Characterization of the P140K,PVP(138-140)MLK,and G156A O 6 -Methylguanine-DNA Methyltransferase Mutants: Implications for Drug Resistance Gene Therapy

The G156A O⁶-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O⁶-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O⁶-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 CD34⁺ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 m M BG, while G156A had a BG ED₅₀ of 15 muM , compared with 0.1 muM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O⁶- methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O⁶-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant 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 (less than or equal to 25 muM ) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.