Inhibition of oncogene-mediated transformation by ectopic expression of p21Waf1 in NIH3T3 cells.

The p53-regulated p21(Waf1) protein is a universal inhibitor of cyclin-dependent kinases (CDKs). To study the potential tumor-suppressive properties of CDK inhibitors, the ability of p21(Waf1) to interfere with oncogene-mediated cellular transformation was analysed in the NIH3T3 cell system. Cotransfection of waf1 together with activated ras or several other oncogenes into NIH3T3 cells potently inhibited the formation of transformed foci in a dose-dependent manner, Expression of the CDK-binding N-terminal half of p21(Waf1) (N-p21(Waf1)) was necessary and sufficient to inhibit Ras-induced focus formation. In contrast, expression of the C-terminal domain (C-p21(Waf1)) had no effect on Ras-induced focus formation. Immunofluorescence analysis revealed that ectopically expressed p21(Waf1) and C-p21(Waf1) were localized in the nucleus, while N-p21(Waf1) was found in the cytoplasm, with the tendency to accumulate around the nuclear membrane. Surprisingly, stable NIH3T3 transfectants expressing ectopic p21(Waf1) grew at the same rate and displayed similar cell cycle distribution as NIH3T3 cells transfected with the same vector containing no insert. However ectopic p21(Waf1) expression did inhibit Ras-mediated anchorage-independent colony formation, indicating that p21(Waf1) can selectively interfere with oncogene-mediated transformation without affecting NIH3T3 cell growth, at least at the levels of p21(Waf1) expression experiments. Transient NIH3T3 cells inhibited Ras-indnced transcription from a E2F-responsive element but not from a serum-responsive element, indicating that p21(Waf1) acts downstream of early transcriptional events induced by Ras but upstream of E2F-controlled gene transcription. These results provide evidence that p21(Waf1) potently suppresses oncogene-mediated cellular transformation of NIH3T3 cells and that it may do so by inhibiting E2F-driven transcription of S phase genes.