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Different effects of methotrexate on DNA mismatch repair proficient and deficient cells
I Frouin1, E Prosperi, M Denegri
1Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., Via Abbiategrasso 207, I-27100, Pavia, Italy.
Abstract:
Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.
Insights
DNA mismatch repair (MMR) does not influence antifolate-induced DNA repair synthesis. However, MMR-proficient cells show increased apoptosis and reduced viability when treated with antifolates like methotrexate.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Antifolates inhibit dihydrofolate reductase, blocking thymidylate synthesis and impairing DNA synthesis.
- DNA damage from antifolates may involve nucleotide misincorporation, potentially repaired by DNA mismatch repair (MMR).
Purpose of the Study:
- To investigate DNA repair mechanisms following antifolate treatment.
- To determine the role of DNA mismatch repair (MMR) in repairing antifolate-induced DNA damage.
- To explore the relationship between MMR status and antifolate-induced cell death.
Main Methods:
- Comparison of MMR-proficient (HeLa, HCT116+chr3) and MMR-deficient (HeLa cell-clone12, HCT116) human cell lines.
- Treatment with methotrexate (MTX) to induce DNA damage.
- Assessment of DNA repair synthesis, apoptosis markers (nuclear shrinkage, chromatin condensation, DNA laddering, PARP proteolysis), and cell viability.
Main Results:
- DNA repair synthesis occurred independently of cellular MMR function.
- Apoptosis was observed in both MMR-proficient and MMR-deficient cells following MTX treatment.
- MMR-proficient cells exhibited lower viability and more efficient apoptosis induction compared to MMR-deficient cells.
Conclusions:
- Cellular MMR is not essential for repairing antifolate-induced DNA damage.
- MMR competence enhances antifolate-induced apoptosis and reduces cell survival.
- These findings highlight the complex interplay between DNA repair pathways and drug-induced cytotoxicity.