Figure 2

Light activated DTIC does not methylate DNA. A, Cells were treated with 300 μM DTIC for 24 h or 1 mM TMZ for 3 h, cells were harvested, genomic DNA purified and equal amounts of gDNA were used to determine the level of DNA methylation. ND: not detectable. B, Cells were treated with 300 μM DTIC or 1 mM TMZ for 24 h and stained for γH2AX by immunofluorescence. C and D, Cell lines were grouped upon their MGMT status (MGMT + ve = MGMT proficient; MGMT-ve = MGMT deficient), and the average GI50 for TMZ or DTIC was compared between both groups. Student’s t test compares the average GI50 for MGMT + ve cells vs MGMT-ve cell. n.s.: not significant, *p < 0.05. MGMT + ve cells are significantly more resistant to TMZ than MGMT-ve cells, p = 0.0116. E, Dose response curve of cell survival to TMZ or DTIC in the absence (DMSO) or presence of the MGMT inhibitor lomeguatrib (LOM, 20 μM). A375 or WM1361 cells were treated with lomeguatrib 1 h before addition of different concentrations of DTIC or TMZ. After 5 days cells were stained with toluidine blue and quantified. DMSO treated cells were set as 100%. The GI50 for each combinatorial treatment is shown.