Expression of SV40 Large T Antigen Stimulates Reversion of a Chromosomal Gene Duplication in Human Cells

Abstract

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare “immortalization”—clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finnet al.(1989)Mol. Cell. Biol.9, 4009–4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexistingHPRT+cells, were confirmed asHPRT+by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicatedHPRTregion, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion ofHPRTmRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion toHPRT+was unaltered during the normalin vitrolifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; eachP< 10−5). Stimulation ofHPRT-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, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.