Intriguingly, while p21WAF1/CIP1 was degraded rapidly 2 to 4 hours post radiation, p27Kip1 maintained a relatively unchanged level http://www.selleckchem.com/products/lapatinib.html . when p27Kip1 was degraded 8 hours post radiation, p21WAF1/CIP1 levels started to restore. It seems these two CDK inhibitors are orchestrated to ensure a G1 arrest in MiTF expressed A375 cells. Previously we showed that MiTF was Inhibitors,Modulators,Libraries temporarily degraded after elevation of cellular reactive oxygen species levels, a process that was also mediated by Erk1/2 Inhibitors,Modulators,Libraries kinase. Considering that both UVC and ROS causes similar DNA damages and therefore may employ similar repair pathways, the Erk1/2 mediated phos phorylation and degradation of MiTF may reflect a gen eral mechanism of MiTF mediated survival pathways which is outlined in Fig 7.
Inhibitors,Modulators,Libraries Upon UVR or ROS stress, MAP kinase is activated which leads to phosphorylation of MiTF on serine 73 and subsequent degradation of MiTF protein. Inhibitors,Modulators,Libraries The temporary degradation was corre lated with a temporary G1 cell cycle arrest, correspond ing with p21WAF1/CIP1 degradation and re activation, which allows sufficient time for DNA damage repair and ensure of a better cell survival. In response to UVB radiation, MiTF levels were not changed at the examined dose and time range, nor its phosphorylation status. However, MiTF was degraded without obvious band shifting after UVA treat ment. Pre treatment with U0126 also did not prevent MiTF degradation after UVA radiation, suggest ing that after UVA MiTF was not phosphorylated by Erk1/2 kinase, nor was the degradation mediated by phosphorylation.
These data indicate that signaling path ways after UVA, UVB and UVC are different, which is consistent with previous observations that different Inhibitors,Modulators,Libraries wavelengths of UV light trigger different cellular responses. The UVA MiTF signaling pathway is still under intensive investigation in our laboratory. Conclusions In summary, our data indicated that MiTF played an active role in response to UVC radiation by directly linking Erk1/2 and p21WAF1/CIP1 activation. Erk1/2 kinase is downstream of BRAF and NRAS pathways, which are frequently mutated in human melanomas. Recently it was reported that the MiTF pathway was also frequently mutated in human melanomas. Taken together, mutations in these pathways may compromise the cellular defense mechanisms against UV mediated DNA damage and therefore increase the genome instability, eventually leading to melanomagenesis. Methods Cell lines and cell culture Normal human melanocytes were isolated from new born foreskin followed the procedure by Eisinger and Marco, and cultured in MCDB153 medium containing 2% FCS, 0. 3% bovine pituitary extract, 10 ng/mL 12 O tetradecanoylphorbol 13 acetate, make it clear 2 mmol/L CaCl2, 5 ug/mL insulin, and 0. 1 mmol/L IBMX.