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DNA mismatch repair-dependent response to fluoropyrimidine-generated damage
Mark Meyers1, Mark W Wagner, Anthony Mazurek
1Department of Radiation Oncology and Case Comprehensive Cancer Center, Laboratory of Molecular Stress Responses, Case Western Reserve University, Cleveland, OH 44106, USA.
The Journal of Biological Chemistry
|December 22, 2004
Summary
DNA mismatch repair (MMR) genes MLH1 and MSH2 are crucial for detecting 5-fluorouracil incorporation during DNA replication, triggering cell cycle arrest and death in cancer cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Previous studies highlighted the necessity of MLH1 for 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity and G2 arrest in cancer cells.
- The role of MSH2, another key DNA mismatch repair (MMR) gene, in these cellular responses remained less understood.
Purpose of the Study:
- To investigate the role of MSH2 in cellular responses to FdUrd, similar to previously observed effects with MLH1.
- To elucidate the mechanism by which MMR influences FdUrd cytotoxicity and DNA incorporation.
Main Methods:
- Restoration of MSH2 expression in MSH2-deficient human endometrial carcinoma cells (HEC59) and murine embryonic stem cells.
- Treatment with FdUrd and assessment of cytotoxicity and G2 arrest.
- Comparison of cellular responses to FdUrd and the thymidylate synthase inhibitor Tomudex.
- Analysis of FdUrd incorporation into DNA using radiolabeled FdUrd and methyl-CpG-binding domain 4 glycosylase assays.
- Investigation of hMutoma-hMSH6 heterodimer's recognition of FdUrd incorporation via ATPase activation assays.
Main Results:
- Restoring MSH2 expression in MMR-deficient cells restored FdUrd-induced cytotoxicity and G2 arrest, mirroring MLH1's effects.
- MMR status had minimal impact on cellular responses to Tomudex, suggesting a specific role in FdUrd response.
- MMR-deficient cells showed higher FdUrd incorporation into DNA compared to MMR-proficient cells.
- The hMutoma-hMSH6 heterodimer specifically recognized 5-fluorouracil (FU) mispaired with guanine (Gua) in DNA, not adenine.
- Increased levels of FU:Gua misincorporation were detected in the DNA of MMR-deficient cells.
Conclusions:
- DNA mismatch repair specifically detects 5-fluorouracil (FU) misincorporated as FU:Gua during the first round of DNA replication.
- This detection by MMR, particularly involving the MSH2-MSH6 complex, signals a sustained G2 arrest and induces cell lethality.
- The findings establish a direct mechanistic link between MMR, FU incorporation, and the cytotoxic effects of FdUrd in cancer cells.