Development of a mammalian cell mutation assay based on flow cytometry and its use in analyzing mutations induced by ionizing radiation and chemicals

Abstract

The purpose of this study is to develop and utilize a mammalian mutation assay to analyze mutations caused by various toxic agents in a Chinese Hamster Ovary-Human hybrid cell line (CHO AL). The CHO AL cell line contains a single unrearranged copy of human chromosome 11 stably transfected for the last 30 yrs. Measuring a loss of CD59, a gene localized to chromosome 11, is the basis of the mutation assay. By treating CHO AL cells with various mutagens, the loss of CD59 can be measured as an indicator of genotoxicity. Linear dose responses from 137Cs γ radiation and MNNG (N-methyl-n-nitrosoguanidine) showed that the flow cytometry mutation assay (FCMA) allows a rapid, sensitive, and inexpensive method for detecting the mutations. The kinetics of CD59 mutant expression was analyzed by flow cytometry. The CD59 protein was removed from the plasma membrane by phosphatidyl inositol phospholipase C (PI-PLC) to measure the rate of exocytosis. At 8 hr, the CD59 expression had returned to control values. CD59 siRNA was also used to knock down CD59 mRNA and determine the duration of the CD59 protein on the cell surface before endocytosis. At 48 hr, CD59 expression had 85% reduction in CD59 expression. Cell cycle analysis was also measured by flow cytometry for lead acetate and MNNG. Lead acetate had a cell cycle delay on day 2 whereas MNNG had a prolonged G2 block until day 4 and fully recovered by day 7. CD59 mutant expression is more likely controlled by cell cycle alterations than protein turnover rate since, through PI-PLC and siRNA treatment, the minimum time before CD59 mutants could be measured was 48 hr. All mutagens measured by FCMA exhibited a peak in CD59 mutant expression at various time points. To examine the mutant peak region, the CHO AL cells were irradiated and sorted on the CD59 mutant peak by flow cytometry. The sorted populations were analyzed for survival, growth rate and phenotypic expression of CD44/CD59/CD90. The peak in CD59 mutant expression may be explained by cell lethality of CD44-CD59-CD90- mutants for radiation. The mutant spectra was determined for irradiated CHO AL cells using flow cytometry to measure cell surface markers CD59/CD44/CD90/CD98/CD151 and GPI-anchor control. PCR results reflected flow cytometry results. The FCMA gives researchers the tools to measure mutant yield and mutant spectra rapidly and efficiently and provides new insights into the mechanisms of action of mutagenic agents such as ionizing radiation. Using this system, researchers could quickly ascertain the mutagenic potential of novel chemicals and pharmaceuticals as well as clarify the method of mutagenesis without large expense. Additionally, the FCMA would be able to quickly analyze mutated populations during extended time periods to evaluate the evolution of phenotypic expression.