Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system): expression in division-arrested cell cultures

Abstract

The phenotypic expression time of ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants was studied with Chinese hamster ovary cells in culture (CHO/HGPRT system). After mutagen treatment of exponential phase cultures, the cells were maintained either in the exponential phase through subculture in medium containing 5% dialyzed fetal bovine serum (FBS) or in a nondividing viable state by use of medium containing 0-1% dialyzed FBS. The time course of expression of the 6-thioguanine-resistant phenotype was similar with both exponential phase and division-arrested cultures showing maximum expression by 9 days after mutagen treatment, and both methods of expression also yielded similar mutant frequencies over a range of EMS concentrations. This study shows that once the mutagenic event is fixed, the expression of the mutant phenotype does not require continued cell division since it occurs in division-arrested cultures. These results also suggest that both dilution of pre-existing hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme by cell division and turnover by protein degradation are involved in the phenotypic expression. Both processes occur in exponential cultures, but only protein turnover in arrested cultures. Consistent with this was the demonstration that the rates of total cell protein turnover increased in division-arrested cultures maintained in serum-free medium. These results separate genetic damage and phenotypic expression in a temporal sense, and point out the need to consider the mechanisms responsible for each process involved in the induction and expression of mutations.