Mechanisms of tolerance to DNA damaging therapeutic drugs
Peter Karran
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK
Email: karran{at}icrf.icnet.uk
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Abstract
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The cytotoxic effect of many anticancer drugs relies on their ability to damage DNA. Drug resistance can be associated with the ability to remove potentially lethal DNA lesions. DNA damage tolerance offers an alternative route to resistance. In a drug-tolerant cell, persistent DNA damage has become uncoupled from cell death. Tolerance to some DNA damaging drugs is accompanied by inactivation of the cell's DNA mismatch repair pathway. This is widely acknowledged as the mechanism underlying resistance to methylating agents and to 6-thioguanine which produce structurally similar types of DNA damage. Defects in mismatch repair are also associated with resistance to numerous drugs that produce a wide variety of structurally diverse DNA lesions. Here I consider possible mechanisms by which mismatch repair might influence drug resistance and the extent to which loss of mismatch repair might be considered to confer a multidrug resistance phenotype.
Abbreviations: 2-AP, 2-aminopurine; DSB, double-strand break; O6-meGua, O6-methylguanine; MMR, mismatch repair; MSI, microsatellite instability; 6-meTG, 6-thiomethylguanine; PhIP, 2-amino 1-methyl-6-phenylimidazo [4,5-b]pyridine; 6-TG, 6-thioguanine.
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Introduction
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Human cells can avoid death after exposure to genotoxic agents by excising potentially lethal DNA adducts and DNA repair significantly influences both the development and the treatment of human cancer. Nucleotide excision repair (NER) removes bulky DNA adducts produced by ultraviolet light, by numerous chemical carcinogens and also by some therapeutic drugs. More specialized pathways, notably base excision repair (BER) and a specific DNA methyltransferase (MGMT) for DNA O6-methylguanine (O6-meGua) remove more subtle, but no less potentially hazardous, forms of DNA damage (for a review of DNA repair, see ref. 1). Because these DNA repair pathways all promote cell survival, the involvement of DNA mismatch repair (MMR) in killing cells with damaged DNA appears paradoxical. Nevertheless, it has become clear that functional MMR is required for the lethality of certain DNA lesions and its inactivation is accompanied by tolerance to the cytotoxic effects of some drugs. DNA damage tolerance offers an alternative survival strategy in which the potentially lethal DNA lesions are not excised from DNA. Instead, the persistent DNA damage has become uncoupled from cell death.
Mismatch repair is principally an editing function (for a review, see ref. 2). The relevant steps in the pathway are outlined in Figure 1
. Recognition and binding of a persistent DNA replication error or template/primer misalignment by hMutS
, an hMSH2/hMSH6 heterodimer, precedes an intervention by the hMutL
heterodimer comprising hMLH1 and hPMS2. Recognition by the hMutS
and hMutL
complexes is followed by excision of a section of the newly synthesized DNA strand that includes the mismatch. The incorrect sequence is replaced with a faithful copy and ligation completes repair. The hMSH2, hMSH6, hMLH1 and hPMS2 proteins are all essential for this branch of the MMR pathway. The importance of MMR in editing replication is exemplified by the mutator phenotype that accompanies inactivation of any one of these functions in human tumour cells. Microsatellite instability (MSI)the hallmark of mismatch repair deficiencyreflects the persistence of numerous uncorrected replication template/primer misalignments in repetitive microsatellite regions.

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Fig. 1. Mismatch repair in DNA replication and recombination. (Left) Replication mistakes at the replication fork generate mismatches in the form of improper base pairs that escape proofreading or small structural anomalies such as unpaired single or double base loops (light shaded loops). These aberrant structures are bound by the hMutS mismatch recognition complex. Mismatch recognition is followed by recruitment of hMutL and the excision and replacement of a tract of DNA that contains the incorrect base is initiated. Ligation of the newly synthesized DNA to the existing strand completes repair. Excision is targeted to the daughter DNA strand by an unknown mechanism. One model for methylation tolerance suggests that hMutS and hMutL processing of mispairs containing O6-meGua remains incomplete because the methylated, or `incorrect', base is in the template DNA strand. These incomplete processing events have a high probability of being lethal. (Right) The events depicted reflect the possible involvement of MMR in preventing recombinational repair of a DSB. The overall impact of MMR would, however, be the same for exchanges between any divergent or homologous DNA molecules. Recombinational exchanges are initiated by invasion of a DNA molecule by single strand tails generated by resection of the ends of a homologous duplex. The ends are bound by the hRad52 and hRad51 proteins that presumably catalyse the invasion and formation of the initial heteroduplex joint. When the recombining molecules are true homologs, the joint molecules are perfectly matched and extensive regions of heteroduplex may be formed. Rare mismatches may be corrected by MMR leading to a gene conversion event. In the case of significantly divergent, or homologous, molecules, numerous mismatches are created. These are processed by hMutS and hMutL and, if the density of mismatches is too high, the processing aborts the joint molecules. This may be due to a physical impediment to the formation of extensive heteroduplexes. Alternatively, the presence of multiple mismatch repair events may simply destabilize the existing heteroduplex regions of the initial joint molecule leading to its collapse. Inactivation of either hMutS or hMutL prevents processing and arrest of the recombination process is avoided.
