In vivo mutagenesis of the reporter plasmid pSP189 induced by exposure of host Ad293 cells to activated polymorphonuclear leukocytes.

Abstract

We measured the mutation frequency and spectrum induced by exposure of the mutation reporter plasmid pSP189 in vivo to phorbol myristate acetate (PMA)-activated human polymorphonuclear leukocytes (PMNs). The mutation frequency induced in the supF tRNA gene of pSP189 transfected into human Ad293 cells by a 30 min exposure to 4 x 10(6) activated PMNs/ml was 3- to 9-fold higher than the background mutation frequency of 0.1-1.8 x 10(-5). The enhanced mutation frequency caused by activated PMNs required replication of the reporter plasmid in host Ad293 cells. Fifty five unique activated PMN-associated mutants characterized by sequencing included base substitutions (55%) and deletions (45%), however, no small (1-3 bp) deletions were observed. Ninety four percent of point mutations occurred at C:G base pairs, with C:G-->T:A transitions (47%) and C:G-->A:T transversions (37%) predominating. A prominent hot-spot was observed at d(pCAGAC) on the tRNA strand. Although H202 generation was required for mutagenesis, the mutation spectrum induced in pSP189 by in vivo exposure to activated PMNs differed from that induced by in vivo exposure to H202. It also differed from the spectrum induced in single-stranded DNA in vitro by activated PMNs, suggesting that the mutational spectrum is a complex function of the kinetics of reactive oxygen generation and factors contributed by the target cell.