DNA Mismatch Repair and p53 Play Independent Roles in DNA Damage-Induced Apoptosis
DNA Mismatch Repair and p53 Play Independent Roles in DNA Damage-Induced Apoptosis
By Ming Zeng, MD, PhD, Latha Narayanan, MS, and Peter M. Glazer, MD, PhD
The dna mismatch repair (mmr) system has been thought to play a key role in maintaining genome integrity, primarily by correcting base pair mismatches and other DNA polymerase errors. However, when bacteria were used to study alkylation damage, it was found that MMR factors were implicated in the cellular response to DNA damage.1 A similar role for MMR-mediated cellular response to DNA damage also was found in mammalian cells. Cells deficient in MMR were found to have resistance to alkylating agents.2,3 This effect was subsequently observed to apply to a variety of DNA-damaging agents, including several used in cancer therapy, such as cis-platinum and temozolomide.4,5 On the basis of available data, we proposed a mechanism of cellular responses to alkylating agents via MMR-mediated cytotoxicity.
MMR-Mediated Cytotoxicity
To test whether other DNA-damaging agents, such as ionizing radiation (IR), also require MMR in cellular response, Fritzell and colleagues used different MMR-deficient cells to study the cellular response to high-dose rate IR.6 The clonogenic survival data suggest a role for the MMR factors MSH2, MLH1, and PMS2 in the cytotoxicity of IR;6 results showed a small but statistically significant increase in clonogenic survival after IR of MMR mutant cells compared to wild type. These studies were carried out in immortalized cell lines established from transgenic mice in which the MSH2, MLH1, and PMS2 genes were mutated by targeted disruption in mouse embryonic stem cells.7
Recently, DeWeese and associates, focusing on cells from MSH2 knockout mice, reproduced and extended the observations to show that at low-dose rates, the survival differences between wild-type and MSH2-deficient cells were even larger than the differences seen at high-dose rates.8 In addition, Zhang and colleagues found that IR-induced apoptosis was reduced in MSH2-nullizygous mouse embryo fibroblasts compared to wild type.9
MMR-Associated Apoptosis
On the basis of these results, a model was proposed that applies radiation-induced oxidized bases to the same MMR-mediated pathway of cytotoxicity as the alkylated bases. However, several other studies using MMR-deficient, human cancer-derived cell lines or immortalized MSH2-deficient mouse lines failed to find substantial or consistent differences in radiation response.7,10 These conflicting reports complicate the understanding of the mechanism of MMR-mediated cellular response to ionizing radiation.
The mechanism by which the MMR complex may influence damage response is not yet fully understood, and different hypotheses have been developed to interpret the available data. Two hypotheses focus on how the signal is initiated and mediated downstream, eventually resulting in cell death or successful repair of the lethal/sub-lethal damage. One proposes that the MMR complex recognizes base damage and then initiates a cycle of futile repair,11 leading to gaps and breaks that ultimately may signal apoptosis. The other hypothesis proposes that the recognition of damage by the MMR complex directly initiates a signal transduction pathway, thus triggering apoptotic pathways. Both hypotheses support the central role for MMR in cellular response to certain types of DNA damage, but debate how the initial signal starts.
The evidence supporting a central role for signal transduction in the MMR-mediated damage response includes the finding that MSH2 function is required in the DNA damage response for both oxidative agents and IR. The role of the MMR complex in the recognition and processing of oxidatively damaged bases has been suggested by the results of several studies. For example, Ni and coworkers observed binding of MSH2/MSH6 complexes to DNA containing 8-oxo-guanine,12 and DeWeese et al reported increased accumulation of 8-oxo-guanine in MSH2-deficient mouse cells.8 Two studies of mutagenesis in yeast also indicated a role for MMR in mutagenesis related to oxidative base damage.12,13 Previous studies of MMR-associated apoptosis have identified MSH2 and MLH1 as key mediators of the process. For example, MSH2-deficient cells exhibited reduced apoptosis after IR in two studies.10,11
In one study, simple overexpression of MSH2 or MLH1, but not PMS2, MSH3, or MSH6, induced apoptosis in human cells.11 These latter observations suggested a special role for MSH2 and MLH1 factors in the apoptotic response, raising questions as to the particular role of PMS2 and other factors in induced apoptosis. In addition, work by DeWeese et al implicated MSH2 in an exaggerated response to IR when delivered at low-dose rates.8 Zeng and associates showed that PMS2 also plays a role in the different effects of low-dose rate IR.14 These latter observations suggest, as above, that the effect of the low-dose rate IR is mediated via recognition and processing by the MutSa (MSH2/MSH6) and MutLa (MLH1/PMS) complexes, not simply by MSH2 or MLH1 alone. These results also directly demonstrate that PMS2 plays a role in damage-induced apoptosis, suggesting that formation and normal functioning of the MutSa and MutLa complexes are required for MMR-dependent, IR-induced apoptosis.
