S1and using activated c-Raf or MEK or were treated with TPA (and and was counted (blue columns) and analyzed for SA–Gal staining (red columns) 12 days after transfection. growth factor receptors to signal receivers in various cell compartments (2). Amplifications of or activating point mutations in family genes are frequently found in many types of human cancers. and its family members, and family genes has been linked to the development of many types of human tumors and often correlates with poor prognosis. In addition, point mutations at or near the phosphorylation site Thr-58 are often found in lymphomas (4). Because phosphorylated Thr-58 is targeted by the SCFFbw7/Cdc4 E3 ligase for rapid turnover via the proteasome pathway, mutations at this site result in stabilization and accumulation of Myc (5). Phosphorylation (+)-Clopidogrel hydrogen sulfate (Plavix) of Thr-58 requires a priming phosphorylation at Ser-62 by proline-directed kinases, such as Erk and cyclin-dependent kinase (Cdk) 1 (6, 7). (+)-Clopidogrel hydrogen sulfate (Plavix) The tumorigenic potentials of Myc and Ras are limited by the activation of an apoptotic response by Myc (3) and by the induction of premature cellular senescence by Ras (8). Cellular senescence is a state of irreversible growth arrest that normal cells undergo eventually as a result of telomere erosion, but it can be induced prematurely during inappropriate activation of oncogenes. This often involves triggering a DNA damage response as a result of replicative stress or generation of reactive oxygen species, and it is associated with increased levels of the tumor suppressor p53 and the Cdk-inhibitor p16INK4a (9, 10). Studies during recent years have, however, revealed that antitumor programs like differentiation, apoptosis, and cellular senescence can still exist latently in tumor cells. Potentially, these processes can be reactivated if the oncogene, which promotes tumorigenesis, Mouse monoclonal to TIP60 is inactivated (11, 12). For instance, in mouse tumor models driven by regulatable Myc, switching off Myc was shown to be sufficient for sustained tumor regression of several types of cancer (for reviews, see refs. 11C13). Recently, Myc inhibition using a dominant-negative approach also resulted in regression of Ras-dependent (+)-Clopidogrel hydrogen sulfate (Plavix) tumors, although normal tissues were spared, substantiating the suitability of Myc as a therapeutical target in Myc- and Ras-driven tumors (14). This emphasizes the urge to find drugs that can target Myc and/or Ras activity. It still remains unclear how Myc and Ras cooperate. Previous work demonstrated that Ras suppresses Myc-induced apoptosis (15). Here, we provide evidence that Myc contributes to malignant transformation by repressing Ras-induced senescence and, furthermore, we define how this is achieved and regulated mechanistically. Results Repression of Ras-Induced Senescence by Myc Depends on Ser-62 Phosphorylation. Using primary rat embryo fibroblasts (REFs), we confirmed that oncogenic H-Ras induced senescence as scored by senescence-associated -Gal (SA–Gal) activity (Fig. 1 and and and and and and S2and and Fig. S1and using activated c-Raf or MEK or were treated with TPA (and and was counted (blue columns) and analyzed for SA–Gal staining (red columns) (+)-Clopidogrel hydrogen sulfate (Plavix) 12 days after transfection. Contr, control. Phosphorylation of Ser-62 Is Mediated by Cyclin E/Cdk2. To identify Ser-62 kinases, U2OS cells were transfected with Myc-T58A to avoid cross-talk between the two sites (Fig. S1and Fig. S2and and and and and and data not shown) nor did CVT-2584 inhibit proliferation in the absence of Myc and Ras (Fig. 2and Fig. S3and and and Fig. S3and and and Fig. S3and Fig. S3and and and is a component of the p16Ink4a-Rb-pathway, one of the major pathways controlling oncogene-induced senescence (9, 10). In the parental U-937-GTB cells, less Myc bound to the promoter, as expected, and the Cdk2 signal was hardly above background (Fig. S4genes and express high levels of c-Myc, Cdk2 clearly associated with chromatin (Fig. S4(promoter, which is repressed by (+)-Clopidogrel hydrogen sulfate (Plavix) Myc (Fig. 5, shutoff may therefore be important for long-term maintenance of oncogene-induced senescence. Moreover, the Cdk2 inhibitor CVT-313 caused reduced association of total and phosphorylated Myc at these promoters, comparable to IFN- + TPA treatment (Fig. 5, promoter after treatment with IFN- + TPA (Fig. S4((((and genes were repressed by IFN- + TPA and by the Cdk2 inhibitor CVT-313 (Fig. 6and genes were strongly induced (Fig. 6and and knockout human fibroblasts expressing lower levels of c-Myc (22). Our finding that Myc represses Ras-induced cellular senescence is probably related to other well-known attributes of Myc, such.