Development of an O-6-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-H-3]O-6-benzylguanine to the protein

摘要

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-(3) H]O-6-benzylguanine ([(3)HIBG) to AGT. The assay involves incubation of intact cells or cell homogenates with [H-3]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 [H-3]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380M(-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. (c) 2008 Elsevier Inc. All rights reserved