Characterization of the P140K, PVP(138-140)MLK, and G156A O-6-methylguanine-DNA methyltransferase mutants: Implications for drug resistance gene therapy

The G156A O-6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encod ed by the G156A O-6-methylguanine-DNA methyltransferase gene (MGMT), is res istant to O-6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD 34(+) cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relativ e to G156A, and suggests that gene transfer of P140K may confer improved he matopoietic cell protection. To address this hypothesis, we measured BG + B CNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGM T-transduced K562 cells. In addition, we performed a detailed characterizat ion of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retrov iral producer cells. In K562 cell extracts, the MLK and P140K mutants retai ned full activity at doses up to 1 mM BG, while G156A had a BG ED50 Of 15 m u M, compared with 0.1 mu M for wtAGT. In the absence of BG, the G156A prot ein possessed a 56% reduction in specific O-6-methyltransferase activity co mpared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific act ivities, although the O-6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stabl e, with half-lives of greater than 18 hr. In contrast, only 20% of the G156 A protein was stable after 12 hr in cycloheximide and, interestingly, the r emaining protein appeared to retain most of the activity present in non-cyc loheximide-treated cells. Differences in BG resistance, activity, and stabi lity between P140k, MLK, and G156A suggest that P140K; may be the optimal m utant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (l ess than or equal to 25 mu M) achieved in clinical trials, suggesting simil ar efficacy in many irt vivo applications.