Inactivation of human O(6)-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O(6)-benzylguanine.
Inactivation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) enhances tumor cell killing by therapeutic alkylating agents. O(6)-Benzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are much more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examined the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide containing b(6)G (11-mpBG) when modified with terminal methylphosphonate linkages to protect it from nucleases. This modification did not reduce the ability to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED(50) value of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligodeoxyribonucleotide containing O(6)-methylguanine (m(6)G) was also found to be a good substrate (ED(50) value of 10 nM), but the benzylated form was repaired more rapidly and preferentially. When added to HT29 cell cultures, 5 microM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 microM, suggesting that the benzyl group allows better uptake into the cell. However, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved.
Pegg
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AE
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA. aep1@psu.edu
Goodtzova
K
K
Loktionova
N A
NA
Kanugula
S
S
Pauly
G T
GT
Moschel
R C
RC
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004794:Enzyme Repression; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D019073:HT29 Cells; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000737:chemistry
D018906:Antineoplastic Agents, Alkylating; D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; C064976:O(6)-benzylguanine; C032627:methylphosphonic acid; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine