Significance of multiple mutations in cancer

Keith R. Loeb and Lawrence A. Loeb1

Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195 and the Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA


    Abstract
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
There is increasing evidence that in eukaryotic cells, DNA undergoes continuous damage, repair and resynthesis. A homeostatic equilibrium exists in which extensive DNA damage is counterbalanced by multiple pathways for DNA repair. In normal cells, most DNA damage is repaired without error. However, in tumor cells this equilibrium may be skewed, resulting in the accumulation of multiple mutations. Among genes mutated are those that function in guaranteeing the stability of the genome. Loss of this stability results in a mutator phenotype. Evidence for a mutator phenotype in human cancers includes the frequent occurrence of gene amplification, microsatellite instability, chromosomal aberrations and aneuploidy. Current experiments have centered on two mechanisms for the generation of genomic instability, one focused on mutations in mismatch repair genes resulting in microsatellite instability, and one focused on mutations in genes that are required for chromosomal segregation resulting in chromosomal aberrations. This dichotomy may reflect only the ease by which these manifestations can be identified. Underlying both pathways may be a more general phenomenon involving the selection for mutator genes during tumor progression. During carcinogenesis there is selection for cells harboring mutations that can overcome adverse conditions that limit tumor growth. These mutations are produced by direct DNA damage as well as secondarily as a result of mutations in genes that cause a mutator phenotype. Thus, as tumor progression selects for cells with specific mutations, it also selects for cancer cells harboring mutations in genes that normally function in maintaining genetic instability.


    Introduction
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
Mutations are not only a hallmark of cancer but may be central to how cancers evolve. Cancer cells divide where normal cells do not; they invade, metastasize and kill the host of origin. The facts that cancer is inheritable at the cellular level and that cancer cells contain multiple mutations, suggest that tumor progression is driven by mutagenesis. Molecular techniques are progressively making it easier to dissect the human genome, from chromosomes down to genes and ultimately nucleotide sequences. With each deeper level of exploration, more and more mutations are being documented in cancer cells. The emerging concept is that genomes of cancer cells are unstable, and this instability results in a cascade of mutations some of which enable cancer cells to bypass the host regulatory processes that control cell location, division, expression, adaptation and death. Genetic instability is manifested by extensive heterogeneity of cancer cells within each tumor. In addition, tumors invariably develop resistance to chemotherapeutic agents. Each of the tumor phenotypes involves, or can be mimicked by, specific mutations introduced in critical genes. These mutations either arise from copying unrepaired DNA damage or from errors committed during DNA synthesis. In this review we will consider the generation of mutations in human cells, the multiplicity of mutations present in human tumors, and the relationship of multiple mutations to tumor progression. Recent studies have suggested two pre-eminent mechanisms for the generation of mutations in cancer cells, one involving deficits in DNA repair and one involving deficits in chromosomal partitioning during cell division. We will consider the hypothesis that there are thousands of mutations in cancer cells and that there are many mechanisms for the generation of a mutator phenotype in cancer cells.


