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.
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.
Ishiguro
Kimiko
K
Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Shyam
Krishnamurthy
K
Penketh
Philip G
PG
Sartorelli
Alan C
AC
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
D011506:Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase
Cancer Res. 1999 Feb 15;59(4):793-7
10029064
Cancer Treat Rev. 1997 Jan;23(1):35-61
9189180
Cancer Res. 2001 Apr 1;61(7):3033-8
11306484
Cancer Res. 1994 Dec 1;54(23):6221-7
7954470
J Invest Dermatol. 2007 Apr;127(4):751-61
17218941
Cancer Res. 1993 Jul 15;53(14):3416-20
8324751
J Cell Physiol. 1984 Nov;121(2):383-90
6593322
Carcinogenesis. 2003 Apr;24(4):625-35
12727789
Cancer Res. 1987 Dec 1;47(23):6229-35
2824027
J Mol Biol. 1993 Jun 20;231(4):950-9
8515475
Biochemistry. 1996 Jan 30;35(4):1328-34
8573590
J Pharmacol Exp Ther. 2001 May;297(2):524-30
11303039
Biochem Pharmacol. 2000 Feb 1;59(3):283-91
10609557
Chembiochem. 2001 Apr 2;2(4):285-7
11828457
Br Med Bull. 2008;85:17-33
18245773
J Med Chem. 1998 Dec 17;41(26):5265-71
9857094
Proc Natl Acad Sci U S A. 1990 Jul;87(14):5368-72
2164681
Cancer Res. 1995 Sep 15;55(18):4085-91
7664284
Anal Biochem. 2003 Oct 1;321(1):38-43
12963053
N Engl J Med. 2005 Mar 10;352(10):997-1003
15758010
Cancer Invest. 1997;15(6):588-98
9412665
J Clin Oncol. 2007 Jan 1;25(1):25-31
17146105
Carcinogenesis. 1986 May;7(5):745-9
3698202
J Biol Chem. 1983 Feb 25;258(4):2327-33
6822564
J Med Chem. 1996 Feb 2;39(3):796-801
8576923
Mutat Res. 1990 Nov-Dec;233(1-2):117-26
2233793
J Clin Oncol. 2002 May 1;20(9):2388-99
11981013
Methods Mol Biol. 2000;152:49-61
10957968
Cancer Lett. 1984 Jun;23(2):213-21
6744245
Carcinogenesis. 2002 May;23(5):823-30
12016156
Cancer Res. 1991 Aug 15;51(16):4131-4
1868433
Cell. 1976 Jun;8(2):263-9
971485
Mutat Res. 2000 Apr;462(2-3):83-100
10767620
Cancer Res. 1997 Sep 1;57(17):3672-7
9288770
Mol Cancer Ther. 2005 Nov;4(11):1755-63
16275997
DNA Repair (Amst). 2007 Aug 1;6(8):1071-8
17482892
J Med Chem. 1996 Dec 20;39(26):5192-201
8978847
Biochemistry. 1993 Nov 16;32(45):11998-2006
8218276
Carcinogenesis. 1996 Feb;17(2):219-24
8625442