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

Paolo Michieli, Weiqun Li, Matthew V. Lorenzi, Toru Miki, Rina Zakut, David Givol, Jacalyn H. Pierce

Research output: Contribution to journalArticle

Abstract

The p53-regulated p21Waf1 protein is a universal inhibitor of cyclin-dependent kinases (CDKs). To study the potential tumor-suppressive properties of CDK inhibitors, the ability of p21waf1 to interfere with oncogenemediated 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 p21waf1 (N-p21waf1) was necessary and sufficient to inhibit Ras-induced focus formation. In contrast, expression of the C-terminal domain (C-p21Waf1) had no effect on Ras-induced focus formation. Immunofluorescence analysis revealed that ectopically expressed p21Waf1 and C-p21Waf1 were localized in the nucleus, while N-p21Waf1 was found in the cytoplasm, with the tendency to accumulate around the nuclear membrane. Surprisingly, stable NIH3T3 transfectants expressing ectopic p21Waf1 grew at the same rate and displayed similar cell cycle distribution as NIH3T3 cells transfected with the same vector containing no insert. However, ectopic p21Waf1 expression did inhibit Ras-mediated anchorage-independent colony formation, indicating that p21Waf1 can selectively interfere with oncogene-mediated transformation without affecting NIH3T3 cell growth, at least at the levels of p21Waf1 expression achieved in these experiments. Transient transfection of waf1 into N1H3T3 cells inhibited Ras-induced transcription from a E2F-responsive element but not from a serum-responsive element, indicating that p21Waf1 acts downstream of early transcriptional events induced by Ras but upstream of E2F-controlled gene transcription. These results provide evidence that p21Waf1 potently suppresses oncogene-mediated cellular transformation of NIH3T3 cells and that it may do so by inhibiting E2F-driven transcription of S phase genes.

Original languageEnglish
Pages (from-to)775-784
Number of pages10
JournalOncogene
Volume12
Issue number4
Publication statusPublished - 1996

Keywords

  • Cellular transformation
  • Cyclin-dependent kinase inhibitors
  • Oncogenes
  • p53 effectors
  • Tumor suppressor genes

ASJC Scopus subject areas

  • Cancer Research
  • Genetics
  • Molecular Biology

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  • Cite this

    Michieli, P., Li, W., Lorenzi, M. V., Miki, T., Zakut, R., Givol, D., & Pierce, J. H. (1996). Inhibition of oncogene-mediated transformation by ectopic expression of p21Waf1 in NIH3T3 cells. Oncogene, 12(4), 775-784.