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 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 [Finn et 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 preexisting HPRT+ cells, were confirmed as HPRT+ by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicated HPRT region, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion of HPRT mRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion to HPRT+ was unaltered during the normal in vitro lifespan of these cells, but increased in 9 clones 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, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.
Cheng
R Z
RZ
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Shammas
M A
MA
Li
J
J
Shmookler Reis
R J
RJ
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000502:physiology*; D002455:Cell Division; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics*; D002999:Clone Cells; D004247:DNA / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D005347:Fibroblasts; D017353:Gene Deletion; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007926:Lesch-Nyhan Syndrome / Q000235:genetics; D008727:Methotrexate / Q000378:metabolism; D005810:Multigene Family; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D005938:Glucocorticoids; D012333:RNA, Messenger; D019271:Hypoxanthine; D003907:Dexamethasone; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D013936:Thymidine; D008727:Methotrexate