational specicity that roughly corresponded to the known chemical specicity of the agents. For example, chemicals that are known to form DNA adducts preferentially on G residues formed mutations preferentially at G:C base pairs, and those known to form DNA adducts preferentially on A residues formed mutations preferentially at A:T base pairs. In cases where whole classes of mutagenic agents form similar mutation spectra, back extrapolation from the characteristics of mutations in tumors to the identity of the causative agent is not possible. However, in cases where the mutational pattern is more unique (e.g., UV and aatoxins), such back extrapolation can provide support with regard to questions of cancer causation. Additional mutation analysis systems have been established that allow determination of mutational specicity in experimental animals [21]. In some of these systems, it is possible to examine target organ specicity as well as muta-

tional patterns. Of these, the most widely used mutagenesis reporter genes are the endogenous APRT [22,23] and HPRT [24] genes, and the Escherichia coli lacI or bacteriophage lambda cII transgenes [25]. The X-chromosomal HPRT gene has been used as a mutagenesis target for somatic mutations in peripheral blood lymphocytes of both humans and animals. Not only is this a useful reporter gene for point mutations, but it also reveals the activation of pathways normally involved in gene rearrangements that can trigger the generation of deletion mutations. The heterozygous APRT mouse has been particularly useful for understanding events that lead to loss of heterozygosity (LOH), a common mechanism for loss of the second allele in many inherited cancers. The Big BlueTM mouse system [25] carries an integrated bacteriophage lambda-based vector, LIZ, which contains the E. coli lacI and lacZ genes; either the lacI or the lambda cII genes can be assayed for mutations in DNA recovered from mouse tissues. This system has been used widely for tissue-specic analysis of mutagenesis [26]. Although each of these systems has unique properties, generally they reveal similar patterns of mutational specicity of environmental carcinogens.

3. Mutation avoidance: DNA repair and checkpoint pathways Cells have elaborate mechanisms for safeguarding the informational integrity of the genome and suppressing mutations. Mutation avoidance mechanisms include: (1) multiple checks and balances within the DNA replication complex that insure high delity DNA replication (<1 error in 106 nucleotides incorporated [27]); (2) pathways that suppress

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Fig. 3. DNA damage responses. Cells respond to DNA damage by activating a variety of DNA damage response pathways. If the DNA damage is excessive, cells die through induction of apoptosis. Alternatively, cell cycle checkpoints are induced that delay cell cycle progression to allow time for DNA repair to occur. Specic DNA repair pathways recognize and repair specic types of DNA damage. In the absence of DNA repair, the DNA damage results in mutations.

oxidative stress, which can result from endogenous metabolic processes and lead to oxidative DNA damage [28]; (3) pathways that regulate cell cycle progression to insure an orderly duplication and segregation of chromosomes [29]; and (4) DNA repair pathways that correct all types of

DNA damage caused by endogenous processes or exogenous agents (Fig. 3) [3032]. More than 130 genes have been identied that contribute to DNA repair. The importance of these mechanisms in cancer prevention is evident from the increased cancer risk associated with disruption of these pathways (Table 1) [33]. This is particularly evident from the study of a wide variety of familial cancers. One of the rst widely studied hereditary diseases associated with increased cancer risk was xeroderma pigmentosum [34,35]. This defect in nucleotide excision repair leads to a dramatic increase in the risk of sunlight-induced skin cancer. Individuals with this condition are unable to excise and repair UV photoproducts in skin DNA, so that mutagenesis and carcinogenesis are increased. Nucleotide excision repair is important for the removal of a wide variety of premutagenic DNA lesions in addition to UV photoproducts, including most bulky DNA adducts. In humans, the process of nucleotide excision repair requires more than 30 proteins [35]. These proteins are involved in DNA damage recognition, single-strand incision on either side of the lesion, excision of the single-stranded region containing the lesion, DNA repair synthesis, and ligation. XPAXPG are required for global genome repair (GGR), which serves to repair lesions throughout the genome. Transcription-coupled repair (TCR), which occurs mainly in transcribed regions of the genome, requires the Cockayne syndrome gene products (CS-A and CS-B), as well as the XP proteins, and is important in cell survival. Increased cancer risk is associated primarily with defects in the XP genes and not the CS genes. The high delity of DNA replication is normally maintained by the accuracy of DNA polymerase ; compromising the DNA polymerase proofreading function can cause an increase in mutation rate, and can lead to an increased cancer risk in transgenic mice [36]. Furthermore, an increase in the expression of the less accurate DNA polymerase , which normally functions in DNA repair, can also increase mutagenesis and is associated with cancer [37]. Certain less