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The hMutS
and hMutL
complexes initiate a similar editing function on recombination intermediates and it is well established that mismatch repair influences recombinational exchanges in prokaryotes and in mammalian cells. At one extreme, mismatch repair is an important barrier against recombination between species whose DNA molecules differ significantly in sequence (3). It prevents exchanges between DNA molecules from unrelated organisms by aborting or destabilizing the mismatched heteroduplexes that are intermediates in the process. MMR also monitors meiosis and the MutS
and MutL
mismatch repair complexes play an important role in both meiotic and mitotic recombination. Homozygous Mlh1 and Pms2 knockout mice are sterile and display defects in meiotic recombination (reviewed in ref. 2). MMR defects also affect mitotic recombination events. Abrogation of MMR facilitates gene targeting (4) and homologous recombination frequencies are significantly increased in MMR-defective mouse (5) and human (6,7) cells. The dramatic replication-related MSI phenotype in MMR-defective tumour cells has rather overshadowed this involvement of MMR in regulating recombination. Nevertheless, inactivation of MMR apparently permits the completion of normally proscribed recombinational exchanges (Figure 1
).
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Methylation tolerance
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Methylating agents are cytotoxic because they methylate DNA guanines. Any persistent DNA O6-meGua that is not demethylated by MGMT is processed by MMR. Processing requires both the hMutS
and hMutL
mismatch repair factors (8) and is lethal. Because O6-meGua is the major toxic DNA lesion produced by methylating agents, cells with hMSH2, hMSH6, hMLH1 or hPMS2 defects are, without exception, highly resistant to killing by these drugs (9). Inactivation of MMR uncouples the persistence of the potentially lethal O6-meGua from cell death and confers tolerance to methylating agents.
The mechanism of methylation tolerance remains incompletely defined. Two models have been put forward to explain the participation of MMR in cell death. In the first of these, cell death is a consequence of misguided attempts by the MMR system to correct base pairs containing damaged bases (8). On this model, engagement of MMR requires that the aberrant base pair exhibits a structural resemblance to mispaired unmodified bases. The second model proposes a new role for MMR as a general DNA damage detector. It suggests that the ability of hMutS
and hMutL
to interact with damaged bases has been adapted as an integral part of the process of programmed cell death (10). Abolition of MMR can therefore be regarded as conferring a multidrug resistance phenotype. There are two significant differences between these two models. The former suggests that only a limited range of DNA damagealtered bases that are replicated and miscode to produce base pairs resembling non-WatsonCrick pairswill be subject to MMR processing. No such limitations are implicit in the latter model and MMR is endowed with the ability to interact with any type of structural DNA perturbation. The second difference is really one of intent. The structure-based model implies that cell death is the consequence of a mistaken identification of damaged base pairs as a MMR substrate. On the general sensor model, cell death is a positive, intended outcome of the DNA damage/MMR interaction.
In this Commentary, I examine some of the evidence for these two models and suggest an alternative that combines elements of both and is compatible with most of the published data.
