EMS mutant spectra generated by multi-parameter flow cytometry

Abstract

The CHO AL cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59− regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59− clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Assessment of the Genotoxic Potential of Azidothymidine in the Comet, Micronucleus, and Pig-a Assay

“…As determined by the Pig-a gene mutation assay, AZT did not cause a statistical significant increase in the number of mutant phenotypes in RBCs or RETs. As expected (Dobo et al, 2011;Keysar and Fox, 2009), the positive control EMS (120 mg/kg/ day) induced gene mutations in both RBC and RET. AZT was shown to be mutagenic in the HPRT test in vitro, although the effect was rather low (Sussman et al, 1999).…”

Hypoxia/Reoxygenation-Induced Mutations in Mammalian Cells Detected by the Flow Cytometry Mutation Assay and Characterized by Mutant Spectrum

“…We have shown that mutants generated by γ radiation are frequently large deletions (20) while those generated by EMS are small, frequently intragenic, mutations (31). We previously compared the mutant spectrum obtained by detecting the presence or absence of CD surface proteins with the presence or absence of flanking genes obtained by PCR analysis, and there was a perfect correlation (20).…”