Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin

Abstract

In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene. © 1999 Cancer Research Campaign

DNA Polymerase ζ Accounts for the Reduced Cytotoxicity and Enhanced Mutagenicity of Cisplatin in Human Colon Carcinoma Cells That Have Lost DNA Mismatch Repair

“…We have previously noted that loss of MMR is associated with enhanced capacity to express a protein from an adducted gene (38). The current results confirm this observation.…”

“…Thus, the effect of loss of REV3 and/or MMR on the function of a gene inactivated by DDP adducts can be examined by comparing the ability of the MMR + subline, the luciferase activity was significantly reduced by 2.6 F 0.05 (SE)-fold. This result is consistent with our prior finding that the MMR-proficient cells are less capable of generating luciferase activity from the platinated vector than the MMR-deficient cells (38). Notably, when REV3 expression was knocked down, luciferase expression was impaired to the same degree in MMR-proficient and MMR-deficient cells.…”

“…This procedure resulted in plasmid DNA that was >90% supercoiled as verified by gel electrophoresis. The platination procedure yielded 1.5 F 1.4 pg/Ag DNA, which is equivalent to 9.3 adducts per plasmid or 3.2 adducts per Luc-coding region and promoter as previously reported (38). Similar levels of platination have previously been shown not to affect the efficiency of transfection (39).…”

DNA Polymerase ζ Regulates Cisplatin Cytotoxicity, Mutagenicity, and The Rate of Development of Cisplatin Resistance

“…The pRL-CMV vector (Promega, Madison, WI) containing Renilla luciferase cDNA was platinated to 1.5 Ϯ 1.4 pg/g DNA, which is equivalent to 9.3 adducts per plasmid or 3.2 adducts per Luc coding region as reported previously (22). Similar levels of platination have been shown previously not to affect the efficiency of transfection (23).…”

A combination of MSH2 DNA mismatch repair deficiency and expression of the SV40 large T antigen results in cisplatin resistance of mouse embryonic fibroblasts

“…Therefore, loss of MMR ability may enable cells to survive DNA damage induced by cisplatin. Indeed, defects in MMR genes have been implicated in the resistance of human tumour cells to cisplatin in vitro (8)(9)(10)(11)(12). However, reports of cisplatin resistance in MMR-deficient murine cells in vitro are conflicting and suggest the joint requirement of MMR deficiency and additional gene alterations for cisplatin resistance.…”