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Structure-related model
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Experiments in Escherichia coli dam mutants provided the initial evidence to implicate MMR in lethal processing of DNA O6-meGua (11). These were guided by similarities between the methylated base and base analogs, such as 2-aminopurine (2-AP) to which dam strains are hypersensitive (12). It was hypothesized that the structural features and ambiguous coding properties of O6-meGua resemble those of a base analog that is delivered to DNA by direct chemical modification rather than during DNA replication. The finding that a mutation in either mutS or mutL (the bacterial counterparts of hMutS
and hMutL
) abrogated the hypersensitivity of dam strains to 2-AP (13) and to MNNG (11) was consistent with this idea. O6-meGua is a lethal DNA lesion in dam strains but is tolerated if the MMR system is inactive. These experiments also showed that interaction with the E.coli MMR system was not a general feature of DNA damage and did not even extend to methylated bases other than O6-meGua. Mammalian cells with an analogous methylation tolerance derived from inactive MMR (8,14) also do not tolerate the N-methylated purines that are produced in DNA simultaneously with O6-meGua. Methylation tolerant mammalian cells are, however, generally cross-resistant to the base analog 6-thioguanine (6-TG). [6-TG but not 2-AP is extensively incorporated into DNA by mammalian cells]. The geometry of the 6-TG and O6-meGua bases is similar. The structural resemblance between O6-meGua and the methylated form of 6-TG, 6-thiomethylguanine (6-meTG), is even more striking. This rare thiomethylpurine is produced by infrequent in situ methylation of DNA 6-TG by endogenous S-adenosylmethionine. Thiomethylguanine-containing base pairs have been identified as the 6-TG DNA lesions that interact lethally with the human MMR system (15). Cross-resistance to DNA O6-meGua and 6-meTG implies that structural ambiguity and an ability to miscode during replication dictate the interaction between MMR and DNA modifications in mammalian cells as well as in bacteria. Tolerance to 6-TG can therefore be regarded simply as another example of methylation tolerance. O6-meGua and 6-meTG are so alike that they provide a compelling argument for a structural basis to the interaction between MMR and DNA damage. One significant advantage of DNA damage recognition based on structure is that it does not imply any new functions. It is simply MMR trying to do its normal job but on inappropriate substrates.
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Mismatch repair as a general DNA damage sensor
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Despite the appeal and simplicity of the structure-based arguments, it is generally considered that MMR can process numerous DNA lesions and mediate the cytotoxicity of many DNA damaging treatments (Table I
is adapted from two recent reviews, refs 16 and 17). The DNA adducts concerned are so disparate that they are unlikely to share common structural features. How do they trigger processing by MMR? Is it necessary to invoke MMR as a general damage detector?
Early experiments in E.coli again provide some clues. Escherichia coli dam strains are also hypersensitive to cisplatin (18). In an exact parallel with methylation tolerance, this sensitivity is partially reversed by a second mutation in either mutS or mutL. The crucial difference is that neither the predominant cisplatin intrastrand dipurinyl DNA adducts nor the minor interstrand cross-links bear any significant resemblance to a methylated base or to a base analog. Recent experiments have re-emphasized the role of recombinational repair in maintaining the viability of E.coli dam strains (19) and the wholesale involvement of recombinational repair in protecting E.coli from the toxic effects of cisplatin has been clarified (20). These observations suggest that cisplatin tolerance in E.coli mutS dam or mutL dam strains might reflect the involvement of MMR in editing recombination, rather than replication. Indeed, because MMR proteins can sabotage recombination-related processes in vitro (21), it was suggested that a role for MMR in editing recombination might underlie its involvement in methylation tolerance as well (22). However, in human cells, MMR defects have contrasting effects on homologous recombination induced by O6-meGua or by bulky DNA lesions. In the former case, MMR is actually required to induce recombination. In the case of bulky benzo[a]pyrene DNA adducts, homologous recombination is generally somewhat higher in the absence of MMR (23). Overall, the experimental data generally conform better to two partially separate pathways of DNA damage tolerance.
A second model organism, the yeast Saccharomyces cerevisiae, provides an unequivocal indication that lethal MMR processing of DNA damage must occur by more than one mechanism. MutS
- or MutL
-defective S.cerevisiae are more resistant than wild-type strains to cisplatin and to doxorubicin (24). Significantly, for resistance to these drugs, mismatch repair defects are epistatic with the RAD52 group of genes. This clearly implicates the homologous recombination pathway in resistance to DNA damage produced by cisplatin and doxorubicin. It is particularly noteworthy that MMR defective yeasts are not tolerant to methylating agents. Their MMR system does not process persistent DNA O6-meGua into a lethal DNA lesion and MNNG resistance in yeast is unaffected by mutations in components of either the MutS
or MutL
complexes (25). Thus, S.cerevisiae provides clear evidence for two distinct mechanisms of tolerance to DNA damage. The important finding that cisplatin and doxorubicin resistance involves homologous recombination is consistent with the involvement of recombinational repair in cisplatin (and other bulky DNA damage) in E.coli. Overall, the evidence supports the idea that tolerance to bulky DNA adducts involves the inactivation of the recombination editing function of MMR. This implies that MMR is preventing recombinational repair that would normally promote cell survival.