Table 1 Hereditary cancer syndromes with defects in DNA repair and checkpoint pathways Pathway Mismatch repair Nucleotide excision repair Replication bypass Replication fork integrity; double-strand break repair Genes MSH1, MSH2, MLH1, MSH6, PMS1, PMS2, XPAXPG XPV NBS1 MRE11 WRN BLM/RECQL3 BRCA1, BRCA2 FANCA-FANCG ATM TP53 RB1 Syndrome HNPCC Xeroderma pigmentosum (XP) XP variant Nijmegen breakage syndrome A-T-like disorder Werners syndrome Blooms syndrome Familial breast cancer Fanconi anemia Ataxia telangiectasia (AT) Li-Fraumeni syndrome Retinoblastoma Cancer type Colon cancer Skin cancer Skin cancer Lymphoma Lymphoma/leukemia Various Leukemia, carcinomas Breast/ovarian cancer Leukemia Lymphoma/leukemia Various Retinoblastoma

DNA crosslink repair DNA damage signaling; cell cycle checkpoints

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accurate bypass polymerases can also cause mutations that contribute to carcinogenesis. In the cancer-prone human genetic disorder, xeroderma pigmentosum variant (XP-V), the function of DNA polymerase , which can accurately bypass UV-induced TT dimers, is replaced by less accurate DNA polymerases, leading to higher UV-induced mutation rates and a higher risk of sunlight-induced cancer [38,39]. The importance of the mismatch repair pathway in the prevention of mutagenesis and carcinogenesis is illustrated by the large increase in cancer risk in individuals with mismatch repair defects. Defects in mismatch repair genes are associated with increased cancer risk in hereditary non-polyposis colon cancer (HNPCC) [40]. The mismatch repair system is responsible for removal of base mismatches caused by base deamination, oxidation, methylation, and DNA replication errors. The mismatch is recognized, and one of the two DNA strands is selectively excised, which is followed by repair synthesis and ligation of the resulting single-stranded DNA gap. Mutation of mismatch repair genes is associated with microsatellite instability and an increased rate of somatic mutations. A number of genes associated with increased cancer risk are important for DNA damage signaling, cell cycle checkpoints, and DNA double-strand break (DSB) repair (Table 1) [30]. The conversion of premutagenic DNA lesions (e.g., UV photoproducts, DNA adducts, etc.) to heritable mutations often requires active DNA replication and/or mitosis. Replication of damaged templates can result in replication errors or replication fork blockage. When progression of replication forks is blocked by DNA damage, a number of recovery mechanisms are induced, some of which are thought to involve RecQ-like helicases, such as BLM [41] and WRN [42]. Blocked replication forks can also result in the induction of DSBs, and the MRN complex (MRE11, RAD50, and NBS) participates in their repair [43]. BRCA1 and BRCA2 are also thought to participate in DSB repair [44]. The FANC genes appear to be required for repair of DNA crosslinks [45]. Human cells have multiple regulatory pathways (called cell cycle checkpoints) that are activated by DNA damage and that arrest cell cycle progression to allow time for DNA repair to occur [46]. For example, the protein kinase encoded by the ATM gene, which is mutated in the human genetic disorder ataxia telangiectasia (AT), has an important regulatory role in DNA damage response [47]. This kinase is activated in response to many types of DNA damage, and it in turn activates other proteins responsible for cell cycle regulation and DNA repair. Individuals with AT are sensitive to certain DNA-damaging agents and exhibit a dramatically increased cancer risk. The ATM kinase phosphorylates a number of proteins required for cell cycle checkpoints (e.g., p53) and DSB repair (e.g., NBS1). Loss of these functions results in genomic instability and increased cancer risk. Given the importance of DNA repair pathways in cancer prevention, it is reasonable to speculate that disruption of these pathways by exogenous agents could contribute to carcinogenesis as well. Such an agent would be expected to act

as a co-carcinogen, and enhance the mutagenic and carcinogenic activity of genotoxic carcinogens. A possible example of this type of co-carcinogen is arsenic [48]. Epidemiological studies show that arsenic exposure is strongly associated with the development of skin lesions, including skin cancers [49,50]. Elevated risk of other malignancies, such as bladder, lung, kidney, and liver carcinomas, is also associated with arsenic exposure. Tests of the mutagenic activity of arsenic in a variety of assay systems have generally been negative (with a few exceptions) [51]. However, in cell culture systems, arsenic has been shown to enhance the mutagenic activity of other carcinogenic agents. This enhancement of mutagenesis was shown to be associated with a suppression of DNA repair [48]. Recently, arsenic was shown to act as a co-carcinogen with UV radiation in the induction of skin tumors in the hairless mouse [52]. These results suggest that the carcinogenic activity of arsenic may be due in part to its ability to suppress DNA repair pathways. Certainly, it is possible that other environmental agents may increase cancer risk by similar mechanisms.

