Protection of CHO cells by mutant forms of O(6-)alkylguanine-DNA alkyltransferase from killing by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) plus O-6-benzylguanine or O-6-benzyl-8-oxoguanine

O-6-Benzylguanine (BG) is an inactivator of human O-6-alkylguanine-DNA alky ltransferase (AGT) currently undergoing clinical trials to enhance cancer c hemotherapy by alkylating agents. Mutant forms of AGT resistant to BG in vi tro were expressed in CHO cells to determine if they could impart resistanc e to killing by the combination of BG and 1,3-bis (2-chloroethyl)-1-nitroso urea (BCNU). All the BG-resistant mutant proteins tested (P140A, P140K, P13 8M/V139L/P140K, G156A, P140A/G160R, and G160R) showed a reduced rate of rea ction with methylated DNA substrates in vitro. However, when expressed in e qual amounts in CHO cells, mutants P140A, P140K, P138M/V139L/P140K, and G16 0R gave levels of protection from the chloroethylating agent BCNU equivalen t to that of wild-type AGT. This indicates that a 10-fold reduction in rate constant did not prevent their ability to repair chloroethylated DNA in th e cell. AGT activity was readily lost when CHO cells expressing wild type A GT were exposed to BG or its 8-oxo metabolite (O-6-benzyl-8-oxoguanine), bu t cells expressing mutants P140A or G160R required 30-fold higher concentra tions and cells expressing mutants P140K or P138M/V139L/P140K were totally resistant. When cells were treated with 80 mu M BCNU plus BG or 8-oxo-BG, t hose expressing wild-type AGT were killed when inhibitor concentrations of up to 500 mu M were used, whereas cells expressing P140K or P138M/V139L/P14 0K showed no effect, and cells expressing P140A or G160R showed an intermed iate resistance. These results suggest that: (i) appearance of BG-resistant mutant AGTs may be a problem during therapy, and (ii) the P140K mutant AGT is an excellent candidate for gene therapy approaches where expression of a BC-resistant AGT in hematopoietic cells is used to reduce toxicity. (C) 1 999 Elsevier Science Inc.