An overview of the mechanisms of mutagenesis and carcinogenesis

Cancer is a genetic disease due to the accumulation of numerous mutations rendering the tumour cell insensitive to control by the local cellular environment and by the whole organism. Analysis of the frequency of appearance of human cancer as a function of age shows that between four and seven mutations in key genes are usually necessary to produce most human cancers. Interesting debates in the literature are concerned with the idea that normal mutation rates followed by selective advantage of mutated clones are enough to produce the numerous mutations found in human cancers. Alternatively, the mutator phenotype hypothesis is based on the idea that the normal mutation rates are insufficient to account for the multiple mutations found in tumours. It is, however, difficult not only to know this exact mutation frequency in cells but also to know the total number of cell divisions giving rise to a cancer. Therefore, during at least one step in the carcinogenic process, a mutator phenotype in target cells may occur due to mutations controlling the fidelity of DNA replication or DNA repair, the apoptosis pathways or the cell cycle checkpoint regulations. Among the multiple mutations found in human cancers such as gene amplification, chromosome alterations and translocations, point mutations are very important and the molecular mechanisms of their production are well documented. I will describe in detail the various mechanisms that a cell can use to produce point mutations due to lower fidelity in the DNA polymerisation step or to inefficient repair pathways. The presence of multiple mutations in human cancer is interesting not only in terms of understanding the carcinogenesis process in humans but also in eventually promoting strategies to decrease the efficiency of this process and to increase cancer therapy regimen.     

Overexpression of human NOX1 complex induces genome instability in mammalian cells

The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2(-/-) mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2(-/-) fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.     

Protein oxidation,UVA and human DNA repair

Solar UVB is carcinogenic. Nucleotide excision repair (NER) counteracts the carcinogenicity of UVB by excising potentially mutagenic UVB-induced DNA lesions. Despite this capacity for DNA repair, non-melanoma skin cancers and apparently normal sun-exposed skin contain huge numbers of mutations that are mostly attributable to unrepaired UVB-induced DNA lesions. UVA is about 20-times more abundant than UVB in incident sunlight. It does cause some DNA damage but this does not fully account for its biological impact. The effects of solar UVA are mediated by its interactions with cellular photosensitizers that generate reactive oxygen species (ROS) and induce oxidative stress. The proteome is a significant target for damage by UVA-induced ROS. In cultured human cells, UVA-induced oxidation of DNA repair proteins inhibits DNA repair. This article addresses the possible role of oxidative stress and protein oxidation in determining DNA repair efficiency – with particular reference to NER and skin cancer risk.     

Implications of genetic heterogeneity in cancer

DNA sequencing studies have established that many cancers contain tens of thousands of clonal mutations throughout their genomes, which is difficult to reconcile with the very low rate of mutation in normal human cells. This observation provides strong evidence for the mutator phenotype hypothesis, which proposes that a genome-wide elevation in the spontaneous mutation rate is an early step in carcinogenesis. An elevated mutation rate implies that cancers undergo continuous evolution, generating multiple subpopulations of cells that differ from one another in DNA sequence. The extensive heterogeneity in DNA sequence and continual tumor evolution that would occur in the context of a mutator phenotype have important implications for cancer diagnosis and therapy.     

Conditional coalescent trees with two mutation rates and their application to genomic instability

Humans have invested several genes in DNA repair and fidelity replication. To account for the disparity between the rarity of mutations in normal cells and the large number of mutations present in cancer, an hypothesis is that cancer cells must exhibit a mutator phenotype (genomic instability) during tumor progression, with the initiation of abnormal mutation rates caused by the loss of mismatch repair. In this study we introduce a stochastic model of mutation in tumor cells with the aim of estimating the amount of genomic instability due to the alteration of DNA repair genes. Our approach took into account the difficulties generated by sampling within tumoral clones and the fact that these clones must be difficult to isolate. We provide corrections to two classical statistics to obtain unbiased estimators of the raised mutation rate, and we show that large statistical errors may be associated with such estimators. The power of these new statistics to reject genomic instability is assessed and proved to increase with the intensity of mutation rates. In addition, we show that genomic instability cannot be detected unless the raised mutation rates exceed the normal rates by a factor of at least 1000.     

