Abstract
To directly examine the cellular roles of the retinoblastoma gene product (pRb), we constructed an effective antisense Rb RNA vector that expressed an antisense Rb-1 sequence directed toward the 3' untranslated region of Rb-1 mRNA. In this study, we introduced the antisense Rb vector into mouse fibroblast SV-T2 cells expressing large amounts of SV40 LT, which inhibits the biological functions of pRb through direct binding. Only a small number of drug-resistant colonies were obtained after drug selection. Co-transfection of SV-T2 cells with pRb expression vectors and the antisense Rb vector resulted in an apparent increase in the number of drug-resistant colonies, suggesting that a decrease in cellular pRb content induced cell death in the cells. The cell death in SV-T2 cells was also abrogated by simultaneous transfection with antisense p53 RNA expression vector. These results indicate that a decrease in the amount of biologically active pRb by both the effects of the transfected antisense Rb RNA and the large amount of endogenous SV40 LT induces cell death in SV-T2 cells through a p53-dependent pathway. The antisense Rb and p53 vectors used in this study are useful to examine the biological functions of these genes in established cell lines.
