Characterization of 6-TG–resistant clones. (A) Expression of MMR genes. RNA from representative 6-TG– and MNU-resistant A2780SCA5 clones was amplified by RT-PCR. RNA was included from control cell lines Raji (MMR proficient), SW48 (hMLH1 deficient), LoVo (hMSH2 deficient), and DLD-1 (hMSH6 deficient). Products were separated using agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. ^*Clones JA5 and JA8 arose in different flasks. (B) MMR protein expression. Extracts from parental A2780SCA5, control MMR-defective cells, and JA5 and JA8 were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), blotted, and probed sequentially with antibody against hMLH1, hMSH2, hMSH6, and hPMS2. (C) Mismatch correction in vitro. Extracts (100 μg) of HeLa, A2780SCA5, JA5, or JA8 were incubated with a standard nicked circular DNA substrate containing a T/C mispair. DNA was recovered and digested with MluI, and products were separated using agarose gel electrophoresis. The arrow indicates a band that was diagnostic for correction. (D) 6-TG (left) and MNU (right) sensitivity of JA5 and JA8. Cells were treated as described in “Patients, materials, and methods,” and survival was determined by clonal assay. A2780SCA5 (♦); A2780MNU1 (▪); JA5 (▴); JA8 (•). Error bars represent standard deviations.