Effect of nuclear localization of large tumor antigen on growth potential of SV40-transformed cells

Transport-defective mutants of PARA, an SV40-adenovirus 7 hybrid virus, induce the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm (cT-ag) of infected and transformed cells. The ability of cT mutants of PARA to induce phenotypic transformation of hamster embryo fibroblasts was examined. The cytoplasmic localization of T-ag was not stable, as extensive subculturing resulted in cell populations comprised of mixtures of cells which expressed T-ag in either the nucleus and/or the cytoplasm. Clonal analysis resulted in the selection of two cell populations: one cell type expressed T-ag solely in the cytoplasm and the second contained a mixture of cells which expressed T-ag in the nucleus and/or the cytoplasm. Serial subculture of clonal cell lines containing T-ag exclusively in the cytoplasm resulted again in the evolution of mixed populations which expressed T-ag in the nucleus and/or the cytoplasm. Analysis of synchronized cultures of such mixed clonal lines revealed that the intracellular distribution of T-ag was under the influence of the cell cycle; T-ag was present in the nucleus during S-phase, the period of maximal DNA synthesis, and in the cytoplasm during other phases of the cell cycle. These results suggest that a selective advantage exists during in vitro culturing for cells with a partial reversion of the cT phenotype. Examination of the growth properties of clonal lines in vitro indicated that cells in which T-ag could be detected in the cytoplasm, either continuously or during certain phases of the cell cycle, exhibited reduced growth potential under stringent culture conditions relative to wild-type transformants, behaving more like minimal transformants. In addition, the capacity for tumor induction in weanling hamsters by PARA(cT)-transformed hamster cells was reduced substantially in comparison to wild-type transformed cells. These results suggest that the constant presence of SV40 T-ag in the nucleus promotes maximal expression of the transformed cell phenotype.