INHIBITION OF ONCOGENE-MEDIATED TRANSFORMATION BY ECTOPIC EXPRESSION OF P21(WAF1) IN NIH3T3 CELLS

The p53-regulated p21(Waf1) protein is a universal inhibitor of cyclin -dependent kinases (CDKs). To study the potential tumor-suppressive pr operties 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 sev eral other oncogenes into NIH3T3 cells potently inhibited the formatio n of transformed foci in a dose-dependent manner, Expression of the CD K-binding N-terminal half of p21(Waf1) (N-p21(Waf1)) was necessary and sufficient to inhibit Ras-induced focus formation. In contrast, expre ssion of the C-terminal domain (C-p21(Waf1)) had no effect on Ras-indu ced focus formation. Immunofluorescence analysis revealed that ectopic ally 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 a ccumulate around the nuclear membrane. Surprisingly, stable NIH3T3 tra nsfectants expressing ectopic p21(Waf1) grew at the same rate and disp layed similar cell cycle distribution as NIH3T3 cells transfected with the same vector containing no insert. However ectopic p21(Waf1) expre ssion did inhibit Ras-mediated anchorage-independent colony formation, indicating that p21(Waf1) can selectively interfere with oncogene-med iated transformation without affecting NIH3T3 cell growth, at least at the levels of p21(Waf1) expression experiments. Transient NIH3T3 cell s inhibited Ras-indnced transcription from a E2F-responsive element bu t 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 transfor mation of NIH3T3 cells and that it may do so by inhibiting E2F-driven transcription of S phase genes.