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Targeted base substitutions and small deletions induced by neocarzinostatin at the APRT locus in plateau-phase CHO
1Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond, USA.
Abstract:
Treatment of confluence-arrested CHO-D422 cells for 48 h with low concentrations (0.5-3 nM) of the radiomimetic antibiotic neocarzinostatin resulted in an increase in up to 11-fold in the frequency of mutations at the hemizygous APRT locus. Analysis by PCR and DNA sequencing revealed that the mutations were a mixture of base substitutions, small deletions, and large-scale rearrangements. base substitutions occurred preferentially at sequence positions where the drug is known to produce abasic sites with closely opposed strand breaks, e.g., AGT, TGT and AGC, where the abasic site occurs at the underlined base and the strand break occurs opposite the first base in each triplet. These results suggest that the substitutions were produced by replicative bypass of the abasic sites, perhaps during attempted repair of the accompanying strand break. Single-base deletions, which comprised nearly half of all deletions, were targeted to these same sequence positions, suggesting that they may have been generated either by replicative bypass of the abasic sites, or by end-joining repair of double-strand breaks, which are induced the same sites. Quantitative analysis of neocarzinostatin-induced damage to APRT DNA in vitro confirmed the association between lesions involving concommitant damage to both DNA strands, and mutations. The results are consistent the hypothesis that agents which induce such bistranded DNA damage can produce biologically significant levels of mutagenesis even in nondividing cells.
Insights
The radiomimetic antibiotic neocarzinostatin significantly increases mutation frequency in cells by causing DNA damage. This damage, particularly bistranded DNA lesions, drives mutagenesis even in non-dividing cells.
Area of Science:
- Molecular Biology
- Genetics
- Toxicology
Background:
- Neocarzinostatin (NCS) is a radiomimetic antibiotic known to induce DNA damage.
- Understanding the mutagenic potential of DNA-damaging agents is crucial for risk assessment.
Purpose of the Study:
- To investigate the mutagenic effects of low concentrations of neocarzinostatin on CHO-D422 cells.
- To characterize the types of mutations induced by neocarzinostatin and their relationship to specific DNA lesions.
Main Methods:
- Treatment of confluence-arrested CHO-D422 cells with neocarzinostatin (0.5-3 nM).
- Analysis of mutations at the APRT locus using Polymerase Chain Reaction (PCR) and DNA sequencing.
- In vitro analysis of neocarzinostatin-induced DNA damage.
Main Results:
- Neocarzinostatin treatment increased mutation frequency up to 11-fold.
- Mutations included base substitutions, small deletions, and large-scale rearrangements.
- Mutations preferentially occurred at sites with abasic sites and closely opposed strand breaks, indicating damage to both DNA strands.
Conclusions:
- Neocarzinostatin induces a significant increase in mutations, particularly at sites of bistranded DNA damage.
- The findings support the hypothesis that agents causing bistranded DNA damage can induce significant mutagenesis in non-dividing cells.
- Replicative bypass of abasic sites and end-joining repair of double-strand breaks are likely mechanisms for neocarzinostatin-induced mutations.