Adherent colonies were stained for 2 to 10 minutes with 1% crystal violet in methanol, rinsed in distilled water, and dried before the adsorbed dye was re solubilized with methanol containing 0. 1% SDS by gentle agitation for 1 to 4 hours at room temperature. Dye concentration was quantified using ELx800 Universal Microplate Reader at 595 nm. For quantitation, technical support readings of absorbance at 595 nm were normalized to those obtained from untreated cells, assumed to yield 100% cell survival, and empty wells, considered to be 0% cell survival. Cytotoxicity results were analyzed as described. Briefly, after each experiment, survival curves were generated, for cisplatin and each FA pathway inhibitor alone and for the drug combinations.

The LD50s for each drug in combination were determined, and LD50/ LD500 units were derived as ratio of LD50 Inhibitors,Modulators,Libraries for cisplatin or IR and the FA pathway inhibitor Inhibitors,Modulators,Libraries relative to LD50 of each drug alone for each cell line. Isobolograms were generated at LD50 levels. Each plot pre sents values generated in at least three independent experi ments. In addition, combination index values were calculated by the use of the Chou and Talladay method. An identical analysis was performed at the 70% killing level. Western blot analysis was done as described. Anti FANCD2 and HRP conjugated ECL anti rabbit IgG were used. Films were digitalized using a standard scanner and images processed using ImageJ. Introduction Most melanomas have mutually exclusive activating muta Inhibitors,Modulators,Libraries tions in the mitogen activated protein kinase path way involving NRAS or BRAF genes in melanomas of skin primary, c Kit in acral and mucosal melanomas, and GNAQ and GNA11 in uveal melanomas.

These mutations render melanoma cells independent of the normal receptor tyrosine kinase mediated pathway regulation, and constitutively drive melanoma cells to oncogenic prolifera tion and survival. The most Inhibitors,Modulators,Libraries common of these mutations is the BRAFV600E mutation, present in approximately Inhibitors,Modulators,Libraries 50% of melanomas of skin origin. BRAFV600E mutant cutaneous melanomas are dependent on MAPK signaling for cell cycle progression and proliferation, and have high sensitivity to type I BRAF inhibitors and to MEK inhibitors. Very high response rates and improved survival have been noted with the administration of the type I BRAF inhibitor vemurafenib to patients with BRAFV600E mutant cutaneous metastatic melanoma. Tumor responses were dependent on the presence of the BRAFV600E oncogene and efficient inhibition of the MAPK pathway as detected by decreased phosphor ylation of ERK. Inhibition of the immediately down stream MEK1/2 kinases in BRAFV600E mutant cutaneous melanoma was shown to lead to www.selleckchem.com/products/wortmannin.html marked inhibition of cell proliferation in cell lines.