    Mutations result from DNA damage
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 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
It has become increasingly recognized that, rather than being inert, cellular DNA undergoes continuous damage and resynthesis. DNA is damaged by both environmental and cellular (endogenous) sources. Many of the environmental agents that damage DNA have been demonstrated to be mutagens. Epidemiologic data indicate that many of these agents are also human carcinogens. The association between environmental chemicals and human cancers was most definitively established in situations where small groups of individuals have been exposed to an inordinately high concentration of a specific chemical that elicited a rare tumor. DNA damage by chemicals can be divided into two categories: (i) those that produce large bulky adducts and are repaired by the nucleotide excision pathway; and (ii) those that cause small alterations, such as alkylating agents that add methyl and ethyl groups onto nucleotides in DNA, and are repaired by the base excision repair pathway. In addition to DNA damage by environmental agents, Ames et al. (1) have emphasized that many foods contain natural chemicals that also damage DNA and produce similar alterations. Moreover, it has been argued that consumption of natural DNA-damaging chemicals from foods is much greater than our exposure to DNA-damaging chemicals produced by industry (2). Cellular metabolic processes also generate reactive chemical intermediates with the potential to damage DNA and, as a result, might also be a source of spontaneous mutations and cancers (3). Even water has the potential to damage DNA, causing hydrolysis of the glycosylic bond and the formation of mutagenic abasic sites in DNA. Based on rates of depurination of DNA in vitro, it has been estimated that each cell undergoes 10 000 depurinations per day (4). DNA damage by reactive oxygen species has been estimated to occur at an equally high frequency (5,6), and many of the damaged bases, such as 8-oxo-deoxyguanosine, miscode (79). Even though deamination of cytosine to thymidine is less frequent than oxidative damage to DNA (10), the result is a change in nucleotide sequence in the DNA. Mechanisms have evolved to repair each DNA lesion, but considering the high frequency at which they occur and the compact and inaccessible structure of human chromatin, it is likely that a significant fraction of these lesions escape DNA repair and produce mutations during replication of the unrepaired DNA by DNA polymerases (Figure 1Go). In general, DNA polymerases copy past small alterations such as methyl or ethyl groups with high efficiency, and thus are likely to produce mutations depending on the miscoding potential of the altered nucleotide (11). Bulky adducts are not easily bypassed by normal cellular polymerases and mutagenesis is dependent on the induction of damage-inducible error-prone pathways frequently involving special error-prone DNA synthesis (12). Taken together, these results suggest that the nucleotide sequence of cellular DNA is maintained at a homeostatic equilibrium, such that an increase in the production of DNA damage or a reduction in DNA repair results in an increased frequency of mutations (Figure 1Go).



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Fig. 1. Factors governing mutation accumulation in cancer cells. A variety of agents have the potential to damage DNA. Mutations are generated by DNA damage that escapes the multiple mechanisms for DNA repair. Thus, there is a dynamic equilibrium between DNA damage and DNA repair and this equilibrium is perturbed in cancer cells.

 

    Mutations arising from misincorporation by DNA polymerases
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
Mutations can also result from nucleotide misincorporation by DNA polymerases in copying non-damaged DNA templates during DNA replication or even during DNA repair synthesis. Studies on the fidelity of DNA synthesis indicate that purified DNA polymerases incorporate non-complementary nucleotides at rates much greater than one would predict based on the rare spontaneous mutations in human cells. The error rates of purified mammalian DNA polymerases in vitro thus far documented (13) vary from one in 5000 for DNA polymerase ß, the enzyme responsible for the synthesis of short segments of DNA during repair synthesis, to one in 10 000 000 for DNA polymerases {delta} and {varepsilon}, enzymes involved in DNA replication (14,15). The latter polymerases contain a 3'->5' exonucleolytic activity that excises non-complementary nucleotides immediately after misincorporation by the polymerase. The frequencies of misincorporation by any DNA polymerase can vary as much as 100-fold as a result of the nucleotide sequence in DNA (16). DNA polymerases pause at sites that have the potential to form secondary structures and these pause sites have been shown to be loci for enhanced misincorporation (17). Blocks to accuracy could be minimized by replication proteins that function in concert with DNA polymerase in DNA replication and/or DNA repair. In addition, misincorporated nucleotides are excised by the mismatch repair system (18) and this system may interact with a variety of adducted nucleotides in DNA (19). Thus, there are multiple systems with overlapping specificities for the repair of DNA.