4. Mutators, cell proliferation, and cancer development how many mutations? Most solid tumors that have been studied cytogenetically appear to be genetically unstable; aneuploidy and chromosomal rearrangements are common features of tumor cells. It has been estimated that some tumor cells may have thousands of mutations. However, it is not clear whether genetic instability is a prerequisite for cancer development or whether it is a consequence of the cancer phenotype. Certainly, as discussed above, defects in cell cycle checkpoints and DNA repair pathways that increase genetic instability also increase cancer risk. Is induction of such a mutator phenotype an essential step in the carcinogenic process? The arguments on both sides of this question [53] depend on assumptions concerning the number of mutations required for cancer development and the rate of cell division. The argument in favor of this hypothesis [54] assumes that at least ve hits (mutations) are required for cancer development. Given a normal somatic mutation rate of about 106 per cell doubling, the probability of the ve independent hits occurring in a single cell is 1030 per cell doubling. Even considering that there are perhaps 1014 cells in the body and perhaps 50 cell divisions during the average life span, this leads to a calculated cancer risk of 1015 . Since cancer is much more prevalent than that, this suggests that an essential step in cancer development is an increase in mutation frequency. This increase could be due to an inhibition of any of the pathways that normally serve to maintain genomic stability. Whether disruption of normal mutation-avoidance pathways (e.g., DNA repair, DNA damage signaling pathways, etc.) is also responsible for such genomic instability remains to be demonstrated. Although it is clear that an increase in mutagenesis can promote cancer development, it

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has been argued that, at least in some highly proliferative tissues, induction of a mutator phenotype would not be a prerequisite for cancer development [3,7]; in these cases, enough cell divisions could occur within a stem cell population to allow for accumulation of mutations that provide a selective advantage and clonal expansion [55]. Thus, the counter argument invokes proliferation and selection of mutant cells. If the rst hit provides a growth advantage to the cell, this cell will proliferate, increasing the probability of a second hit within the expanded population. Either argument is consistent with the observed increase in cancer incidence with age a longer time and more cell divisions increase the probability of generating the mutator state or allowing for rounds of mutagenesis and clonal expansion. It has been postulated that stimulation of cell proliferation by tumor promoters, natural hormones, or as a result of cell injury can enhance the mutagenic effects of endogenous or exogenous genotoxic agents [56,57]. A wide variety of non-mutagenic agents that stimulate cell proliferation can increase cancer risk, perhaps through the enhancement of mutagenesis. The link between cell proliferation and mutagenesis has been demonstrated in mouse model systems. For example, a classical tumor promoter, phorbol 12-myristate 13-acetate (TPA), increased the frequency of benzo[a]pyrene-induced mutations in the non-selected lacI gene in the Big BlueTM mouse by promoting cell division in the damaged cell population [58]. Likewise, the mutagenicity of N-ethyl-N-nitrosourea was dramatically enhanced in the liver by partial hepatectomy, which stimulates cell proliferation [59]. It is possible that cell turnover caused by chronic infections, proliferation of target tissues induced by hormones, such as estrogen, or hyperplasia due to exposure to environmental agents, such as arsenic, may all enhance mutagenesis. In addition, such agents likely stimulate expansion of mutant cell populations that may have a selective advantage. Both these effects likely contribute to the link between stimulation of cell proliferation and increased cancer risk.

times leading to chromosome condensation. There appears to be a relationship between DNA methylation and histone modications; patterns of histone deactylation and histone methylation are associated with DNA methylation and gene silencing. Interestingly, these epigenetic changes in chromatin can also alter the sensitivity of DNA sequences to mutation, thus rendering genes more susceptible to toxic insult. The relationship of chromatic structure to gene expression, DNA repair, and mutagenesis is an important area for further study in carcinogenesis.

6. Concluding remarks Given the importance of preserving genomic stability in cancer prevention and the critical role that DNA damage signaling and repair pathways play in mutation avoidance, it may be important to ask whether these pathways may offer a potential site for intervention in the carcinogenic process. Many of the damage response pathways have only recently been identied, and aspects of these pathways remain to be elucidated. Although it is clear that protein kinases such as ATM are important in DNA damage sensing and activation of downstream effectors, it is not well understood how the various responses (i.e., cell cycle checkpoints, replication fork maintenance, DNA repair, etc.) are activated, and how these are coordinated to minimize mutagenesis. Indeed, in some cases, mutagenesis may be the lesser of the two evils when the alternative is wholesale cell death brought about by excessive DNA damage. In any case, it would seem reasonable to postulate that by somehow enhancing the mutation avoidance pathways, it might be possible to mitigate the carcinogenic effects of environmental agents. Perhaps, opportunities for intervention will be suggested as further research reveals the details of these elaborate cellular DNA damage response pathways.

Acknowledgements 5. Epigenetic mechanisms of gene activation and silencing A discussion of genetic alterations in human cancer would be incomplete without addressing the role of epigenetic phenomena in regulation of gene expression. While activation of proto-oncogenes and inactivation of tumor suppressor genes by mutations (base substitutions, deletions, DNA rearrangements, etc.) are certainly well documented, alterations in expression of cancer genes can also occur by epigenetic mechanisms [4,60,61] [62]. The most well-understood epigenetic mechanisms involve DNA methylation and histone acetylation, methylation, and phosphorylation. Demethylation of promoter regions at the CpG sequences can lead to over-expression of proto-oncogenes, and silencing of gene expression can occur as a result of hypermethylation, someThe helpful suggestions of Joseph R. Testa is gratefully acknowledged. This work was supported by grants R01-NS34782, P42-ES04908, and P30-ES06096 from the National Institutes of Health.

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