title: Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-benzylguanine to the protein.
abstract: Although it is known that (i) O(6)-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O(6)-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-3H]O(6)-benzylguanine ([3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M(-1)s(-1), respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ishiguro
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Shyam
authors.forename: Philip G
authors.initials: PG
authors.lastname: Penketh
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D057075:Enzyme Assays / Q000379:methods*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D011506:Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase
references.citation: Cancer Res. 1999 Feb 15;59(4):793-7
references.pmid: 10029064
references.citation: Cancer Treat Rev. 1997 Jan;23(1):35-61
references.pmid: 9189180
references.citation: Cancer Res. 2001 Apr 1;61(7):3033-8
references.pmid: 11306484
references.citation: Cancer Res. 1994 Dec 1;54(23):6221-7
references.pmid: 7954470
references.citation: J Invest Dermatol. 2007 Apr;127(4):751-61
references.pmid: 17218941
references.citation: Cancer Res. 1993 Jul 15;53(14):3416-20
references.pmid: 8324751
references.citation: J Cell Physiol. 1984 Nov;121(2):383-90
references.pmid: 6593322
references.citation: Carcinogenesis. 2003 Apr;24(4):625-35
references.pmid: 12727789
references.citation: Cancer Res. 1987 Dec 1;47(23):6229-35
references.pmid: 2824027
references.citation: J Mol Biol. 1993 Jun 20;231(4):950-9
references.pmid: 8515475
references.citation: Biochemistry. 1996 Jan 30;35(4):1328-34
references.pmid: 8573590
references.citation: J Pharmacol Exp Ther. 2001 May;297(2):524-30
references.pmid: 11303039
references.citation: Biochem Pharmacol. 2000 Feb 1;59(3):283-91
references.pmid: 10609557
references.citation: Chembiochem. 2001 Apr 2;2(4):285-7
references.pmid: 11828457
references.citation: Br Med Bull. 2008;85:17-33
references.pmid: 18245773
references.citation: J Med Chem. 1998 Dec 17;41(26):5265-71
references.pmid: 9857094
references.citation: Proc Natl Acad Sci U S A. 1990 Jul;87(14):5368-72
references.pmid: 2164681
references.citation: Cancer Res. 1995 Sep 15;55(18):4085-91
references.pmid: 7664284
references.citation: Anal Biochem. 2003 Oct 1;321(1):38-43
references.pmid: 12963053
references.citation: N Engl J Med. 2005 Mar 10;352(10):997-1003
references.pmid: 15758010
references.citation: Cancer Invest. 1997;15(6):588-98
references.pmid: 9412665
references.citation: J Clin Oncol. 2007 Jan 1;25(1):25-31
references.pmid: 17146105
references.citation: Carcinogenesis. 1986 May;7(5):745-9
references.pmid: 3698202
references.citation: J Biol Chem. 1983 Feb 25;258(4):2327-33
references.pmid: 6822564
references.citation: J Med Chem. 1996 Feb 2;39(3):796-801
references.pmid: 8576923
references.citation: Mutat Res. 1990 Nov-Dec;233(1-2):117-26
references.pmid: 2233793
references.citation: J Clin Oncol. 2002 May 1;20(9):2388-99
references.pmid: 11981013
references.citation: Methods Mol Biol. 2000;152:49-61
references.pmid: 10957968
references.citation: Cancer Lett. 1984 Jun;23(2):213-21
references.pmid: 6744245
references.citation: Carcinogenesis. 2002 May;23(5):823-30
references.pmid: 12016156
references.citation: Cancer Res. 1991 Aug 15;51(16):4131-4
references.pmid: 1868433
references.citation: Cell. 1976 Jun;8(2):263-9
references.pmid: 971485
references.citation: Mutat Res. 2000 Apr;462(2-3):83-100
references.pmid: 10767620
references.citation: Cancer Res. 1997 Sep 1;57(17):3672-7
references.pmid: 9288770
references.citation: Mol Cancer Ther. 2005 Nov;4(11):1755-63
references.pmid: 16275997
references.citation: DNA Repair (Amst). 2007 Aug 1;6(8):1071-8
references.pmid: 17482892
references.citation: J Med Chem. 1996 Dec 20;39(26):5192-201
references.pmid: 8978847
references.citation: Biochemistry. 1993 Nov 16;32(45):11998-2006
references.pmid: 8218276
references.citation: Carcinogenesis. 1996 Feb;17(2):219-24
references.pmid: 8625442