There is increasing evidence for the interchangeability of DNA polymerases (20) and thus mutation rates might be altered by varying the relative expression of different DNA polymerases (21) as well as by mutations that render these enzymes error-prone (22). The increase in DNA polymerase ß in certain tumors suggests the possibility that polymerase ß could substitute for a more accurate DNA polymerase, resulting in increased mutagenesis (23). In Escherichia coli, DNA damage results in the induction of an error-prone response referred to as the SOS pathway (24). It has long been suspected that this pathway facilitates the bypass of blocking lesions during DNA replication through induction of proteins that render the normal DNA polymerases error-prone. Recently, one of the proteins involved in the SOS response, UmuD'2C, has been identified as an error-prone DNA polymerase that was designated as Pol V (25,26). Moreover, a similar error-prone DNA polymerase has been identified in yeast (27) and found to be mutated in Xeroderma pigmentosum variant cells (XP-V) (28,29). Conceivably, error-prone DNA polymerases are elevated in certain cancers and contribute to the accumulation of DNA mutations in these tumors.

The aforementioned results lead one to conclude that mutation rates in cells are maintained at a homeostatic equilibrium in which the extensive DNA damage is counterbalanced by multiple mechanisms for DNA repair (Figure 1Go). In normal cells, only infrequently do lesions in DNA escape the screen provided for by DNA repair and direct misincorporation at the time of DNA synthesis. In eukaryotic cells there is the emerging concept of checkpoints in which cells sterilize the DNA immediately prior to the onset of DNA replication (30). Mutations in G1/S checkpoint genes allow DNA replication in the presence of unrepaired lesions (31) and result in enhanced mutagenesis.


    Multiple chromosomal aberrations in many human cancers
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
One of the hallmarks of cancer cells is genetic instability. This is manifested both at the single nucleotide level, resulting in point mutations, or at the chromosomal level resulting in translocations, deletions, amplifications and whole chromosome aneuploidy. Both of these forms of genetic instability can lead to a mutator phenotype via altered protein expression, function or gene dosage effects. There is a growing debate in the literature whether single nucleotide changes or large macromolecular chromosomal abnormalities are more prevalent in cancers or constitute a causative factor in cancer formation (32). Aneuploidy, a change in chromosome number, is a defining characteristic of many human cancers and is one of the most frequent changes observed in cancer. Since each chromosome contains thousands of genes, it is amazing that changes in chromosome number are compatible with cellular viability. Specific chromosomal exchanges occur at high frequencies and are diagnostic of certain tumors. In general, there is a positive correlation between the number of chromosomal alterations within a tumor and the malignant potential of that cancer. This suggests that a progressive diminution in chromosome maintenance pathways leads to an increasing number of errors during carcinogenesis, and promotes the development of highly anaplastic tumors. Chromosomal changes visible by light microscopy must involve deletions, additions or translocations of DNA segments that span millions of nucleotide bases. Conceivably, these visible chromosomal aberrations are the tip of an iceberg: hidden amongst these large rearrangements could be an even greater number of smaller changes in the nucleotide sequence of DNA in tumor cells.


    Cancer cells exhibit a mutator phenotype
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
The large number of chromosomal aberrations and the striking heterogeneity of tumors suggest that cancer cells are genetically unstable. Two overlapping mechanisms were initially and independently proposed to account for this instability. First, it was proposed that cancer cells exhibit a mutator phenotype based on the postulate that there was an increase in the rate of errors in DNA synthesis during tumor cell division (33). These errors could arise from mutations in DNA polymerases that render them error-prone, or from mutations in DNA repair proteins that render them defective. Mutations arising from either process would occur throughout the genome. Some of these mutations would be in additional genes normally required for maintaining genetic stability. As a result, there would ensue a cascade of mutations as tumor cells undergo successive cell divisions. Second, in 1976, Nowell analyzed the sequence of chromosomal aberration in tumors, and proposed a model for mutation accumulation based on successive waves of clonal selection (34). Recent studies suggest the possibility that these models are not only tightly coupled but may be interdependent (vide infra).


