Sustained post transcriptional p21WAF1 expression is dependent on ERK activation In order to establish whether sustained activation of the MEK/ERK pathway plays a role in post transcriptional mediated p21WAF1 accumulation, RD cells, pre treated with U0126, were Sorafenib VEGFR-2 treated with TPA for different time intervals. p21WAF1 protein accumulation was insensitive to MEK inhibitor treatment for Inhibitors,Modulators,Libraries up to 2 days, after which it dropped to the level of untreated control cells, paralleling U0126 treatment alone. These results suggest that the post transcriptional mechanism of p21WAF1 induction might be strictly dependent on activated MEK/ ERK pathway. Constitutively active MEK1 or MEK2 and ERK1 and ERK2 siRNA transient trans fection experiments demonstrated that activated MEKs/ ERKs are upstream pathways of p21WAF1 expression.

The forced expression of MEK1 or MEK2 induced both ERK phosphorylation and p21WAF1 increased expression. Inhibitors,Modulators,Libraries RNA interference experiments were performed with both ERK1 and ERK2 siRNA in order to prevent TPA Inhibitors,Modulators,Libraries induced ERK1/2 activation. After 4 days of TPA treatment, we observed a lack of p21WAF1 expression combined with the down regulation of total and phospho ERKs in ERK1 and ERK2 siRNA co transfected cells, while ERK activation and p21WAF1 expression were present in control trans fected cells. All these experiments demonstrate that the post transcriptional mechanism of p21WAF1 expression is dependent on the ERK pathway in RD cells.

Dependence of p21WAF1 transcriptional expression and myogenic differentiation on the p38 pathway We have previously shown that the p38 pathway is acti vated concomitantly in both the activation and down reg ulation of the Inhibitors,Modulators,Libraries ERK pathway, and that its inhibition prevents myogenic differentiation and reverts the growth arrest state after prolonged treatment. Therefore, we investigated whether ERKs and p38 cooperate, as has recently been demonstrated in CC139 and Rat 1 cells, or act separately to induce p21WAF1 and growth arrest. For this purpose, RD cells were treated with TPA and U0126 in the presence or absence of the p38 specific inhibitor SB203580. SB203580 treatment did not alter TPA induced p21WAF1 expression, but it did reduce U0126 induced p21WAF1 expression, particularly after pro longed treatments . alone it only slightly enhanced the p21WAF1 basal level.

Furthermore, p38 inhibition affected neither the TPA induced expression of cyclin D1 nor the decrease medi ated by MEK/ERK inhibition. The pRb Inhibitors,Modulators,Libraries phosphorylation www.selleckchem.com/products/chir-99021-ct99021-hcl.html status is, moreover, modulated by SB203580 after 2 days in U0126 treated cells. Conversely, pRb phosphorylation in TPA treated cells is insensitive to SB203580. SB203580 alone does not affect pRb phosphorylation and the slight increase in p21WAF1 expression at prolonged treatments is not suffi cient to induce hypo phosphorylation of pRb.