Effect of O6-benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells.
O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.
Cai
Y
Y
Section of Hematology-Oncology, Department of Medicine, Committee on Clinical Pharmacology, Cancer Research Center, University of Chicago, Chicago, Illinois 60637, USA.
Ludeman
S M
SM
Wilson
L R
LR
Chung
A B
AB
Dolan
M E
ME
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D016466:CHO Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002699:Chlorambucil / Q000633:toxicity; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D008558:Melphalan / Q000633:toxicity; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors; D010752:Phosphoramide Mustards / Q000633:toxicity*
D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; D010752:Phosphoramide Mustards; C064976:O(6)-benzylguanine; C030090:phosphoramide mustard; D002699:Chlorambucil; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D008558:Melphalan