Department of Pharmacology, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, United States.
Biochem Pharmacol. 2010 Nov 1;80(9):1317-25. doi: 10.1016/j.bcp.2010.07.022. Epub 2010 Jul 21.
O(6)-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O(6)-chloroethyl (Onrigin™ and carmustine) and O(6)-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O(6)-methylguanines/cell and ∼300 O(6)-chloroethylguanines/cell, respectively. AGT repaired O(6)-methylguanines until the AGT pool was exhausted, while its repair of O(6)-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O(6)-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O(6)-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.
O(6)-烷基鸟嘌呤-DNA 烷基转移酶（AGT）介导肿瘤对烷化剂的抗性，这些烷化剂会产生鸟嘌呤 O(6)-氯乙基（Onrigin™和卡莫司汀）和 O(6)-甲基（替莫唑胺）损伤；然而，AGT 对这些损伤的保护效率以及给定数量的 AGT 分子产生的对这些试剂的抗性程度尚不清楚。从含有 17000 个 AGT 分子/细胞的 AGT 缺失和 AGT 完整的 HL-60 细胞的细胞毒性差异测量中，AGT 分别对氯乙基化（90CE）和甲基化（KS90）Onrigin™类似物产生 12 倍和 24 倍的抗性。对于 50%的生长抑制，KS90 和 90CE 分别产生 5600 个 O(6)-甲基鸟嘌呤/细胞和约 300 个 O(6)-氯乙基鸟嘌呤/细胞。AGT 修复 O(6)-甲基鸟嘌呤，直到 AGT 池被耗尽，而其对 O(6)-氯乙基鸟嘌呤的修复是不完全的，因为损伤进展到 AGT 无法修复的链间 DNA 交联。因此，对于细胞毒性所需的 O(6)-氯乙基鸟嘌呤损伤的数量较少，这解释了 AGT 对 90CE 产生的明显抗性（12 倍）。将人或鼠 AGT 转染到 AGT 缺陷的可移植肿瘤细胞（即 EMT6、M109 和 U251）中，产生了表达 AGT 的转染子，其表达 AGT 的分子数从 4000 到 700000 个/细胞不等。使用这些转染子进行的 90CE 体外生长抑制测定表明，AGT 引起浓度依赖性抗性，最高可达约 10000 AGT 分子/细胞。这一发现得到了体内研究的证实，在这些研究中，表达 4000 和 10000 个鼠 AGT 分子/细胞使 EMT6 肿瘤分别对 Onrigin™部分和完全耐药。这些研究表明，Onrigin™的抗肿瘤活性源于鸟嘌呤 O(6)-氯乙基化，并定义了否定其抗肿瘤活性的 AGT 阈值浓度。