    Evidence for a mutator phenotype in human cancers
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
The hypothesis that cancer cells exhibit a mutator phenotype predicts that by the time cancers are detected clinically, they already contain enormous numbers of mutations. Recent evidence indeed indicates that human cancers harbor thousands of mutations. Perhaps the earliest hint of the large number of mutations in tumor cells were studies indicating that gene amplification in tumor cells accounts for the rapid emergence of their resistance to the drug PALA (35). Amplification of resistant genes was undetectible (<10–9) in primary diploid cells but was observed at frequencies as large as 10–3 to 10–4 in transformed cells derived from a variety of tumors. The extensiveness of point mutations in tumor cells was initially delineated by studies on microsatellite instability in colon cancer. Perucho and colleagues, using arbitrary primers for PCR amplification, detected multiple bands at different loci of DNA from colon tumors but not from adjacent normal cells (36,37). Based on the changes detected in the limited number of nucleotide sequences that were sampled, these authors calculated that the tumors they examined contained more than 100 000 mutations (38). The mutations these authors observed were in microsatellites, repetitive nucleotide sequences located between genes. However, it is becoming increasingly apparent that repetitive sequences are also present within genes and also undergo expansion or contraction specifically in cancer cells at very high frequencies (39,40). Contraction or expansion of repeats within genes provides an attractive mechanism for inactivation of tumor suppressor genes during tumor progression. High levels of microsatellite instability are especially prominent in hereditary non-polyposis colon cancer (HNPCC). In this syndrome, mutation in one allele of the genes involved in DNA mismatch repair is inherited (41) and loss of the second wild-type allele occurs with high frequency. In other cancers that exhibit expansion or deletion of repetitive sequences, diminished DNA repair appears to be mediated by methylation with reduced expression of a least one of the genes involved in mismatch repair (42,43).

It has been assumed that the changes in the length of repetitive sequences are mediated by slippage of DNA polymerases. We recently reported that damage to plasmids containing repetitive sequences by reactive oxygen species results in enhanced slippage specifically within the repetitive sequences suggesting a role of oxygen free radicals in enhancing microsatellite instability (44). In this system the preference for the induction of frameshifts in microsatellite sequences by reactive oxygen species is 70-fold greater than in non-repetitive sequences. It is now important to establish if microsatellite instability in tumors results from DNA damage by reactive oxygen species. If so, this instability might then be diminished by drugs that scavenge reactive oxygen species; this approach may be particularly relevant for tumor prevention in HNPCC families.

Microsatellite instability in tumors may be a harbinger for other types of mutations that are also present in tumors in large numbers. Tumors that exhibit microsatellite instability frequently contain alterations in the lengths of repetitive sequences within a variety of cancer-associated genes including: APC, IGF, TGF-ß, hMSH3 and hMSH6 (39,40). Tissue-culture cells defective in mismatch repair also exhibit elevations in non-selectable genes such as hgprt and oubain resistance (45). Nearly all breast (46) and ovarian tumors (47) studied utilizing comparative genomic hybridization have been shown to contain multiple changes in gene copy number. This technique only scores for changes that involve thousands of nucleotides. Studies using PCR-amplified gene fragments have revealed as many as 56 regions that exhibit loss of heterozygosity in breast cancer (48). This technique scans only a minute fraction of the genome and thus a much larger number of alterations are likely to be present throughout the genome. From these data we have calculated that a tumor with 50 regions of loss of heterozygosity detected by PCR-amplification of a limited number of gene segments might contain as many as 100 000 other alterations throughout the entire genome (49).

Cell cycle checkpoints, apoptosis and the mutator phenotype
Alterations in the timing of cell-cycle phases provide another mechanism for the generation of a mutator phenotype. During cell-cycle progression there are several regulatory pathways that function as checkpoints and monitor the repair of damage before proceeding to the next stage in the cell cycle (30). If activated, these checkpoints function to transiently arrest cell-cycle progression so that damage can be repaired or that the proper assembly of cell components can be completed. The elimination of cell-cycle checkpoints is a facile way to develop a mutator phenotype. Mutations in p53, for example, result in decreasing the stringency of the DNA damage checkpoint, allowing cancer cells to replicate damaged DNA, and to accumulate mutations in daughter cells that lead to genomic instability (50). In the presence of unrepairable damage, apoptosis is triggered as a final means to halting the spread of mutations. Again, many cancers contain mutations that delay or prevent the apopotic response and thus promote the survival of genetically unstable malignant cells (51).

