The contribution of endogenous sources of DNA damage to the multiple mutations in cancer

There is increasing evidence that most human cancers contain multiple mutations. By the time a tumor is clinically detectable it may have accumulated tens of thousands of mutations. In normal cells, mutations are rare events occurring at a rate of 10(-10) mutations per nucleotide per cell per generation. We have argued that the mutation rates exhibited by normal human cells are insufficient to account for the large number of mutations found in human cancers, and therefore, that an early event in tumorigenesis is the development of a mutator phenotype. In normal cells, spontaneous and induced DNA damage is balanced by multiple pathways for DNA repair, and most DNA damage is repaired without error. However, in tumor cells this balance may be shifted such that damage overwhelms the repair capacity, resulting in the accumulation of multiple mutations. Our hypothesis is that multiple random mutations occur during carcinogenesis. The sequential mutations that are observed in some human tumors result from selective events required for tumor progression. We consider the possibility that endogenous sources of DNA damage, in particular oxidative DNA damage, may contribute to genomic instability and to a mutator phenotype in some tumors. Endogenous and environmental sources of reactive oxygen species (ROS) are abundant. In tumor cells, antioxidant or DNA repair capacity may be insufficient to compensate for the production of ROS, and these endogenous ROS may be capable of damaging DNA and inducing mutations in critical DNA stability genes. The possibility that oxidative DNA damage could be a significant source of the genomic instability characteristic of human cancers is exciting, because it may be feasible to modulate the extent of oxidative damage through antioxidant therapy. The use of antioxidants to reduce the extent of molecular damage by ROS could delay the progression of cancer.     

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.     

Too many mutants with multiple mutations

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These "multiples" appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts.     

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.     

Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair

The MPH1 gene from Saccharomyces cerevisiae, encoding a member of the DEAH family of proteins, had been identified by virtue of the spontaneous mutator phenotype of respective deletion mutants. Genetic analysis suggested that MPH1 functions in a previously uncharacterized DNA repair pathway that protects the cells from damage-induced mutations. We have now analyzed genetic interactions of mph1 with a variety of mutants from different repair systems with respect to spontaneous mutation rates and sensitivities to different DNA-damaging agents. The dependence of the mph1 mutator phenotype on REV3 and REV1 and the synergy with mutations in base and nucleotide excision repair suggest an involvement of MPH1 in error-free bypass of lesions. However, although we observed an unexpected partial suppression of the mph1 mutator phenotype by rad5, genetic interactions with other mutations in postreplicative repair imply that MPH1 does not belong to this pathway. Instead, mutations from the homologous recombination pathway were found to be epistatic to mph1 with respect to both spontaneous mutation rates and damage sensitivities. Determination of spontaneous mitotic recombination rates demonstrated that mph1 mutants are not deficient in homologous recombination. On the contrary, in an sgs1 background we found a pronounced hyperrecombination phenotype. Thus, we propose that MPH1 is involved in a branch of homologous recombination that is specifically dedicated to error-free bypass.     

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.     

Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Polkappa). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks.Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.     

Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.     

On the absence of mutations in nucleotide excision repair genes in sporadic solid tumors

In general, stochastic tumors show genomic instability associated with the proliferation of DNA point mutations, that is, a mutator phenotype. This feature cannot be explained by a dysfunctional mismatch repair alone, and indicates that nucleotide excision repair (NER) and/or base excision repair should be suppressed. However, mutations in NER genes are not causally implicated in the oncogenesis of sporadic solid tumors, according to the Cancer Gene Census at http://www.sanger.ac.uk/genetics/CGP/Census/. This brings up an apparent paradox: how to explain the recurrent non-existence in NER genes of somatic mutations causally related to cancer? In a recent study, we have shown that the origin of point mutations in cancer cell genomes can be explained by a structurally conserved NER with a functional disorder generated from its entanglement with a disabled apoptosis gene network. In the present study, we further characterize NER gene network properties and show that it has a highly connected architecture. This feature suggests that the absence of mutations in NER genes in sporadic solid tumors is a result of their participation in many essential cellular functions.     

