Development of an O6-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O6-benzylguanine to the protein
Development of an O6-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O6-benzylguanine to the protein
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DOI：
10.1016/j.ab.2008.08.009
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发表时间：
2008-12-01
影响因子：
2.9
通讯作者：
Sartorelli, Alan C.
中科院分区：
文献类型：
--
作者：
Ishiguro, Kimiko;Shyam, Krishnamurthy;Sartorelli, Alan C.
尽管已知（i）O-6-烷基鸟嘌呤-DNA烷基转移酶（AGT）赋予肿瘤细胞对鸟嘌呤O-6-靶向药物（如氯雷他嗪、卡莫司汀和替莫唑胺）的抗性，并且（ii）肿瘤中的AGT水平高度可变，但在治疗前测量肿瘤中的AGT活性不是常规的临床实践。这部分源于缺乏可靠的临床AGT试验;因此，基于放射性苄基残基从[苯-（3）H]O-6-苄基鸟嘌呤（[（3）HIBG）转移至AGT，设计了一种简单的AGT试验。该试验包括用[H-3]BG孵育完整细胞或细胞匀浆，并测量70%甲醇可沉淀部分中的放射性。在细胞匀浆中回收了完整细胞中约85%的AGT。通过Western印迹分析检查AGT水平确认AGT测定的准确性，但由于[H-3]BG与黑色素结合而导致含黑色素细胞中出现假阳性结果除外。人和鼠AGT的二级动力学常数分别为1100和380 M（-1）s（-1）。各种人类细胞系中的AGT水平范围从小于500分子/细胞（检测限）到45，000分子/细胞。啮齿动物细胞系通常缺乏AGT表达，并且啮齿动物细胞中的AGT水平远低于人类细胞。(c)2008年爱思唯尔公司版权所有
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