In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEKextracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.

Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.

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), 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), 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 (3-8). In addition, ras genes have been found to be activated in a variety of carcinogen-induced tumors in animals, suggesting that ras genes are targets of carcinogens and that they are likely to participate in the initiation of neoplastic transformation (9). The transforming activity of activated ras depends on its interaction with multiple downstream effectors, which mediate different aspects of oncogenic transformation (10, 11). One of the best characterized ras downstream effectors is ....

Several genes that contain the PR (PRDI-BF1 and RIZ) domain have been linked with human cancers. We describe here a new PR-domain-containing gene designated as PRDM5 (PFM2). A PRDM5 cDNA was isolated based on its homology to the PR domain of RIZ1 (PRDM2). The gene encodes an open reading frame of 630 amino acids and contains a PR domain in the NH-terminal region followed by 16 zinc finger motifs. Radiation hybrid analysis mapped PRDM5 to human chromosome 4q26, a region thought to harbor tumor suppressor genes for breast, ovarian, liver, lung, colon, and other cancers. The gene has a CpG island promoter and is silenced in human breast, ovarian, and liver cancers. A recombinant adenovirus expressing PRDM5 caused G2/M arrest and apoptosis upon infection of tumor cells. These results suggest that inactivation of PRDM5 may play a role in carcinogenesis.

Oncogene-induced senescence is a tumor-suppressive defense mechanism triggered upon activation of certain oncogenes in normal cells. Recently, the senescence response to oncogene activation has been shown to act as a bona fide barrier to cancer development in vivo. Multiple previous studies have implicated the importance of the p38 MAPK pathway in oncogene-induced senescence. However, the contribution of each of the four p38 isoforms (encoded by different genes) to senescence induction is unclear. In the current study, we demonstrated that p38␣ and p38␥, but not p38␤, play an essential role in oncogenic ras-induced senescence. Both p38␣ and p38␥ are expressed in primary human fibroblasts and are activated upon transduction of oncogenic ras. Small hairpin RNA-mediated silencing of p38␣ or p38␥ expression abrogated ras-induced senescence, whereas constitutive activation of p38␣ and p38␥ caused premature senescence. Furthermore, upon activation by oncogenic ras, p38␥ stimulated the transcriptional activity of p53 by phosphorylating p53 at Ser 33 , suggesting that the ability of p38␥ to mediate senescence is at least partly achieved through p53. However, p38␣ contributed to ras-inducted senescence via a p53-indepdendent mechanism in cells by mediating ras-induced expression of p16 INK4A , another key senescence effector. These findings have identified p38␣ and p38␥ as essential components of the signaling pathway that regulates the tumor-suppressing senescence response, providing insights into the molecular mechanisms underlying the differential involvement of the p38 isoforms in senescence induction.The ras proto-oncogenes encode small GTP-binding proteins that transduce growth signals from cell surface (1-3). Aberrant activation of ras is a crucial step in tumor formation. Constitutive activation of ras genes, either through point mutations or overexpression, is associated with a wide variety of human tumors at high frequency and contributes to the initiation and maintenance of multiple tumorigenic phenotypes in these cancers (4 -11). However, in early-passage primary human and rodent cells, activated ras causes a permanent proliferative arrest known as premature senescence, because of its phenotypic similarities to replicative senescence observed in late-passage cells (12). Other oncogenes, such as E2F1 and raf, or inactivation of certain tumor suppressor genes, also induce senescence in normal human cells (13-15). The existence of the premature senescence response to oncogene activation implies that like apoptosis, oncogene-induced senescence serves as an anti-tumorigenic defense mechanism. Indeed, it has been well documented that cellular transformation by ras requires cooperation from immortalizing oncogenes that overcome the senescence response, such as those inactivating p53 (8,16,17). Recent studies have also demonstrated that senescent cells can be detected in early-stage premalignant lesions of lung, pancreas, skin, and prostate in both human cancer patients and mouse tumor models and that disruption of s...