X-ray induction of microsatellite instability at autosomal loci in human lymphoblastoid WTK1 cells

Many models of carcinogenesis posit that multiple genetic events are required for a normal cell to become cancerous. As the mutation rate of a single gene is in the range of 10(-8) to 10(-5) per cell division, a central question remains, how does a single cell acquire multiple mutations? One hypothesis, originally articulated by Loeb [10], proposed that some mutations may not be isolated events, but are associated with a mutator phenotype that leads to the occurrence of additional mutations elsewhere in the cellular genome. To test this hypothesis, we utilized a human lymphoblastoid cell line (WTK1) that is known to be hypermutable at the autosomal thymidine kinase (TK) locus. We isolated 139 independent clones which were selected for new TK mutations that arose either spontaneously or as the result of a single X-ray exposure of 1.5Gy. These clones were examined for second-site alterations in several microsatellite loci scattered throughout the genome using polymerase chain reaction (PCR) amplification followed by both denaturing gel electrophoresis and single-stranded conformational polymorphism (SSCP) analysis. Of these clones, 21 exhibited second-site mutations primarily involving loss of heterozygosity, 17 arose from irradiated cells whereas the remaining four arose from non-irradiated cells. We further examined the 17 clones which exhibited alterations specifically at the D16S265 locus; alterations at this site were associated with an enhanced frequency of mutations at other loci in the same region of chromosome 16q, but were not associated with additional mutations at other sites in the genome. Furthermore, new mutations arose in loci on 16q when these clones were propagated for 6 months in culture. Overall, these results support the hypothesis that radiation can induce a type of genetic instability which may facilitate the occurrence of multiple mutations throughout the genome in a small population of exposed cells. Furthermore, some cells may possess localized regions in the genome which are highly sensitive to the induction of instability.     

MSH2 missense mutations alter cisplatin cytotoxicity and promote cisplatin-induced genome instability

Defects in the mismatch repair protein MSH2 cause tolerance to DNA damage. We report how cancer-derived and polymorphic MSH2 missense mutations affect cisplatin cytotoxicity. The chemotolerance phenotype was compared with the mutator phenotype in a yeast model system. MSH2 missense mutations display a strikingly different effect on cell death and genome instability. A mutator phenotype does not predict chemotolerance or vice versa. MSH2 mutations that were identified in tumors (Y109C) or as genetic variations (L402F) promote tolerance to cisplatin, but leave the initial mutation rate of cells unaltered. A secondary increase in the mutation rate is observed after cisplatin exposure in these strains. The mutation spectrum of cisplatin-resistant mutators identifies persistent cisplatin adduction as the cause for this acquired genome instability. Our results demonstrate that MSH2 missense mutations that were identified in tumors or as polymorphic variations can cause increased cisplatin tolerance independent of an initial mutator phenotype. Cisplatin exposure promotes drug-induced genome instability. From a mechanistical standpoint, these data demonstrate functional separation between MSH2-dependent cisplatin cytotoxicity and repair. From a clinical standpoint, these data provide valuable information on the consequences of point mutations for the success of chemotherapy and the risk for secondary carcinogenesis.     

Negative clonal selection in tumor evolution

Development of cancer requires the acquisition of multiple oncogenic mutations and selection of the malignant clone. Cancer evolves within a finite host lifetime and mechanisms of carcinogenesis that accelerate this process may be more likely to contribute to the development of clinical cancers. Mutator mutations are mutations that affect genome stability and accelerate the acquisition of oncogenic mutations. However, mutator mutations will also accelerate the accumulation of mutations that decrease cell proliferation, increase apoptosis, or affect other key fitness parameters. These "reduced-fitness" mutations may mediate "negative clonal selection," i.e., selective elimination of premalignant mutator clones. Target reduced-fitness loci may be "recessive" (both copies must be mutated to reduce fitness) or "dominant" (single-copy mutation reduces fitness). A direct mathematical analysis is applied to negative clonal selection, leading to the conclusion that negative clonal selection against mutator clones is unlikely to be a significant effect under realistic conditions. In addition, the relative importance of dominant and recessive reduced-fitness mutations is quantitatively defined. The relative predominance of mutator mutations in clinical cancers will depend on several variables, including the tolerance of the genome for reduced-fitness mutations, particularly the number and potency of dominant reduced-fitness loci.     

Mutator Mutations Enhance Tumorigenic Efficiency across Fitness Landscapes

Background

Tumorigenesis requires multiple genetic changes. Mutator mutations are mutations that increase genomic instability, and according to the mutator hypothesis, accelerate tumorigenesis by facilitating oncogenic mutations. Alternatively, repeated lineage selection and expansion without increased mutation frequency may explain observed cancer incidence. Mutator lineages also risk increased deleterious mutations, leading to extinction, thus providing another counterargument to the mutator hypothesis. Both selection and extinction involve changes in lineage fitness, which may be represented as “trajectories” through a “fitness landscape” defined by genetics and environment.

