Optimal phenotypic expression times for HPRT mutants induced in foreskin-, skin-, and lung-derived human diploid fibroblasts

Abstract

A series of paired lung- and skin-derived fibroblast cultures has been established from human embryonic tissues under carefully controlled, identical conditions, providing the unique opportunity to study differences between normal diploid fibroblast populations from skin and lung without the confusion of genetic differences between donors. To reliably assess differences in the induced mutation frequencies observed among different cell populations, optimal phenotypic expression times in the HPRT mutagenesis system were determined for neonatal foreskin, fetal skin, and fetal lung cultures. Cell populations were mutagenized with several doses of N-methyl-N'-nitro-N-nitrosoguanidine and were replated in 6-thioguanine selective medium at intervals over 14 days. Survivals following MNNG exposure ranged from 1.6% to 45.5%. For all doses and survivals tested a 7-day expression period was the optimal value for cultures from the three different tissue sources in six independent experiments. Mutant frequency data derived from untreated control populations confirmed that spontaneous mutations during the expression period contributed negligibly to the final mutant frequency. Differences between the mutation frequencies obtained using an in situ and a replating protocol were approximately twofold for lung-, skin-, and foreskin-derived cultures.