Owing to ERK inhibition, the level of phosphorylated c Myc was markedly reduced before c Myc down regulation began. That ERKs are upstream kinases of c myc in RD cells, as suggested by U0126 experiments, was further demonstrated by RNA interference experiment with ERK1, ERK2, screening library ERK1/ERK2 siRNA in transient transfection. After 3 days of transfec tion, we observed a down regulation Inhibitors,Modulators,Libraries of total and phos pho ERKs and a lack of c Myc phosphorylation particularly in ERK2 and ERK1/ERK2 siRNA transfected cells. While the expression level of Max isoforms, which heterodimerize with c Myc, was unaf fected, the amount of c Myc associated with Max was dramatically reduced in U0126 treated cells, as shown by immunoprecipitation experiments. Equal amounts of Max were detected in the immunocom plex.

Taken together, these results indicate that c Myc is a down stream target of ERKs and MEK/ERK inhi bition mediates loss of c Myc and of the c Myc/Max het erodimer, Inhibitors,Modulators,Libraries providing one possible molecular mechanism of growth arrest i. e. Inhibitors,Modulators,Libraries that induced by the MEK inhibitor U0126. Effects of U0126 on G0/G1 arrest and cell cycle regulator expression in RD cell lines Since c Myc expression is well known to be down regu lated during inhibition of cell growth we addressed whether the observed c Inhibitors,Modulators,Libraries Myc down regulation is simply a consequence of cessation of cell growth due to U0126 treatment. To this purpose we performed a time course experiment with or without U0126 treatment of RD cells that were subsequently processed for FACS and immunoblotting analysis.

As shown in Figure 2A, while c Myc level was already significantly reduced at 3 hrs of U0126 treatment the percentage of cells in G0/G1 was unchanged compared to untreated cells. Subsequently at 12 hrs, the percentage of cells in G0/G1 phase was highly Inhibitors,Modulators,Libraries increased by U0126 treatment, thus fol lowing of several hours the c Myc down regulation. This result demonstrated that, since in the U0126 treated cells the loss of c Myc pre ceded their withdrawal from cell cycle, c Myc down regu lation is not a consequence of the cessation of cell growth but rather it might cause growth arrest. In light of these results we hypothesised that c Myc dependent cell cycle proteins expression was altered too. In fact, it has been suggested that c Myc mediated cell transformation involves modulation of cell cycle protein expression.

Thus, we investigated whether cell cycle pro teins were modulated in U0126 treated RD cells by immunoblotting experiments. Figure 2B shows that U0126 treatment induced a decrease in cyclin E2, which was stronger than that observed in cyclin E1, from 12 hrs up to 4 days, and a decrease in cyclin A and B accumula tion which started at 1 day former and persisted thereafter. Moreover, a reduction in CDK2, which forms com plexes with cyclin E, A and B, started one day after treat ment. Of note, we have recently shown that U0126 induced a decrease in cyclin D1 and an increase in CKI, p21WAF1 and p27.