Protection of CHO cells by mutant forms of O6-alkylguanine-DNA alkyltransferase from killing by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine or O6-benzyl-8-oxoguanine.
O6-Benzylguanine (BG) is an inactivator of human O6-alkylguanine-DNA alkyltransferase (AGT) currently undergoing clinical trials to enhance cancer chemotherapy by alkylating agents. Mutant forms of AGT resistant to BG in vitro were expressed in CHO cells to determine if they could impart resistance to killing by the combination of BG and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). All the BG-resistant mutant proteins tested (P140A, P140K, P138M/V139L/P140K, G156A, P140A/G160R, and G160R) showed a reduced rate of reaction with methylated DNA substrates in vitro. However, when expressed in equal amounts in CHO cells, mutants P140A, P140K, P138M/V139L/P140K, and G160R gave levels of protection from the chloroethylating agent BCNU equivalent 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 the cell. AGT activity was readily lost when CHO cells expressing wild-type AGT were exposed to BG or its 8-oxo metabolite (O6-benzyl-8-oxoguanine), but cells expressing mutants P140A or G160R required 30-fold higher concentrations and cells expressing mutants P140K or P138M/V139L/P140K were totally resistant. When cells were treated with 80 microM BCNU plus BG or 8-oxo-BG, those expressing wild-type AGT were killed when inhibitor concentrations of up to 500 microM were used, whereas cells expressing P140K or P138M/V139L/P140K showed no effect, and cells expressing P140A or G160R showed an intermediate 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 BG-resistant AGT in hematopoietic cells is used to reduce toxicity.
Loktionova
N A
NA
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, 17033, USA.
Xu-Welliver
M
M
Crone
T M
TM
Kanugula
S
S
Pegg
A E
AE
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D016466:CHO Cells; D002330:Carmustine / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004347:Drug Interactions; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; C097065:O(6)-benzyl-8-oxoguanine; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine