Figure 9.

Drug resistance after repeated low-dose TMZ plus O6BG treatment. (A) Treatment schedule was as follows: 3 × 10⁶ A2058 cells were seeded into a 25-cm plate and treated with 10 µM TMZ in the presence of O6BG once daily over 5 days. After about 15 days, there were approximately 250 colonies, which were pooled and expanded. After sufficient proliferation, 3 × 10⁶ of these cells were seeded again into a 25-cm plate and exposed to a single treatment of 100 µM TMZ in the presence of O6BG. Another 15 days later, approximately 2000 colonies emerged, which were sub-cultured to allow for the isolation of 12 individual clones (numbered 71 through 82), which were analyzed by WB and CFA. (B) Cell lysates were prepared from all clones and analyzed by WB for MGMT and MSH6 protein levels. Lysates from A375 and A2058 cells were included for comparison. (Clones 81 and 82 are not included in this figure; clone 76 was lost.) Numbers under the blot indicate quantification of MGMT bands, with reference to the actin signal, and relative to Clone 79 (set at 1.0); <0.1 indicates below detection limit. (C) To confirm drug sensitivity in correlation with MGMT or MMR deficiency, clones 71 and 79 were treated with increasing concentrations of TMZ in the presence or absence of O6BG, and CFA was performed 12 days later (n = 3, ±SE).