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.

Chemical and toxicological characterization of slurry reactor biotreatment of explosives-contaminated soils

“…Although the cells are exposed to the compounds for only 5 h, residual amounts of some compounds or their metabolites may stay cell-associated for some time. If the mutation is cell-cycle dependent (18), the number of cells at risk during the exposure may be Iess than the total ceIIs present. While every effort was taken to assure that cells were in a reproducible log phase growth period, minor variations may affect results.…”

“…One mammalian cell assay uses the CHO ovary cell line (1 7) as a test cell. Analyses are done to identi@ mutations destroying activity of the enzyme hypoxanthine-guanine phosphoribosyltransferase coded in the hprt locus (18,19). This is an established assay that has been used to evaluate compounds either for their direct mutagenic activity or after "activation" with liver enzyme extracts -so called "S9 preparations."…”

Proliferation is necessary for both repair and mutation in transgenic mouse cells

“…Earlier work directed at elucidating the links among DNA synthesis, cell division, and mutation induction was conducted in arrested Chinese hamster ovary cells by O'Neill and colleagues (3)(4)(5). They treated cells that had been arrested by serum deprivation with the alkylating agent ethyl methanesulfonate (3) and compared the results with those obtained with proliferating Chinese hamster ovary cells.…”

Induction of chromosome aberrations, sister chromatid exchanges, and specific locus mutations in Chinese hamster ovary cells by 5-bromodeoxyuridine

“…We have previously shown that a 7-9 day expression time allows maximal expression of the TGr phenotype (O'Neill et al, 1977;O'Neill and Hsie, 1979) and that greater than 98% of the variant TGr clones contained altered or reduced HGPRT enzyme activity (Hsie et al, 1975;O'Neill et al, 1977).…”