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Involvement of the mismatch repair system in temozolomide-induced apoptosis
Abstract:
Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generating O6-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence of O6-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient in O6-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G2/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2'-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response to O6-guanine methylation involves the mismatch repair system.
Insights
A functional mismatch repair system is crucial for temozolomide-induced apoptosis. This DNA repair pathway is required for triggering programmed cell death and p53 induction following O6-guanine methylation.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Postreplicative mismatch repair (MMR) is vital for DNA damage response.
- Agents like temozolomide generate O6-methylguanine, a DNA lesion that can trigger apoptosis.
- The role of MMR in temozolomide-induced cytotoxicity and apoptosis requires further elucidation.
Purpose of the Study:
- To investigate the dependence of temozolomide-induced apoptosis on a functional mismatch repair system.
- To determine if MMR is necessary for the p53 and p21/waf-1 protein induction triggered by O6-guanine methylation.
Main Methods:
- Utilized two human lymphoblastoid cell lines: MMR-proficient TK6 and MMR-deficient MT1.
- Treated cells with temozolomide and etoposide to assess cytotoxicity, apoptosis, and protein expression.
- Employed cytofluorimetric analysis for cell cycle arrest, DNA content, and DNA fragmentation.
Main Results:
- MMR-proficient TK6 cells exhibited significant growth inhibition, G2/M arrest, and apoptosis upon temozolomide treatment.
- MMR-deficient MT1 cells were resistant to temozolomide, with no observed apoptosis or p53/p21/waf-1 induction.
- Both cell lines underwent apoptosis with p53/p21/waf-1 induction when treated with etoposide, indicating intact p53-dependent pathways in MT1 cells.
Conclusions:
- A functional mismatch repair system is essential for triggering apoptosis in response to temozolomide.
- The induction of p53 and p21/waf-1 proteins by O6-guanine methylation is dependent on the mismatch repair system.
- These findings highlight the critical role of MMR in mediating the cytotoxic effects of temozolomide.