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O6-methyltransferase-deficient and -proficient CHO cells differ in their responses to ethyl- and
M Bignami1, E Dogliotti, G Aquilina
1Istituto Superiore di Sanita, Roma, Italy.
Abstract:
The mutagenic and cytotoxic effects of N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) were compared in two isogenic Chinese hamster ovary (CHO) cell lines differing for the expression of the repair function for O6-methylguanine (O6-meGua), the O6-methyl-DNA-methyltransferase (MT). Survival and ouabain resistance (ouar) mutation frequency were similar in the two cell lines after treatment with ENU while both effects were strongly reduced in the MT-proficient (MT+) CHO cells after exposure to MNU. The slow repair kinetics of O6-ethylguanine (O6-etGua) when compared to O6-meGua, i.e. 25% versus 88% removal at 20 h after treatment, could still account for the similar mutational curves reported in the two cell lines after ENU treatment. The number of ENU-induced sister chromatid exchanges (SCE) was slightly reduced in the MT+ as compared to MT-deficient CHO cells suggesting a role for O6-etGua in SCE formation. Comparison of survival after exposure to ENU and MNU showed that, at similar levels of O6-alkylguanine on DNA, the ethyl- is more tolerated than the methyl-adduct. These data focus the attention on the importance of DNA damage processing in the cytotoxic response to alkylating agents.
Insights
DNA repair efficiency influences cell response to mutagenic agents. O6-methylguanine-DNA methyltransferase (MT) proficient cells show reduced mutations from N-methyl-N-nitrosourea (MNU) but not N-ethyl-N-nitrosourea (ENU), highlighting DNA repair
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are alkylating agents with mutagenic and cytotoxic effects.
- O6-methylguanine-DNA methyltransferase (MT) is a key enzyme for repairing O6-alkylguanine DNA adducts.
Purpose of the Study:
- To compare the mutagenic and cytotoxic effects of ENU and MNU in Chinese hamster ovary (CHO) cells with differing MT expression.
- To investigate the role of DNA repair in cellular responses to these alkylating agents.
Main Methods:
- Comparison of survival and ouabain resistance (ouar) mutation frequency in MT-proficient (MT+) and MT-deficient (MT-) isogenic CHO cell lines after ENU and MNU exposure.
- Analysis of O6-ethylguanine (O6-etGua) and O6-methylguanine (O6-meGua) repair kinetics.
- Assessment of ENU-induced sister chromatid exchanges (SCE).
Main Results:
- Survival and mutation frequency were similar between MT+ and MT- CHO cells after ENU treatment.
- Both survival and mutation frequency were significantly reduced in MT+ CHO cells after MNU treatment compared to MT- cells.
- O6-etGua repair was slower than O6-meGua repair.
- ENU-induced SCE were slightly reduced in MT+ cells.
- Ethyl adducts were better tolerated than methyl adducts at equivalent DNA damage levels.
Conclusions:
- DNA damage processing, specifically MT-mediated repair, is crucial for cellular resistance to alkylating agents like MNU.
- The differential repair kinetics of O6-etGua and O6-meGua contribute to the distinct cellular responses observed.
- O6-etGua may play a role in sister chromatid exchange formation.