EXPRESSION OF SV40 LARGE T-ANTIGEN STIMULATES REVERSION OF A CHROMOSOMAL GENE DUPLICATION IN HUMAN-CELLS

Transformation of human cells is characterized by altered cell morphol ogy, frequent karyotypic abnormalities, reduced dependence on growth f actors and substrate, and rare ''immortalization''-clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segment s in a transfected plasmid substrate, for five immortal human cell lin es relative to three normal fibroblast strains [Finn et al. (1989) Mel . Cell. Biol. 9, 4009-4017]. We have now assessed reversion of a 14-ki lobase-pair duplication within the hypoxanthine phosphoribosyl transfe rase (HPRT) gene locus, in a fibroblast strain during its normal repli cative lifespan and after stable transformation with SV40 large-T anti gen. Revertants, selected under HPRT-dependent growth conditions immed iately after purging preexisting HPRT+ cells, were confirmed as HPRTby hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restrictio n fragment representing the duplicated HPRT region, as predicted for h omologous recombination between the 14-kilobase-pair repeats. Amplific ation of a subregion of HPRT mRNA implicated deletion of duplicated ex ons in 93% of revertant colonies. Reversion to HPRT+ was unaltered dur ing the normal in vitro lifespan of these cells, but increased in 9 cl ones stably transformed with large-T antigen (mean = 3.8-fold; each P < 10(-5)). Stimulation of HPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10-30 doublings before replicative senescence, Since no immortal subclones arose from these clones, elev ated reversion must precede immortalization. Increased DNA rearrangeme nts, in cells expressing T-antigen, could facilitate the rare concurre nce of multiple mutations necessary for immortalization.