High Intensity ras Signaling Induces Premature Senescence by Activating p38 Pathway in Primary Human Fibroblasts

Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence. Although oncogenic ras plays a pivotal role in neoplastic transformation, it triggers an anti-oncogenic defense mechanism known as premature senescence in normal cells. In this study, we investigated the induction of cellular responses by different expression levels of oncogenic ras in primary human fibroblasts. We found that a moderate, severalfold increase in ras expression promoted cell growth. Further elevation of ras expression initially enhanced proliferation but eventually induced p16INK4A expression and senescence. The induction of these opposing cellular responses by ras signals of different intensity was achieved through differential activation of the MAPK pathways that mediated these responses. Whereas moderate ras activities only stimulated the mitogenic MEK-ERK pathway, high intensity ras signals induced MEK and ERK to higher levels, leading to stimulation of the MKK3/6-p38 pathway, which had been shown previously to act downstream of Ras-MEK to trigger the senescence response. Thus, these studies have revealed a mechanism for the differential effects of ras on cell proliferation. Furthermore, moderate ras activity mediated transformation in cooperation with E6E7 and hTERT, suggesting that a moderate intensity ras signal can provide sufficient oncogenic activities for tumorigenesis. This result also implies that the ability of ras to promote proliferation and oncogenic transformation can be uncoupled with that to induce senescence in cell culture and that the development of tumors with relatively low ras activities may not need to acquire genetic alterations that bypass premature senescence. Neoplastic transformation is caused by multiple and successive genetic alterations (1.Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar), among which the oncogenic activation of ras genes seems to be one of the critical steps. The ras proto-oncogenes encode a family of small GTP-binding proteins that transduce mitogenic signals from the cell surface. Amplification of the ras genes and constitutively active ras mutant alleles has been frequently found in a wide variety of tumors (2.Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar), suggesting that the growth signals elicited by ras are crucial for tumorigenesis. Whereas wild-type ras transduces mitogenic signals in response to extracellular stimuli, activated ras provides cells with constitutive growth signals. The key role of activated ras in promoting oncogenic transformation has been established by numerous studies. In both in vitro cell culture systems and animal models, activated ras genes can cooperate with other oncogenic genetic alterations to lead to transformation (3.Weinberg R.A. Cancer Res. 1989; 49: 3713-3721PubMed Google Scholar, 4.Ruley H.E. Cancer Cells. 1990; 2: 258-268PubMed Google Scholar, 5.Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1982) Google Scholar, 6.Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. W.C. Weinberg R.A. PubMed Scopus Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar, B. Full Text PDF PubMed Scopus Google In ras genes have been found to be activated in a variety of tumors in suggesting that ras genes are of and that are to in the of neoplastic transformation PubMed Scopus Google The activity of activated ras on with multiple downstream which different of oncogenic transformation PubMed Scopus Google Scholar, 2000; Full Text Full Text PDF PubMed Scopus Google of the ras downstream is L. PubMed Scopus Google Scholar, B. M.D. Nature. PubMed Scopus Google Scholar, D. PubMed Scopus Google Scholar, F. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Counter C.M. 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In cell that established oncogenic ras with other in the transformation of primary human and cells (3.Weinberg R.A. Cancer Res. 1989; 49: 3713-3721PubMed Google Scholar, 4.Ruley H.E. Cancer Cells. 1990; 2: 258-268PubMed Google Scholar, 5.Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1982) Google Scholar, 6.Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. W.C. Weinberg R.A. PubMed Scopus Google Scholar, Weinberg R.A. Nature. PubMed Scopus Google In studies on primary human cells have that in to transformation that these cells be and that both and pathways be E6E7 other W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1982) Google Scholar, 6.Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. W.C. Weinberg R.A. 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