Chromosomal instability
Aneuploidy, frequently present in many cancers, presents another manifestation of genetic instability. Some argue that aneuploidy is the defining characteristic of cancer and serves as the predominant mode of genetic instability, since the majority of human cancers fail to exhibit detectable microsatellite instability (38,52). Although aneuploidy is an easily recognized feature of cancers, little is known about its development or the selective advantage it provides for the tumor. Presumably, altered gene dosage enhances genetic instability and thus represents another form of the mutator phenotype. Aneuploidy can arise from chromosomal fragmentation, translocation, amplification or non-disjunction (resulting in the mis-segregation of entire chromosomes). The generation of aneuploidy can be an early or even an initial event in the development of genetic instability. It is conceivable that progressive increases in aneuploidy represent a predominant pathway for the development of genetic instability and it is independent of the accumulation of other types of mutations.

There is evidence suggesting that aneuploidy first develops from a transient tetraploid intermediate that exhibits profound genetic instability and rapidly accumulates additional chromosomal abnormalities (53). To achieve a tetraploid intermediate, one requires defective mitosis or endoreduplication: two rounds of DNA synthesis without an intervening mitosis. For this reason and because of the presence of morphologically abnormal mitosis in tumors, many have predicted cancer cells to be defective in mitotic pathways. Lengauer et al. (54) studied a set of aneuploid colorectal carcinoma cell lines and demonstrated a pronounced chromosome instability (CIN) phenotype that encompassed gains or losses of chromosomes in excess of 10–2/cell/generation. Chromosomal instability was considered as a causative factor leading to aneuploidy rather than a result of aneuploidy, since diploid cell lines that were genetically modified to produce an aneuploid population did not exhibit chromosome instability. Fusion experiments between diploid and aneuploid cell lines suggested that the molecular basis underlying CIN is dominant. One mechanism for the development of chromosomal instability is a defective mitotic checkpoint, the regulatory pathway that monitors the assembly of the mitotic spindles. In support of this hypothesis, Cahill et al. (55) identified heterozygous mutations in hBub1, a protein that participates in the mitotic checkpoint, in 5% of the aneuploid cell lines that exhibited CIN. Since the heterozygous allele resulted in a chromosome instability phenotype, these investigators surmised that the mutation functioned in a dominant-negative manner inactivating the mitotic checkpoint. This prediction was supported by additional studies in which expression of mutant proteins was shown to alter the mitotic checkpoint in diploid cell lines.

The precise manner by which mutations in hBub1 promote aneuploidy has not been established. Bub1 was first identified along with several other mitotic checkpoint genes [Bub1–3 (56) and Mad1–3 (57)] in genetic screens for yeast genes required for pre-anaphase delay in response to spindle disruption. Additional studies suggest that Bub1 acts as a sensor that binds unattached kinetochores and delays the onset of mitosis until all chromosomes are properly attached to the mitotic spindle (58). The presence of unrepairable mitotic spindle damage normally activates a checkpoint-mediated cell-cycle arrest, which leads to cell elimination via apoptosis. Cells expressing the Bub1 mutant proteins have a compromised mitotic checkpoint and escape the subsequent apoptosis normally induced by severe spindle damage (58). The role of apoptosis in the elimination of cells arising from a defective mitosis is supported by studies in which the expression of Bcl-XL, an anti-apoptotic gene, is sufficient to induce polyploidy in a murine prolymphocytic cell line (59). That Bub1 is essential in higher eukaryotes is supported by the fact that homozygous Bub1 mutations are embryonic lethal in Drosophila (60). Cells from these larvae show severe mitotic abnormalities including accelerated exit from metaphase, chromosome missegregation and fragmentation.

