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Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C
A E Maccubbin1, A Mudipalli, S S Nadadur
1Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Environmental and Molecular Mutagenesis
|January 1, 1997
Summary
Mitomycin C DNA adducts were measured using a 32P-postlabeling technique. Monoadduct formation, particularly at guanine, correlated with increased mutation frequency, suggesting it causes mutagenicity.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mitomycin C (MMC) is a chemotherapeutic agent that forms DNA adducts.
- Cytotoxicity is linked to DNA cross-links, while mutagenicity is attributed to monoadducts.
Purpose of the Study:
- To develop and utilize a 32P-postlabeling technique for quantifying MMC DNA monoadducts.
- To investigate the mutagenic potential of MMC monoadducts in a shuttle vector system.
Main Methods:
- Incubation of shuttle vector plasmid pSP189 with monofunctionally activated MMC.
- Replication of modified plasmid in human Ad293 cells and subsequent bacterial rescue.
- Analysis of mutations in the supF tRNA gene using 32P-postlabeling and sequencing.
Main Results:
- A major MMC/DNA monoadduct involving guanine was identified.
- Increased MMC concentration led to higher adduct levels and mutation frequencies.
- Mutations were predominantly base substitutions (59.1%), with a bias towards G:C base pairs (nearly 80%).
Conclusions:
- The 32P-postlabeling technique effectively measures MMC monoadducts.
- Monoadduct formation, especially at guanine, is strongly implicated in MMC-induced mutagenesis.
- The findings highlight the role of monoadducts in the genotoxicity of mitomycin C.