Characterization of E6201 response in a subset of melanoma cell lines. A. DNA content as a measure of cell cycle progression with E6201 treatment. A subset of sensitive and resistant melanoma cell lines were treated with 200 nM E6201 for 48 hours, after which cell cycle analysis was performed by propidium iodide staining and flow cytometry. The percent increase in cells in G1 phase with E6201 therapy is shown for 13 sensitive and 2 resistant melanoma cell lines. E6201 treatment resulted in an accumulation of cells in G1 phase of the cell cycle in all sensitive melanoma lines studied. No such accumulation was noted in E6201-resistant melanoma lines B. Cell death as assessed by Annexin V positivity after treatment of E6201. After 72 hours of 200 nM E6201 treatment, melanoma cells were analysed for Annexin V-FITC–positive cells by flow cytometry. MEK inhibition by E6201 resulted in a greater than 2-fold increase in Annexin V–positive cells, indicative of apoptosis, in most (11 out of 13) sensitive melanoma lines. No such increase was observed in 2 melanoma cell lines previously shown to be resistant to E6201. C. Determination of DNA fragmentation after E6201-induced cell death. After 72 hours of 200 nM E6201 treatment, a Cell Death Detection ELISA (Roche) was performed as per the manufacturer’s instructions. Treatment with E6201 resulted in increased DNA fragmentation (greater than 2-fold) in 10 out of 13 sensitive melanoma lines and did not induce significant DNA fragmentation in any of the E6201-resistant melanoma cell lines.