During replication, O6-meGua and 6-meTG both produce structures that resemble mismatches. Indeed, O6-meGua:T and 6-meTG:T base pairs are bound efficiently by hMutS
(22,26,27). In cell extracts, MMR can remove O6-meGua from the nicked strand of model DNA substrates (28). The biological relevance of this observation is questionable because O6-meGua would normally be in the uninterrupted template DNA strand. It does, however, provide evidence that hMutS
can successfully recruit hMutL
to O6-meGua-containing base pairs and initiate processing by the full MMR system. The hMutS
complex also binds to cisplatin (22,29), benzo[a] pyrene (30) or aminofluorene (31) DNA adducts. This has been adduced as evidence that MMR processes these bulky DNA lesions and methylated base pairs in a similar fashion. This is not necessarily the case. Although recognition by a mismatch binding complex is necessary for initiation of MMR processing, it is not sufficient. Despite indications that cells deficient in either hMutS
or hMutL
may be resistant to these structurally diverse agents, there is no direct evidence that recognition of the DNA lesions by hMutS
is followed by hMutL
recruitment and further processing. In other words, there is nothing to suggest that the primary damage induced by bulky DNA damaging compounds is processed by MMR.
There is a simple alternative. It is well established that bulky cisplatin (32), benzo[a]pyrene (33) or aminofluorene (34) DNA adducts are poorly bypassed by replicative DNA polymerases. In contrast, O6-meGua (35) and 6-meTG (15) have a greater tendency to miscode rather than to prevent progression of the replication fork. The most likely outcome of an arrested replication fork is the generation of a DNA double-strand break (DSB) (36). Recombination is an acknowledged route for DSB repair and bacteria, yeast and mammalian cells with genetic defects in recombination are extremely sensitive to bulky DNA damaging agents of this type. It seems plausible that involvement of MMR in the lethality of this type of replication-arresting DNA damage reflects its ability to edit, and to abort, intermediates generated during the recombinational repair of DSBs.
Thus, MMR promotes cell death by two replication-related mechanisms. In the first, MMR attempts to process the products of improper coding such as O6-meGua:T, and 6-meTG:T base pairs. The attempt is initiated because of their similarity to A:C or G:T mispairs. The second mechanism may be triggered by collapsed replication forks at non-instructional DNA lesions. It involves attempted recombinational repair of DSBs. In the second case, MMR may intervene to abort inappropriate intermediates in the DSB rejoining process. Participation of MMR in processing DSBsthe secondary DNA lesions generated by replication/recombinationabsolves MMR complexes of a requirement to act as general detectors of DNA damage. MMR does not directly `sense' structurally disparate primary DNA lesions. It recognizes instead a common intermediate in the processing of secondary DNA damage. The most likely candidate for this secondary damage is a DSB.
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Extent of DNA damage tolerance and possible confounding factors
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There is currently some confusion as to the extent to which MMR defects are causally involved in drug resistance. Firstly, it should be noted that there are many disadvantages in working with established cultures of MMR deficient cells. One obvious one is that a mutator phenotype allows cells to evolve rapidly. Maintenance of cells in vitro introduces a selective pressure for improved proliferative fitness that may inadvertently select for traits that modify drug resistance. Their high spontaneous mutation rate makes this a particular problem for MMR deficient cells. It is quite likely that, despite identical origins, the genotypes of established MMR deficient cells differ between laboratories.
Factors other than MMR clearly influence resistance to drugs. These factors affect the response to agents that produce bulky DNA damage to a greater extent than they influence resistance to methylation damage. This differential involvement may underlie some of the apparent discrepancies between MMR deficiencies and drug tolerance. The methylation resistance of MMR-defective cells is extensive (~50-fold). This degree of tolerance is a predictable outcome of MMR deficiency and exceptions are unknown (9). In general, MMR-associated resistance to damage other than O6-meGua (or 6-meTG) is relatively minor. Typical examples for MMR-related cisplatin resistance range from 1.3- to 2-fold (for a review, see ref. 16). Occasionally, MMR-defective cells do not appear to be particularly resistant to bulky DNA damaging drugs (37). It is probably significant in this regard that only two factorsthe level of MGMT expression and MMRare known to have a significant impact on resistance to methylating agents (38). Numerous other effectors of drug resistance have no significant influence on methylation sensitivity. These include p53 status (9,39), drug detoxification or activation pathways and relative drug influx/efflux. Thus, the possible differential involvement of these confounding factors in the two pathways of drug tolerance may underlie the rather unpredictable impact of MMR on non-methylating drug resistance.
