Expression of SV40 large T antigen stimulates reversion of a chromosomal gene duplication in human cells

Exp Cell Res. 1997 Aug 1;234(2):300-12. doi: 10.1006/excr.1997.3649.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, Polyomavirus Transforming / genetics
  • Antigens, Polyomavirus Transforming / physiology*
  • Cell Division
  • Cell Line, Transformed
  • Cell Transformation, Viral / genetics*
  • Clone Cells
  • DNA / analysis
  • Dexamethasone / pharmacology
  • Fibroblasts
  • Gene Deletion*
  • Glucocorticoids / pharmacology
  • Humans
  • Hypoxanthine / metabolism
  • Hypoxanthine Phosphoribosyltransferase / genetics*
  • Lesch-Nyhan Syndrome / genetics
  • Methotrexate / metabolism
  • Multigene Family
  • RNA, Messenger / analysis
  • Recombination, Genetic
  • Simian virus 40 / physiology*
  • Thymidine / metabolism


  • Antigens, Polyomavirus Transforming
  • Glucocorticoids
  • RNA, Messenger
  • Hypoxanthine
  • Dexamethasone
  • DNA
  • Hypoxanthine Phosphoribosyltransferase
  • Thymidine
  • Methotrexate