Oxygen as a friend and enemy: How to combat the mutational potential of 8-oxo-guanine

The maintenance of genetic stability is of crucial importance for any form of life. Prior to cell division in each mammalian cell, the process of DNA replication must faithfully duplicate the three billion bases with an absolute minimum of mistakes. Various environmental and endogenous agents, such as reactive oxygen species (ROS), can modify the structural properties of DNA bases and thus damage the DNA. Upon exposure of cells to oxidative stress, an often generated and highly mutagenic DNA damage is 7,8-dihydro-8-oxo-guanine (8-oxo-G). The estimated steady-state level of 8-oxo-G lesions is about 10³ per cell/per day in normal tissues and up to 10⁵ lesions per cell/per day in cancer tissues. The presence of 8-oxo-G on the replicating strand leads to frequent (10–75%) misincorporations of adenine opposite the lesion (formation of A:8-oxo-G mispairs), subsequently resulting in C:G to A:T transversion mutations. These mutations are among the most predominant somatic mutations in lung, breast, ovarian, gastric and colorectal cancers. Thus, in order to reduce the mutational burden of ROS, human cells have evolved base excision repair (BER) pathways ensuring (i) the correct and efficient repair of A:8-oxo-G mispairs and (ii) the removal of 8-oxo-G lesions from the genome. Very recently it was shown that MutY glycosylase homologue (MUTYH) and DNA polymerase λ play a crucial role in the accurate repair of A:8-oxo-G mispairs. Here we review the importance of accurate BER of 8-oxo-G damage and its regulation in prevention of cancer.     

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.     

Genomic Instability and Carcinogenesis: An Update

Cancers arise as a result of stepwise accumulation of mutations which may occur at the nucleotide level and/or the gross chromosomal level. Many cancers particularly those of the colon display a form of genomic instability which may facilitate and speed up tumor initiation and development. In few instances, a “mutator mutation” has been clearly implicated in driving the accumulation of other carcinogenic mutations. For example, the post-replicative DNA mismatch repair deficiency results in dramatic increase in insertion/deletion mutations giving rise to the microsatellite instability (MSI) phenotype and may predispose to a spectrum of tumours when it occurs in the germline. Although many sporadic cancers show multiple mutations suggesting unstable genome, the role of this instability in carcinogenesis, as opposed to the power of natural selection, has been a matter of controversy. This review gives an update of the latest data on these issues particularly recent data from genome-wide, high throughput techniques as well as mathematical modelling. Throughout this review, reference will be made to the relevance of genomic instability to the pathogenesis of colorectal carcinoma particularly its hereditary and familial subsets.     

Genomic instability in Multiple Myeloma-relevance for Clinical Outcome and Efficacy of Therapy

Genomic instability is a driving force in the natural history of blood cancers including multiple myeloma,an incurable neoplasm of immunoglobulin producing plasma cells that reside in the hematopoietic bone marrow. Long recognized manifestations of genomic instability in myeloma at the cytogenetic level include abnormal chromosome numbers (aneuploidy) caused by trisomy of odd-numbered chromosomes; recurrent oncogene-activating chromosomal translocations that involve immunoglobulin loci; and large-scale amplifications, inversions, and insertions / deletions (indels). Catastrophic genetic rearrangements that either shatter and illegitimately reassemble a single chromosome (chromotripsis) or lead to disordered segmental rearrangements of multiple chromosomes (chromoplexy) also occur. Genomic instability at the nucleotide level results in base substitution mutations and small indels that affect both the coding and non-coding genome. Distinctive signatures of somatic mutations that can be attributed to defects in DNA repair pathways, the DNA damage response or aberrant activity of mutator genes including members of the APOBEC family have been identified. Here we review recent findings on genomic stability control in myeloma that are not only relevant for myeloma development and progression, but also underpin disease relapse and acquisition of drug resistance in patients with myeloma.     

Mechanism of oxidative DNA damage repair and relevance to human pathology

Since DNA is prone to oxidative attack cells have evolved multiple protective strategies to prevent the deleterious effects of DNA oxidation. Base excision repair is the major mechanism for repair of DNA base damage by reactive oxygen species but recent evidence indicate that nucleotide excision repair proteins, that are mutated in human syndromes, are involved too. The mechanisms of repair dealing with the direct oxidation of DNA will be reviewed taking as prototype the oxidized base 7,8-dihydro-8-hydroxyguanine. The function of the individual repair components as inferred from model mice indicate that the ablation of two gene functions is mostly required to lead to accumulation of oxidative DNA damage, mutagenesis and cancer development. The recent identification of human diseases associated with mutations in oxidative damage repair show that defects in this pathway may lead to increased cancer but their major causative role seems to be in neurological diseases.     

Generation of mutator mutants during carcinogenesis

Mutations are rare in normal cells. In contrast, multiple mutations are characteristic in most tumors. Previously we proposed a "mutator phenotype" hypothesis to explain how pre-cancer cells may acquire large number of mutations during carcinogenesis. Here we extend the "mutator phenotype" hypothesis considering recently discovered biochemical activities whose aberrant expression may result in genome-wide random mutations. The scope of this article is to emphasize that simple random point mutations can drive carcinogenesis and highlight new emerging pathways that generate these mutations. We focus specifically on random point mutations generated by replication errors, oxidative base damage, covalent base modifications by enzymes, and spontaneously generated abasic sites as a source of mutator mutants.     

