IDENTIFICATION OF SV40 T-ANTIGEN MUTANTS THAT ALTER T-ANTIGEN-INDUCEDCHROMOSOME-DAMAGE IN HUMAN FIBROBLASTS

The SV40 T antigen causes numerical (aneuploidy) and structural (aberr ations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum grow th factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could pro vide a mutational mechanism, To determine hom the T antigen causes chr omosome damage, point mutations were constructed that altered previous ly defined biochemical functions of the T protein, Mutant T antigen co nstructs were introduced into human diploid fibroblasts and selected b y using G418. Clones of G418(r) cells that expressed mutant T antigens were expanded and scored for chromosome damage, Most of these mutant T antigens caused by levels of chromosome damage similar to those caus ed the wild-type T antigen. However, some T-antigen mutants induced fe wer chromosome changes, A subset of these clones that induced less chr omosome damage than wild-type T were examined further. Mutant T-antige n protein levels from this subset were quantified with flow cytometry and compared with wildtype protein expression levels, Mutations of T a ntigen shown previously to form less stable complexes with p53 caused less chromosome damage, A mutation in the zinc finger domain of T anti gen also caused less chromosome damage, Interestingly, a mutant that c aused loss of tile ATPase activity of T antigen caused an increase in endoreduplicated cells, Also, a correlation was noted between cells ex pressing very low levels of T antigen (below detection limits when usi ng nom cytometry) and an undamaged karyotype. This correlation indicat es that there is a threshhold level of T-antigen expression that induc es chromosome damage and that expression levels on a per-cell basis ra ther than on a population basis should be considered in subsequent stu dies. (C) Wiley-Liss, Inc.