Figure 3

Determination of the influence of MGMT on alkylation at position O⁶ of guanine by treatment with TMZ and fotemustine. (A) Determination of MGMT activity (fmol mg⁻¹ protein) in 11 melanoma cell lines. Time dependence of the apoptotic response as determined by quantifying the sub-G₁ population by flow cytometry after treatment with 50 μ M TMZ with or without co-treatment with 10 μ M O⁶BG in MeWoF40 cells (B, left panel). Apoptotic response of D05, G361, A375, Malme 3M, and RPMI7951 cells to 50 μ M TMZ with or without co-treatment with O⁶BG 144 h later. For D05, A375, Malme 3M, and RPMI7951 the sensitisation was significant (P<0.05) (B, right panel). Time dependence of the apoptotic response as determined by quantifying the sub-G₁ population by flow cytometry after treatment with 32 μ M fotemustine with or without co-treatment with 10 μ M O⁶BG in MeWoF40 cells (C, left panel). Apoptotic response of D05, G361, A375, Malme 3M, and RPMI7951 cells to 32 μ M fotemustine with or without co-treatment with O⁶BG 144 h later. For D05 and RPMI7951 the sensitisation was significant (P<0.05) (C, right panel). (D) Frequency of apoptosis as determined by quantifying the sub-G₁ population by flow cytometry after treatment with TMZ and fotemustine for 120 h in non-transfected and MGMT transient transfected SK29 cells (^*decrease of apoptotic response in MGMT transfected cells in comparison to control is significant, P<0.01). Expression of MGMT protein was determined by western blot analysis in non-transfected (lane 1) and MGMT transient transfected SK29 cells (lane 2) at the time of drug treatment. (E) Induced level of apoptosis after TMZ (50 μ M) or fotemustine treatment (32 μ M) as a function of MGMT activity level. Data points were fitted to a line depicting an exponential decrease with plateau at 0% induced apoptosis. The correlation is significant.