Following a transient mitotic arrest in the presence of spindle damage, some cancer cells are able to resume the cell cycle by entering a second round of DNA synthesis leading to endoreduplication (two rounds of DNA synthesis without an intervening mitosis). Cells lacking functional gene products involved in the G1/S checkpoint such as p53 (61,62), pRb (62), p16INK4A (62) or p21Waf1/Cip1 (61,63), as well as cells that over-express myc (64) have been shown to undergo endoreduplication following a transient mitotic arrest in the presence of spindle damage.

Whether a mitotic checkpoint defect really plays a role in the development of aneuploidy has been questioned by two recent studies. In one, no mutations were detected in five additional mitotic checkpoint genes surveyed from the 19 aneuploid colorectal tumors described above (65). In the other, no hBub1 mutations were detected amongst 31 head and neck carcinomas (66). However, there are many proteins that participate in the segregation of chromosomes; mutations in any one of them can contribute to the formation of aneuploidy. A recent study by Lee et al. (67) detected mutations in p53, Bub1 or Mad3L (mitotic checkpoint) in four spontaneous thymic lymphomas arising in mice homozygous for Brca2 truncation. These authors further showed that a dominant-negative Bub1 can reverse a growth defect and initiate transformation in mouse embryonic fibroblasts homozygous for the Brca truncation. These combined results suggest that deficiencies in the mitotic checkpoint promote chromosomal aberrations, aneuploidy and transformation, but it may occur only in cells harboring other mutations.


    Types of genetic instability in cancer
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
It has been suggested that there are two major mechanisms for the generation of genetic instability (Figure 2Go). One mechanism involves mutations in DNA mismatch correction genes and is manifested by microsatellite instability. The other involves mutations in genes required for chromosomal segregation and is manifested by fragmentation of chromosomes and/or duplication and deletions of whole chromosomes, rather than by sequence changes involving only a few nucleotides. In the first mechanism, two allelic mutations are required to generate microsatellite instability. In the second mechanism, only one mutation may be required, since the dominant nature of chromosome instability phenotype observed in cell fusion (54) suggests that modification in only one allele can result in chromosomal instability. Thus, it could be argued that a mutator phenotype is more likely to be manifested by chromosomal instability than by point mutations. However, this dichotomy may be artificial and simply reflect the relative sensitivities of both assays and the difficulties inherent in detection of random point mutations other than changes in the lengths of microsatellites. Chromosomal alterations involve millions of nucleotides and can be readily demonstrated by cytogenetic techniques. There are hundreds of thousands of microsatellites in the human genome and each target microsatellite contains many nucleotides; slippage at any single position within a repeat sequence should result in a detectable change in its length after PCR amplification and gel electrophoresis. Thus, scoring for mutations in only a few microsatellite sequences may not be adequate for determining whether or not a specific tumor exhibits microsatellite instability. Mutations generated by genetic instability are likely to be random events and only a few would result in a sufficiently selective advantage to generate a clonal population of cancer cells. Methods have been developed to detect clonal mutations. However, we still lack methods for quantitating random nucleotide changes in DNA sequences in tumors, particularly when they involve only a few nucleotide substitutions.



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Fig. 2. Different pathways that could result in a mutator phenotype in tumor cells.

 

