Different mechanisms of reversion of HPRT-deficient V79 Chinese hamster cells

The revertibility of three spontaneous hypoxanthine phosphoribosyltransferase (HPRT)-deficient V79 cell lines has been determinedafter exposure to a number of alkylating agents. TGll and 19reverted at frequences ranging from 1 x 10"5 to 1 x 10"4 afterexposure to doses of ethyl-methane sulphonate (EMS) N-methyl-N-nitrosourea(MNU) and N-ethyl-N-nitrosourea (ENU) resulting in survivingfractions between 1.0 and 0.1. Reversion frequencies in TG15ranged from 10"7 to 5 x 10"6 over a similar dose range. Therelative efficiencies of different monofunctional alkylatingagents in causing reversion of TGll at equitoxic doses wereENU > EMS > N-ethyl-N-nitroso-guanidine MNU > N-methyl-N-nitrosoguanidine> methylmethane sulphonate. Revertant frequencies for allthree cell lines were maximal immediately after treatment anddeclined thereafter at a rate inversely proportional to dose.Such kinetics are explicable if reversion is due to miscodingopposite alkylated guanines. Reversion frequencies after N-butyl-N-nitrosoureaexposure were 100-fold lower than after MNU and kinetics ofexpression of revertant colonies differed. Frequencies werelow immediately after treatment, increased between 0 and 24h then remained at a plateau. Similar kinetics were observedafter chlorozotocin and bis-chloroethylnitrosourea exposure.This difference in expression kinetics suggests that reversionin this case is not the result of direct miscoding but of errorsin excision repair. TGll, 15 and 19 had low spontaneous mutantfrequencies which were either unaffected or only marginallyincreased by treatment with 5-azacytidine. The three HPRT" mutantsand revertants of TGll and 15 were subjected to Southern analysisusing a Chinese hamster HPRT cDNA as a probe. No differencesin restriction fragment patterns were observed and there wasno detectable amplification of HPRT sequences indicating thatthe HPRT" mutants and their revertants are the result of pointmutations. ²Present address: Baylor College of Medicine, Texas MedicalCenter, Houston, TX 77030, USA³present address: Corporate Biosciences Laboratory. ICI plc,The Heath, Runcorn, Cheshire, UK