Fig. 6.

Cotreatment with O⁶-benzylguanine (O⁶-BG) sensitizes cells expressing wild-type (WT) and variant eGFP (enhanced green fluorescent protein)-MGMT (O⁶-methylguanine-DNA methyltransferase) constructs to temozolomide (TMZ) in a dose-dependent manner. (A) MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays were performed as described for Fig. 4A, except that cells were treated with 25 μM O⁶-BG in addition to the indicated concentration of TMZ. WT and variant eGFP-MGMT all conferred a similar degree of resistance to the combination of TMZ and O⁶-BG. Survival is expressed as percentage of untreated cells (0 μM TMZ, 0 μM O⁶-BG [dimethyl sulfoxide (DMSO) only]) for the same corresponding cell line after subtracting background absorbance from all values. A total of at least three separate experiments were performed in multiples of six each on two clones for each eGFP-MGMT variant or eGFP control, and data are expressed as means ± SEMs (n = 3). There was no significant effect of O⁶-BG by itself on survival of the various cell lines. Resistance to TMZ was maintained when cotreated with 100 μM for eGFP-MGMT–containing cell lines versus eGFP control cells (*p < 0.05). However, when cell lines were cotreated with 500 and 1,000 μM TMZ, there was no significant difference between eGFP-MGMT–containing cell lines and eGFP control cells, or between WT and the variant constructs. (B and C) Clonogenic assays were performed as described for Fig. 4B except that cells were treated with 25 μM O⁶-BG in addition to the indicated concentrations of TMZ. (B) Survival is expressed as percentage of untreated cells (0 μM TMZ, 0 μM O⁶-BG [DMSO only]) for the same corresponding cell line. A total of at least three separate experiments were performed in duplicate on two clones for each eGFP-MGMT variant or eGFP control, and data are expressed as mean ± SEM (n = 3). This shows that O⁶-BG alone has modest toxicity. (C) To visualize the isolated effects of TMZ, we renormalized the same data from A and expressed survival as percentage of the 0 μM TMZ/25 μM O⁶-BG–treated value for the same corresponding cell line after subtracting background absorbance from all values. Resistance to TMZ was maintained when cotreated with O⁶-BG and 100 μM and 500 μM for eGFP-MGMT–containing cell lines versus eGFP control cells (*p < 0.05). However, when cell lines were cotreated with O⁶-BG and 1,000 μM TMZ, there was no significant difference between eGFP-MGMT–containing cell lines versus eGFP control cells or between WT and the variant constructs.