MMR-Mediated Cellular Response Pathway
To further define the MMR-mediated cellular response pathway, it was shown that the MSH2/MSH6 and the MLH1/PMS2 complexes are required for the phosphorylation of p53 at serines 15 and 392, following treatment of cells with alkylating agents.15 Consistent with this observation, an MLH1-dependent induction of p53 following IR was observed in human colon cancer cell lines,10 and a MMR-deficient lymphoblastoid cell line showed reduced accumulation of p53 following temozolomide exposure.16 MMR-dependent induction of p53 also was seen in response to a variety of carcinogens.17 These observations raise the important question of the proposed role of p53 in the MMR-mediated damage response pathway for a variety of reasons: p53 is mutated in a large number of human cancers and is involved in cell cycle regulation, transcription, and apoptosis.18,19 Evidence also implicates p53 as a central factor in the cellular response to IR, leading to cell cycle checkpoint activation and apoptosis.20 However, recent work using a series of Chinese hamster fibroblast and human lymphoblastoid cell lines suggests that the MMR-mediated apoptotic response to the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine may be independent of p53,21 which raises questions regarding the functional importance of MMR-dependent signaling of alkylation damage through p53 phosphorylation.
Irradiation-Induced Apoptosis Assays
To further examine the potential relationship between MMR and p53 and their putative interdependent role in the cellular response to IR, Zeng and colleagues used genetically well-defined cells to study the question. They interbred mice carrying targeted disruptions at the PMS2 and p53 loci to produce primary embryo fibro-blasts with defined genotypes at these loci (including wild type, p53 null, PMS2 null, and double null).14,22-24 First, the role of p53 in the MMR-mediated response of cells to IR was investigated using an apoptotic assay as an end point to study cellular response. Primary cells carrying targeted disruptions of p53 and/or the MutL homologue MMR gene PMS2 were used to perform IR-induced apoptosis assays. The results showed that deficiencies in either p53 or PMS2 genes were associated with reduced levels of IR-induced apoptosis compared to the wild type, and were consistent with roles for both factors in the cellular response to IR. In cells deficient in both p53 and PMS2, even lower levels of apoptosis were observed, indicating that MMR and p53 mediate IR-induced apoptosis via separate and apparently additive pathways.
Secondly, by extending these results to an examination of clonogenic survival, it was found that lack of PMS2 rendered cells more resistant to IR regardless of p53 status. These results indicate that the MMR-mediated apoptotic and cytotoxic response to IR does not depend on p53, and were consistent with a recent report that the MMR-mediated apoptotic response to N’-methy1-N’nitro-N-nitrosoguanidine is not dependent on p53.21 Moreover, a recent study in MSH2- and p53-deficient mice and mouse cells suggested that temozolomide-induced apoptosis mediated by MSH2 may proceed via both p53-dependent and p53-independent pathways.25 In addition, Wu and associates reported that the induction of p53 in response to selected carcinogens was dependent on functional MMR and that the MMR-dependent apoptotic response to chemicals was mediated through both p53-dependent and p53-independent pathways.17 Finally, a report from Hickman and Samson further supports the observation of p53-independent MMR-mediated pathways.21 Whether these results reflect inherent differences between cellular responses to IR vs. various chemicals or to differences in the sensitivities of the assays remains to be determined. Whether cell cycle checkpoint regulation is associated with MMR-mediated signaling through p53 also remains to be established.
Nevertheless, it has been shown that p53 phosphorylation is affected by certain MMR factors,15 and there does seem to be some cross-talk between the MMR and the p53 pathways. Although it is possible that some small degree of IR-induced apoptosis depends on this cross-talk, Zeng’s report suggests that the majority of the detectable IR-induced, MMR-dependent apoptosis and cytotoxicity is independent of it. IR generates a large number of lesions in DNA, including double-strand breaks, single-strand breaks, and a wide variety of base and sugar damage. It is likely that cell death from strand breaks is independent of MMR. However, IR-induced base damage, or at least some subset of it, may be subject to MMR recognition.6 On the basis of the emerging model for the alkylation damage-response pathway,26 it is further proposed that this recognition initiates a signal transduction pathway that leads to apoptosis. More and more data suggest that this pathway does not require p53, although MMR recognition of base damage may signal p53 for other purposes. Aside from p53, recent work suggests that MMR-associated signaling involves a number of other factors, including c-abl and p73.26 p73 is a homologue of p53, and one possibility is that MMR signals apoptosis following IR via a p73-dependent pathway. Such a role for p73 in the case of cis-platinum exposure was proposed.26
Conclusion
Aside from the well-established concept that p53 can mediate apoptosis following IR, emerging data also support the concept that MMR can trigger apoptosis in a p53-independent pathway. Additional research to more fully understand the MMR-mediated apoptotic signaling is very important, not only to enhance molecular understanding of DNA repair and apoptosis, but also for the future development of better and more effective cancer treatments. (Dr. Zeng is a Resident and Ms. Narayanan is a Research Associate in Therapeutic Radiology, and Dr. Glazer is Professor of Therapeutic Radiology and Genetics, Yale School of Medicine, New Haven, CT.)
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