The colorectal carcinoma cell line HCT116 provides a different example of a possible confounding factor. These cells are a widely used model for the effects of MMR defects. The hMLH1 gene of HCT116 is inactivated by mutation. The cells do not produce active hMLH1 protein and are completely MMR deficient. Consistent with this, HCT116 cells are methylation tolerant (40) and are resistant to 6-TG (41). HCT116 is a particularly favoured model because of the availability of the related HCT116 + Ch3 cells into which a normal copy of chromosome 3, bearing a wild-type copy of hMLH1, has been introduced (42). The presence of a functional copy of hMLH1 restores MMR activity. It reverses methylation tolerance and HCT116 + Ch3 cells are sensitive to MNNG and to 6-TG. The different methylation and 6-TG sensitivity of HCT116 and HCT116 + Ch3 cells almost certainly reflects their different hMLH1 status. These paired cell lines have been used to furnish support for the possible involvement of hMLH1 deficiency in drug tolerance (reviewed in ref. 16), in p73-mediated signalling (43,44), cell cycle checkpoints (41,45) and other phenotypic traits. Despite the `isogenic' nature of these cells, it is possible that some phenotypic properties are only indirectly, or sometimes not at all, related to the cells' MMR status. For example, HCT116 is one of the many MMR-defective colorectal carcinoma cell lines in which the Type II TGF-ß receptor is inactivated by a frameshift (46). TGF-ß is an important mediator of cell proliferation and loss of responsiveness to this growth factor is important during the development of colorectal and other tumours. Chromosome 3 contains the genes for both TGF-ß and the Type II TGF-ß receptor. In addition to complementing the MMR defect of HCT116 by providing a functional copy of hMLH1, introduction of this chromosome also restores the autocrine TGF-ß response loop (47). This is known to affect the sensitivity of cells to numerous DNA damaging drugs, and in particular to cisplatin (48,49). Restoration of MMR may indeed contribute to the increased cisplatin sensitivity of HCT116 + Ch3 cells. The possible contribution of other genes encoded by chromosome 3 cannot, however, be excluded. When MMR is completely restored in a variant of the A2780 ovarian carcinoma line by expression of a transfected hMLH1 cDNA, the resulting sensitization to cisplatin is extremely limited (37).
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How predictable are tolerance effects due to MMR defects?
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A critical test of the relevance of MMR in generating lethal DNA damage is whether treatment provides a selective pressure to allow the emergence of MMR-defective clones. This criterion provides strong support for a causal involvement of MMR in methylation (and 6-TG) sensitivity. Selection of numerous different cultured cell lines in vitro for resistance to methylating agents almost invariably isolates cells with MMR defects. Similarly, most cells selected for resistance to 6-TG that retain HPRT are tolerant to methylating agents (50) consistent with MMR defects. Methylating agents also provide a significant selective pressure in vivo. Treatment with the methylating agent procarbazine of human glioma xenografts established in athymic mice resulted in selection of resistant cells displaying a methylation tolerant phenotype. The tolerant cells were hMSH2-defective (51). Finally, mgmt-/- mlh1-/- mice are much more resistant to methylating agents than their mgmt-/- mlh1+/+ counterparts (52).
