To the editor:
The MMR system has evolved to increase the fidelity of DNA replication and homologous recombination1. MMR is also implicated in the processing of other types of DNA damage, as mammalian cells with defective MMR are tolerant to SN1 type methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine and to 6-thioguanine and cisplatin2.
Reports describing the differential sensitivity of MMR-proficient and -deficient cells to ionizing radiation raised some controversy, as MMR-deficient cells were found to be slightly more resistant to ionizing radiation in some laboratories3 but either equally4 or less resistant5 in others. The survival differences were also questioned, because MMR status was reported to affect the length of the G2-M checkpoint rather than cell viability6. A report by Brown et al.7 has reopened this discussion by describing the requirement of a functional MMR system for activating the S-phase checkpoint and signaling of ionizing radiation–induced damage.
The aforementioned studies used matched MMR-proficient and -deficient mouse or human cell lines. Given that the establishment of these lines involved long periods of growth in cell culture, and that the MMR-deficient cells have a mutator phenotype, we considered the possibility that the differential responses of these cells to DNA-damaging agents could be linked to phenotypic traits other than MMR. We therefore examined the response to ionizing radiation of the strictly isogenic 293T La+ and 293T Lα− cell pair, in which the MMR-proficient 293T Lα+ cells differ from the MMR-deficient 293T Lα− cells solely by expression of the MMR protein MLH1 (ref. 8) and in which switching the MMR status does not involve clonal selection.
We exposed the 293T Lα+ and 293T Lα− cells to ionizing radiation and monitored their viability and progress through the cell cycle for 72 h. Ionizing radiation arrested both cell types in G2-M after ∼20 h, and we observed no differences in clonogenic survival (data not shown). We also observed no MMR-dependent differences in phosphorylation of the checkpoint kinases CHK1 and CHK2 (activation of which is required for triggering the arrest; Fig. 1a), of NBS1 (data not shown) or of BRCA1 (implicated in the processing of ionizing radiation-induced strand breaks; Fig. 1a). Thus, the MMR status per se did not affect DNA damage signaling in these cells.
We observed no MMR-dependent differences in early post-translational modification of CHK2 in other matched MMR-proficient and -deficient cell line pairs (Fig. 1b), some of which were also used by Brown and colleagues7. When we measured radiation-resistant DNA synthesis in some of these cell lines, we also observed no MMR-dependent differences (Fig. 1c). Although the clones used in our laboratory may not be identical to those examined by Brown et al.7, analysis of extracts of our cell lines showed that MMR protein levels (Fig. 1a,b) and MMR capacity measured by an in vitro MMR assay8 (data not shown) correlated with their MMR status.
Ionizing radiation induces different types of damage in DNA. The most common by far is oxidation and fragmentation of DNA bases, and the MMR system is involved in processing 8-oxoguanosines (GOs) incorporated into DNA during replication9. This type of damage could only signal during the S phase, however, which is inconsistent with the experimental findings, given that the cells analyzed by Brown et al.7 were confluent. It seems more likely that any differences in ionizing radiation–induced DNA damage signaling should be linked with the processing of double-strand breaks, the most deleterious kinds of ionizing radiation–induced DNA damage, as they are processed by recombination, a process in which MMR is involved1. Because we observed no differences in our strictly isogenic cell system, however, MMR does not seem to be required to repair ionizing radiation–induced cytotoxic double-strand breaks. This implies that the small differences in response to ionizing radiation described by others were linked either with small variations in experimental procedures or with phenotypic traits of MMR-deficient cells other than MMR.
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Nature Reviews Molecular Cell Biology (2004)