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Updated: Aug 17, 2026
Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells
Published on: June 9, 2020
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.
Abstract:
Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.
Insights
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.
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