[Mutation of human O6-alkylguanine-DNA alkyltransferase confers resistance to O6-benzylguanine].

OBJECTIVE

O6-alkylguanine-DNA alkyltransferase (O6-AGT), capable of repairing DNA damage, is responsible for tumor cell resistance to nitrosourea. While O6-benzylguanine as a selective inhibitor of AGT helps reverse drug resistance, it would aggravate myelo-suppression. This investigation is to generate AGT mutant and see if it would confer resistance to O6-benzylguanine-induced inhibition but leave its alkyltransferase activity intact.

METHODS

Human O6-methylguanine-DNA methyltransferase (MGMT) cDNA was mutated by site-directed mutagenesis. The mutant cDNA was transferred into E. coli and the protein expressed was purified. The activity of the mutant MGMT was determined in vitro with O6-(3H)-methylguanine-DNA as substrate.

RESULTS

Two mutant MGMT proteins were obtained: G156A and P140A, with glycine-to-alanine mutation at position 156 and proline-to-alanine mutation at position 140, respectively. The AGT activity of both mutants was similar to that of the wild type MGMT. However, their resistance to O6-benzylguanine was significantly increased up to 105.8 and 13.5 fold, respectively as compared to that of the wild type MGMT.

CONCLUSION

The results suggested that transduction of the mutant MGMT herein reported into hematopoietic progenitor cells may lead to their selective resistance to the combined use of O6-benzylguanine and alkylating agents designed to overcome tumor resistance to nitrosourea treatment.