    Coupling of increased mutagenesis and clonal selection
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
Initially, two mechanisms, increased mutations and clonal selection, were invoked to account for a mutator phenotype in cancer. Recent experiments in bacteria (68) and modeling studies (69) provide evidence that both mechanisms are operative and, moreover, intradependent. Mao et al. (68) showed that the spontaneous mutation rate in bacteria progressively increases from 1/100 000 to 1/200 after four rounds of selection for mutations in different genes. Moreover, exposure to a mutagen followed by subsequent rounds of selection for different mutations resulted in a population that was 100% mutators. The inference is that with each round of selection, one not only selects for a particular mutation, such as resistance to rifamycin, but one also selects for mutations that increase the mutation rate throughout the genome, i.e. mutators. The progressive enrichment of mutator mutations with successive rounds of clonal selection may be relevant to tumor progression. As cancers expand, they encounter a series of restrictive blockades that limits further growth. These limitations to growth might include interference by surrounding tissues, reduced nutrition, reduced oxygen levels, need for growth factors, inadequate blood supply, etc. Each of these blockades might be overcome by mutations that provide a growth advantage and establish new clonal populations (Figure 3Go). With each round of selection there would be a `piggy-backing' of mutator mutants and increase in mutation frequency. At some step in tumor progression the mutator phenotype might be detrimental and be lost during clonal selection. However, the multiple mutations created would be persistent and present even in later-stage tumors.



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Fig. 3. Evolution of a tumor based on selection for mutator mutations. As cancers develop there is likely to be a series of barriers that prevent further growth. Only rare mutant cells within the tumor can overcome each of these barriers. Some of these mutant cells would be generated by mutations in genes required for the maintenance of genetic stability in normal cells. Thus, with each round of selection there would be a progressive enrichment for mutations in mutator genes.

 

    Future directions
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 
The findings that multiple mutations are present in human tumors brings forth a series of important questions: what is the origin of these mutations? If they result from random events, how can we develop quantitative assays for their assessment? Are they phenomena that parallel tumor progression or are they rate limiting for tumor progression? If they are rate limiting, can we reduce the rate of accumulation and thus prevent cancer by delay? Since it takes ~20 years from the time of exposure to a carcinogen to the development of a clinically detectable tumor, even a 2-fold decrease in the rate of tumor progression would reduce cancer death in adults (70).

So far, only a limited number of mutator genes have been identified based on their presence in inherited human diseases that exhibit a high proclivity towards the development of specific tumors, or based on the production of mutators in bacteria or in cell culture. Considering the large number of genes involved in DNA replication, recombination and DNA repair, and those required for the correct partitioning of chromosomes during cell replications, it seems likely that many other mutator genes will soon be discovered, and that some of these will have an important role in the pathogenesis of specific cancers (Figure 4Go). Interestingly, the concept of a mutator phenotype was based on mutations in DNA polymerases that render them error-prone (33); yet, we still have only fragmentary evidence for the presence of mutations in DNA polymerases in tumors (71). If a mutator phenotype is important in the development of cancer it is likely to be an early event. Mutations in many of the oncogenes found in different tumors are likely to occur later. As a result, we would argue that drugs directed against many cancer-associated genes are unlikely to reverse the malignant phenotype.



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Fig. 4. Timing of mutations during tumor progression. The mutator phenotype hypothesis proposes that genes required for the maintenance of genetic stability are among the early targets of DNA damage by carcinogens. Mutations in these genes would result in other mutations throughout the genome. Mutations in cancer associated genes would occur later.

 
DNA sequencing provides a powerful tool to establish whether specific mutations are present in a tumor. However, this and related technologies are inadequate for detecting random mutations within a cell population. New techniques are required to detect random mutations and to determine if they accumulate during tumor progression. Stratification of normal individuals based on accumulation of random mutations may be predictive of the development of certain tumors. Stratification of tumors on the basis of accumulated mutations may be predictive of malignant potential.


    Notes
 
1 To whom correspondence should be addressed Email: laloeb{at}u.washington.edu Back


    Acknowledgments
 
This work was supported by grants from the National Cancer Institute (R35 CA 39903, CA 80993) and the National Institute of Aging (AG 01751). K.R.Loeb is a research fellow of the College of American Pathologists.


    References
 Top
 Abstract
 Introduction
 Mutations result from DNA...
 Mutations arising from...
 Multiple chromosomal aberrations...
 Cancer cells exhibit a...
 Evidence for a mutator...
 Types of genetic instability...
 Coupling of increased...
 Future directions
 References
 

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Received September 8, 1999; accepted September 20, 1999.