Mitochondrial genome instability in human cancers

Malfunction of mismatch repair (MMR) genes produces nuclear genome instability (NGI) and plays an important role in the origin of some hereditary and sporadic human cancers. The appearance of non-inherited microsatellite alleles in tumor cells (microsatellite instability, MSI) is one of the expressions of NGI. We present here data showing mitochondrial genome instability (mtGI) in most of the human cancers analyzed so far. The mtDNA markers used were point mutations, length-tract instability of mono- or dinucleotide repeats, mono- or dinucleotide insertions or deletions, and long deletions. Comparison of normal and tumoral tissues from the same individual reveals that mt-mutations may show as homoplasmic (all tumor cells have the same variant haplotype) or as heteroplasmic (tumor cells are a mosaic of inherited and acquired variant haplotypes). Breast, colorectal, gastric and kidney cancers exhibit mtGI with a pattern of mt-mutations specific for each tumor. No correlation between NGI and mtGI was found in breast, colorectal or kidney cancers, while a positive correlation was found in gastric cancer. Conversely, germ cell testicular cancers lack mtGI. Damage by reactive oxygen species (ROS), slipped-strand mispairing (SSM) and deficient repair are the causes explaining the appearance of mtGI. The replication and repair of mtDNA are controlled by nuclear genes. So far, there is no clear evidence linking MMR gene malfunction with mtGI. Polymerase gamma (POLgamma) carries out the mtDNA synthesis. Since this process is error-prone due to a deficiency in the proofreading activity of POLgamma, this enzyme has been assumed to be involved in the origin of mt-mutations. Somatic cells have hundreds to thousands of mtDNA molecules with a very high rate of spontaneous mutations. Accordingly, most somatic cells probably have a low frequency of randomly mutated mtDNA molecules. Most cancers are of monoclonal origin. Hence, to explain the appearance of mtGI in tumors we have to explain why a given variant mt-haplotype expands and replaces part of (heteroplasmy) or all (homoplasmy) wild mt-haplotypes in cancer cells. Selective and/or replicative advantage of some mutations combined with a severe bottleneck during the mitochondrial segregation accompanying mitosis are the mechanisms probably involved in the origin of mtGI.     

Genomic instability and cancer

Tumorigenesis can be viewed as an imbalance between the mechanisms of cell-cycle control and mutation rates within the genes. Genomic instability is broadly classified into microsatellite instability (MIN) associated with mutator phenotype, and chromosome instability (CIN) recognized by gross chromosomal abnormalities. Three intracellular mechanisms are involved in DNA damage repair that leads to mutator phenotype. They include the nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). The CIN pathway is typically associated with the accumulation of mutations in tumor suppressor genes and oncogenes. Defects in DNA MMR and CIN pathways are responsible for a variety of hereditary cancer predisposition syndromes including hereditary non-polyposis colorectal carcinoma (HNPCC), Bloom syndrome, ataxia-telangiectasia, and Fanconi anaemia. While there are many genetic contributors to CIN and MIN, there are also epigenetic factors that have emerged to be equally damaging to cell-cycle control. Hypermethylation of tumor suppressor and DNA MMR gene promoter regions, is an epigenetic mechanism of gene silencing that contributes to tumorigenesis. Telomere shortening has been shown to increase genetic instability and tumor formation in mice, underscoring the importance of telomere length and telomerase activity in maintaining genomic integrity. Mouse models have provided important insights for discovering critical pathways in the progression to cancer, as well as to elucidate cross talk among different pathways. This review examines various molecular mechanisms of genomic instability and their relevance to cancer.     

A biological network in Saccharomyces cerevisiae prevents the deleterious effects of endogenous oxidative DNA damage

In this study, we used Saccharomyces cerevisiae to identify a biological network that prevents the deleterious effects of endogenous reactive oxygen species. The absence of Tsa1, a key peroxiredoxin, caused increased rates of mutations, chromosomal rearrangements, and recombination. Defects in recombinational DNA double strand break repair, Rad6-mediated postreplicative repair, and DNA damage and replication checkpoints caused growth defects or lethality in the absence of Tsa1. In addition, the mutator phenotypes caused by a tsa1 mutation were significantly aggravated by defects in Ogg1, mismatch repair, or checkpoints. These results indicate that increased endogenous oxidative stress has broad effects on genome stability and is highly sensitive to the functional state of DNA repair and checkpoints. These findings may provide insight in understanding the consequences of various pathophysiological processes in regard to genomic instability.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Coordinated action of the Fanconi anemia and ataxia telangiectasia pathways in response to oxidative damage

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H(2)O(2)) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H(2)O(2)-induced oxidative stress requires the coordinated action of FANCD2 and ATM.     

Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions

Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome – mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.