Time versus replication dependence of EMS-induced delayed mutation in Chinese hamster cells

We have previously observed in Chinese hamster cells that ethyl methane sul fonate (EMS) induces mutations which are distributed over at least 10-14 ce ll divisions following treatment. This delayed appearance of mutations coul d be explained by EMS-induced lesions which remain in DNA and have a probab ility that is significantly less than 1.0 of producing base mispairing erro rs during successive replication cycles (replication-dependent). Alternativ ely, delayed mutation may be a time-dependent process in which a slow actin g or damage inducible error-prone repair process removes persistent DNA les ions and replaces them with an incorrect base during the course of 7-10 day s of colony growth following EMS exposure. To address this question, the di stribution of HGPRT delayed mutation events (fifth division or later) in ce lls plated immediately for exponential growth after EMS treatment was compa red with the distribution in cells which remained under confluent growth co nditions for 8 days and then were replated. Both the distribution and rate of accumulation of delayed mutations (mutations/cell division) were similar in the two culture conditions. In contrast, the frequency of early mutatio ns (before the fifth division) in the confluent population was reduced more than 2-fold compared to dividing cells. A comparison of the frequency of E MS-induced DNA lesions in the two populations revealed that the density inh ibited population contained one third the DNA lesions of the exponential po pulation. These results argue against a time-dependent process since, if th is mechanism applies, one would expect an increase in early mutant events a nd a decrease in delayed events in the confluent population. The results, h owever, are consistent with a replication model in which potential early mu tant lesions are preferentially removed in the density inhibited culture du ring the 8 days of incubation while lesions producing late mutants are not removed. (C) 1999 Elsevier Science B.V. All rights reserved.