Phenotypic Conversion of SV40-Immortalized HumanDiploid Fibroblasts to Senescing Cells by Introduction of an Antisense Gene for SV40-T Antigen

Abstract

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70%as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13and about 50% of cells were at G_1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (PRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total PRB was much higher than that in VA-13. The PRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and PRB or the underphosphorylation of PRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.