Methodology/Principal Findings

Here I systematically analyze the relative efficiency of tumorigenesis with and without mutator mutations by evaluating archetypal fitness trajectories using deterministic and stochastic mathematical models. I hypothesize that tumorigenic mechanisms occur clinically in proportion to their relative efficiency. This work quantifies the relative importance of mutator pathways as a function of experimentally measurable parameters, demonstrating that mutator pathways generally enhance efficiency of tumorigenesis. An optimal mutation rate for tumor evolution is derived, and shown to differ from that for species evolution.

Conclusions/Significance

The models address the major counterarguments to the mutator hypothesis, confirming that mutator mechanisms are generally more efficient routes to tumorigenesis than non-mutator mechanisms. Mutator mutations are more likely to occur early, and to occur when more oncogenic mutations are required to create a tumor. Mutator mutations likely occur in a minority of premalignant lesions, but these mutator premalignant lesions are disproportionately likely to develop into malignant tumors. Tumor heterogeneity due to mutator mutations may contribute to therapeutic resistance, and the degree of heterogeneity of tumors may need to be considered when therapeutic strategies are devised. The model explains and predicts important biological observations in bacterial and mouse systems, as well as clinical observations.     

Mutational clusters generated by non-processive polymerases: A case study using DNA polymerase β in vitro

Available DNA mutational spectra reveal that the number of mutants with multiple mutations (“multiples”) is usually greater than expected from a random distribution of mutations among mutants. These overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity DNA synthesis. Excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the DNA polymerases of phages T4 and RB69 in vitro. In the simplest case of a purified polymerase, non-random clusters may be generated by a subfraction of phenotypic variants able to introduce more errors per cycle of DNA synthesis than the normal enzyme. According to this hypothesis, excess multiples are not expected with non-processive polymerases even if they harbor rare mutator variants. DNA polymerase β (Pol β) is a mammalian DNA-repair polymerase with very low processivity. Although several Pol β mutational spectra have been described, there is conflicting evidence on whether or not excess multiples occur, with spectra based on the HSV-tk system tending to show excess multiples. Excess multiples generated by Pol β or any of its mutants might imply that the excesses of multiples observed in numerous other systems, especially those with processive polymerases, could be artifactual. Here, the distributions of mutations generated by native and recombinant rat Pol β and by the Pol β^Y265C mutator were analyzed in the M13mp2 lacZα system. Our results present no evidence for a significant excess of multiples over the expected numbers with any of the Pol β enzymes tested in this system. The reported excess of Pol β-generated multiples in the HSV-tk system may reflect a reduced efficiency of detection of base substitutions that cause weak phenotypes, which in turn may artifactually increase the frequency of multiples.     

Too Many Mutants with Multiple Mutations

ABSTRACT

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These “multiples” appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts.     

Bacterial stationary-state mutagenesis and Mammalian tumorigenesis as stress-induced cellular adaptations and the role of epigenetics

Mechanisms of cellular adaptation may have some commonalities across different organisms. Revealing these common mechanisms may provide insight in the organismal level of adaptation and suggest solutions to important problems related to the adaptation. An increased rate of mutations, referred as the mutator phenotype, and beneficial nature of these mutations are common features of the bacterial stationary-state mutagenesis and of the tumorigenic transformations in mammalian cells. We argue that these commonalities of mammalian and bacterial cells result from their stress-induced adaptation that may be described in terms of a common model. Specifically, in both organisms the mutator phenotype is activated in a subpopulation of proliferating stressed cells as a strategy to survival. This strategy is an alternative to other survival strategies, such as senescence and programmed cell death, which are also activated in the stressed cells by different subpopulations. Sustained stress-related proliferative signalling and epigenetic mechanisms play a decisive role in the choice of the mutator phenotype survival strategy in the cells. They reprogram cellular functions by epigenetic silencing of cell-cycle inhibitors, DNA repair, programmed cell death, and by activation of repetitive DNA elements. This reprogramming leads to the mutator phenotype that is implemented by error-prone cell divisions with the involvement of Y family polymerases. Studies supporting the proposed model of stress-induced cellular adaptation are discussed. Cellular mechanisms involved in the bacterial stress-induced adaptation are considered in more detail.     

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.     

Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations

Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.