DNA Mismatch Repair

Depletion of MGMT activity by promoter methylation or the use of inhibitors is not always sufficient to ensure alkylating agent sensitivity [52]. In the absence of functional MGMT, an important additional mechanism of resistance to alkylating agents is DNA mismatch repair (MMR) deficiency. At least 5 proteins organized as heterodimers are involved in MMR: MutSa (a heterodimer of hMSH2 and hMSH6), MutSp (a heterodimer of hMSH2 and hMSH3) and MutLa (a heterodimer of hMLHl and hPMS2). Mismatched heteroduplexes are recognized by MutSa or MutSp which bind to the DNA. MutLa then binds to the complex and the segment of the DNA strand containing the mismatched base is excised. The gap is then filled in by DNA polymerase and ligation (Fig. 6.2) (for reviews see [19,104]).

MMR functions in resistance to alkylating agent chemotherapy when the O6-methylguanine adduct is not repaired by MGMT prior to DNA replication, resulting in a O6-meG:C base pair that is typically replicated as a O6-meG:T. In the case of mismatch repair of O6-meG:T the DNA gap is created but it cannot be correctly filled in, resulting in a ''futile repair loop'' that leads to additional DNA double strand breaks and apoptotic cell death [10,52]. Thus, a defect in mismatch repair can lead to alkylating agent resistance in a cell that lacks MGMT activity. It does not however, change the incidence of mutations that occur due to the insertion of a ''T'' in the daughter DNA strand. Thus, defective mismatch repair may also contribute to the genetic instability seen in these tumors by promoting additional muta-tional events.

Analysis of MMR in brain tumors and other cancers has included studies of the proteins, gene mutations, mRNA, and MMR activity. These studies have identified a link between MMR deficiency and resistance to a number of therapeutic agents. In fact, some studies suggest that intact MMR is required for cell death in MGMT deficient cells; however, the literature is not always consistent with respect to the contribution of MMR. For example, while a number of studies have established a relationship between resistance to alkylating agents such as BCNU and temozolomide with MMR, others have suggested that this relationship is true for some, but not all alkylating agents [10,57,66,70,87,104-112]. Similarly, some reports demonstrate a clear relationship between MMR deficiency and cisplatin resistance [20,22,106,113-115], others suggest that MMR may play a relatively small role in cisplatin resistance [104,116]. Houghton et al., [58] used temozolomide in combination with irinotecan on mouse xenografts and found that while the 2 drugs did not appear to interact directly, their action was synergistic and was independent of both MGMT and mismatch repair.

Regulation of the genes involved in MMR occurs at a number of levels. Rellecke et al., [108] demonstrated that all 5 MMR genes are transcribed in de novo GBMs prior to treatment. Gene transcription was approximately equal for all but hMSH2. The expression of this gene was high in resistant tumors. Srivastava et al., [117] found higher expression of hMLH2 in high grade tumors when compared to low grade tumors, with a higher number of cells showing positive by immunohistochemistry. MSH2 and MSH6 were found to be regulated through post-translational

FIGURE 6.2 Mammalian Mismatch Repair (MMR). Heterodi-mers of hMSH2/6 (called hMutSa) focus on mismatches and singlebase loops (stage I in the figure, upper strand), whereas hMSH2/3 dimers (hMutSb) recognize insertion/deletion loops (II, lower strand). Heterodimeric complexes of the hMutL-like proteins hMLH1/hPMS2 (hMutLa) and hMLH1/hPMS1 (hMutLb) interact with MSH complexes and replication factors. Strand discrimination may be based on contact with the nearby replication machinery. A number of proteins are implicated in the excision of the new strand past the mismatch and resynthesis steps, including polS/s, RPA, PCNA, RFC, exonuclease 1, and endonuclease FEN1 (II, III). MMR components also interact functionally with NER and recombination. Recent crystallographic studies have revealed that a MutS dimer detects the structural instability of a heteroduplex by kinking the DNA at the site of the mismatch, which is facilitated when base pairing is affected. However, DNA damage with similar characteristics, such as that caused by alkylating agents and inter-calators, may fool MutS, triggering erroneous or futile MMR. (Reprinted from [122]). See Plate 6.2 in Color Plate Section.

FIGURE 6.3 Nucleotide Excision Repair (NER). The globalgenome (GG)-NER-specific complex XPC-hHR23B screens first on the basis of disrupted base pairing, instead of lesions per se. In transcription-coupled repair (TCR), the ability of a lesion (whether of the NER- or BER-type) to block RNA polymerase seems critical (stage I in this figure). The stalled polymerase must be displaced to make the injury accessible for repair, and this requires at least two TCR-specific factors: CSB and CSA. The subsequent stages of GG-NER and TCR may be identical. The XPB and XPD helicases of the multi-subunit transcription factor TFIIH open ~30 base pairs of DNA around the damage (II). XPA probably confirms the presence of damage by probing for abnormal backbone structure, and when absent aborts NER. The single-stranded-binding protein RPA (replication protein A) stabilizes the open intermediate by binding to the undamaged strand (III). The use of subsequent factors, each with limited capacity for lesion detection in toto, still allows very high damage specificity. The endonuclease duo of the NER team, XPG and ERCC1/XPF, respectively cleave 3"Snd 5' of the borders of modification in that they are translocated from the cytoplasm to the nucleus following DNA damage, particularly in the absence of MGMT [107,118]. Finally, the gene encoding hMSH6 is thought to be regulated in part by methylation of CpG islands in the gene's promoter [110] in a manner similar to that seen for MGMT. In addition, hypermethylation of the hMLH1 gene promoter has recently been found to predict a patient's clinical response to nitrosoureas in much the same way as have been found for MGMT promoter methylation. Since cells with deficient MMR and MGMT tend to be drug resistant, the analysis of hMLH1 expression in combination with MGMT expression may provide a more robust clinical test to predict therapy resistance.

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