The evidence for a consistent causal involvement of MMR defects to resistance to other drugs is much less compelling. MMR defects are sometimes present in the selected cells and may be a factor in their drug resistance. In many cases, however, these are not the principal, or even a major, contributor to resistance. Selection of human cells for resistance to cisplatin and other non-methylating drugs almost invariably involves other alterations such as defects in the p53-mediated DNA damage response pathway, factors controlling cell death, such as Bax/Bcl-2, or modifications in drug uptake (for examples, see refs 5355). Significantly, a recent study of resistance profiles among the NCI panel of 60 cell lines that are used to evaluate anticancer drugs identified five as MMR deficient. As expected, all five were resistant to the methylating agent temozolomide. There was, however, no apparent correlation between MMR status and resistance to numerous other drugs, including cisplatin and doxorubicin (56). The same panel of cell lines provided a clear indication that mutant p53 status is a significant factor in cisplatin resistance (57). These findings underline the overall impression that MMR defects are unlikely to be a significant factor in resistance to most drugs. Along similar lines, although an epigenetically silenced hMLH1 gene is associated with resistance to cisplatin and doxorubicin in variants of the A2780 ovarian carcinoma cell line (54,58), the association is not necessarily causal. Instead, it reflects a pre-existing subpopulation of variant cells (59) in which a drug resistance-enhancing p53 mutation accompanies the inactive hMLH1 gene (37,60). Indeed, none of 12 independent cisplatin resistant variants selected from an A2780 cell population from which the p53-defective cells had been eradicated are MMR defective (Massey,A. and Karran,P., unpublished data). These apparently conflicting observations simply emphasize the multifactorial nature of resistance to non-methylating drugs. They do not preclude a contribution of MMR to cisplatin and doxorubicin toxicity in human tumour cells. They do indicate that the effects of MMR are likely to be minor compared with those of other acknowledged resistance mechanisms and are unlikely to provide a significant selective growth advantage following drug exposure. The experience with cisplatin accentuates the need for caution in attributing a significant causal role for MMR defects in resistance to non-methylating drugs.
This point is emphasized by the response of HCT116 + Ch3 cells to the heterocyclic amine 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Resistance to PhIP has been previously ascribed to MMR deficiency (61). HCT116 + Ch3 clones isolated for stable resistance to PhIP were not, however, MMR defective. The resistant cells exhibited a chromosome instability phenotype (rather than MSI) perhaps indicating that some other important control of genome stability had been abrogated (62). Significantly, the same HCT116 + Ch3 cells selected for MNNG resistance were defective in either hMutS
or hMutL
. As with cisplatin, a modest contribution of MMR defects to PhIP resistance is not ruled out by these data. It is clear, however, that in contrast to methylation or 6-TG this does not provide a significant selective advantage to promote the emergence of MMR-defective clones. For PhIP, as with other non-methylation DNA damage, other resistance factors are likely to predominate.
Most of the drugs involved in the studies described herecisplatin, procarbazine, doxorubicinare used in the clinic. One of the principal reasons for interest in DNA damage tolerance is its possible predictive value for clinical response. Overall, data from established cell lines indicate that there is really no convincing evidence to implicate MMR defects in a multidrug resistance phenotype. With the obvious exceptions of methylating agents and 6-TG, MMR status is unlikely to have predictive value for responsiveness of tumours to therapeutic DNA damaging drugs. This conclusion does not necessarily imply that drug resistant tumours will not have MMR defects. There is evidence that resistance in tumours can be associated with changes in expression of MMR genes, although this again seems to be somewhat unpredictable (63,64). These observations clearly have important clinical implications and there is every reason to pursue a full understanding of the relationship between MMR defects and drug resistance. Our models do seem to indicate, however, that drug resistance in these cases has not arisen by a simple selection and that the resistance may not be due to inactivation of MMR alone.
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Conclusion
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In summary, inactivation of MMR provides significant resistance to a limited range of DNA lesions. In the case of O6-meGua, methylation tolerance conferred by inactivation of MMR is extensive and predictable. To a large degree, this is also true for the closely related 6-meTG which might best be considered as a special example of methylation tolerance. Methylation tolerance fits a model in which MMR is activated to process aberrant base pairs that structurally mimic true mispairs but contain a methylated base. This occurs immediately after replication and the presence of the methylated base precludes a satisfactory resolution to this processing. The outcome of this unresolved processing is cell death. Inactivation of MMR confers a significant and predictable degree of resistance. Experiments in bacteria and yeast suggest that MMR may be implicated in processing the DSBs that arise as a secondary consequence of DNA damage introduced by bulky, non-methylating drugs. In this case, it is possible to infer a link between MMR defects and processing of these secondary lesions. Despite suggestions to the contrary (65), there is little evidence that MMR defects predictably confer significant levels of multiple drug resistance in human cells. The reason for this uncertainty is the multifactorial nature of much drug resistance. Indeed, early studies of methylation tolerance and development of models to explain it were greatly helped by the relative simplicity of the response of mammalian cells to methylating agents. Pragmatically, the inability to process DSBs by MMR may be associated with resistance to multiple DNA damaging agents. The trivial extent and the uncertainty of the effects observed suggest that, on present evidence, it is inadvisable to equate MMR defects with a multidrug resistance phenotype.
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Received July 12, 2001;
accepted August 29, 2001.