ERK1 2 isoforms are the immediate downstream substrates and best studied effectors of dual specificity kinases MEK1 2. To assess the effect of MEK1 2 inhibition on ERK1 2 ac tivation state, mel anoma cultures were treated with MEK162 and compared with untreated controls. We selected one sen sitive and one resistant culture in the WT and B RAF mutant categories. Seeing that all N RAS mutant selleckchem Tipifarnib cul tures were sensitive to MEK162, we selected two sensi tive cultures for these studies. WT, B RAF mutant and N RAS mutant cells were treated with increasing doses of MEK162 or left untreated for 4 and 24 hours. Western blot analysis was performed using phospho ERK1 2, total ERK1 2 and B actin antibodies, and results are shown in Figure 2A.

In the MEK162 resistant melanoma cultures, the baseline level of phospho ERK1 2 and the ratio of phospho ERK1 Inhibitors,Modulators,Libraries 2 to total ERK1 2 was lower compared to sensitive cultures. In MEK162 sensitive melanomas Inhibitors,Modulators,Libraries exposure to MEK162 resulted in a significant decrease in the level of ERK1 2 phosphorylation. Clonogenic assays We next examined the effect of MEK162 on clonogeni city of this panel of six melanoma cultures. Inhibition of colony formation corresponded well to the viability Inhibitors,Modulators,Libraries studies conducted on cells. Among the sensitive melanoma cultures, YUROB was somewhat re sistant at 10 nM MEK162, retaining 80% clonogenicity of the control level, whereas the ability of other sensitive cultures to form colonies at this concentration of MEK162 dropped below 50% of control. The least inhibition was seen with the MEK162 resistant YUVON and YUKSI cells.

Induction of apoptosis by MEK162 The MAPK cascade plays a major role in cell Inhibitors,Modulators,Libraries survival and proliferation. Hence, MEK162 mediated inhibition Inhibitors,Modulators,Libraries of MAPK signaling may result in either cell death, or in hibition of proliferation, or both. Microscopic assess ment of sensitive melanoma cell cultures suggested that MEK162 treatment affects cell survival. Lysates http://www.selleckchem.com/products/brefeldin-a.html prepared from MEK162 treated and vehicle treated cells were resolved by SDS PAGE and probed with an antibody detecting a cleavage product of a known caspase substrate, PARP. Cultures were treated with MEK162 for 72 hours or left untreated. Cell lysates were ana lyzed by western blotting using an antibody recognizing cleaved PARP. Increased levels of cleaved PARP were seen in the sensitive cultures. The most abundant PARP cleavage was seen in the sensitive cul tures, YUMAC, YUDOSO and YUKIM, and to a much lesser extent in YUROB, whereas no accumulation of cleaved PARP was detected in the resistant cultures. To further examine the inhibitory effect of MEK162 on this panel of melanoma cultures, we assessed apoptosis by annexin V propidium iodide labeling.