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Alkylation damage, DNA repair and mutagenesis in human cells
V M Maher1, J Domoradzki, N P Bhattacharyya
1Department of Microbiology, Michigan State University, East Lansing 48824-1316.
Abstract:
17 human cell lines that differ significantly in level of O6-alkylguanine-DNA alkyltransferase (AGT) activity were identified by comparing their sensitivity to the cytotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and determining the level of AGT activity in cell extracts from the various lines by measuring the decrease in radiolabeled O6-methylguanine from DNA, using high-performance liquid chromatography. 9 lines exhibited high levels of AGT activity, 2 showed an intermediate level (25-50% of the mean of those with the higher levels), and 6 exhibited very low or virtually undetectable levels of AGT. Included were several lines that are very deficient in capacity for nucleotide excision repair. When representatives from the 3 categories of cell lines defined by the level of AGT activity were compared for sensitivity to the cytotoxic and mutagenic effect of MNNG, they showed an inverse correlation between the degree of cell killing and frequency of mutants induced and the level of AGT activity. The cells' capacity for nucleotide excision repair did not affect these results. Exposure of cells with a high level of AGT activity to O6-methylguanine in the medium reduced the AGT activity 60-80%. These pre-treated cells exhibited a significantly higher frequency of MNNG-induced mutants than did cells that were not pre-treated, suggesting that the O6-methylguanine lesion in DNA is responsible for a significant proportion of the mutations induced. Cell strains containing substrates for assaying intrachromosomal homologous recombination were constructed using parental cell lines from each of the 3 categories of AGT activity. These strains showed an inverse correlation between the level of AGT activity and the frequency of MNNG-induced recombination. When various cell lines representing the 3 categories of AGT activity were compared for sensitivity to ethylnitrosourea, the results were consistent with AGT and nucleotide excision repair playing a role in preventing cell killing and mutation induction by this agent.
Insights
O6-alkylguanine-DNA alkyltransferase (AGT) activity protects human cells from DNA damage caused by MNNG. Higher AGT levels correlate with lower mutation and cell killing rates, indicating its crucial role in DNA repair.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- O6-alkylguanine-DNA alkyltransferase (AGT) is a key DNA repair protein.
- AGT activity varies significantly across human cell lines.
- Understanding AGT's role in DNA repair is crucial for cancer therapy and toxicology.
Purpose of the Study:
- To investigate the relationship between AGT activity levels and cellular sensitivity to DNA damage.
- To determine the role of AGT in preventing mutations and cell death induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
- To assess the contribution of AGT and nucleotide excision repair (NER) to cellular resistance against genotoxic agents.
Main Methods:
- Assessed AGT activity in 17 human cell lines using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) cytotoxicity and DNA repair assays.
- Quantified AGT levels via high-performance liquid chromatography (HPLC) measuring O6-methylguanine decrease from DNA.
- Compared sensitivity to MNNG and ethylnitrosourea (ENU) across cell lines with varying AGT and NER capacities.
Main Results:
- Cell lines were categorized into high, intermediate, and low AGT activity groups.
- Inverse correlation observed between AGT levels and MNNG-induced cell killing and mutation frequency.
- Pre-exposure to O6-methylguanine reduced AGT activity, increasing MNNG-induced mutations, suggesting O6-methylguanine lesions are mutagenic.
- Homologous recombination frequency also inversely correlated with AGT activity.
- Results for ethylnitrosourea were consistent with AGT and NER roles in preventing genotoxicity.
Conclusions:
- AGT activity is a major determinant of cellular resistance to MNNG-induced DNA damage, mutations, and recombination.
- The O6-methylguanine lesion in DNA is a significant mutagenic lesion.
- Both AGT and nucleotide excision repair contribute to cellular defense against genotoxic agents like ethylnitrosourea.