Ishiguro Kimiko, Seow Helen A, Penketh Philip G, Shyam Krishnamurthy, Sartorelli Alan C
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Mol Cancer Ther. 2006 Apr;5(4):969-76. doi: 10.1158/1535-7163.MCT-05-0532.
Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O6-alkylguanine-DNA alkyltransferase-transfected L1210 cells (LC10, 1.4 versus 31 micromol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 micromol/L, respectively, produced similar degrees of G2-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O6-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses.
氯雷他嗪是一种抗肿瘤磺酰肼前药，可产生氯乙基化和氨基甲酰化物质。使用仅具有氯乙基化或氨基甲酰化功能的类似物，在L1210白血病细胞中分析了这些物质的细胞毒性效力。克隆形成试验表明，仅具有氯乙基化作用的试剂1,2-双（甲基磺酰基）-1-（2-氯乙基）肼（90CE）对野生型和O6-烷基鸟嘌呤-DNA烷基转移酶转染的L1210细胞产生明显的差异细胞毒性（LC10，1.4对31 μmol/L），表明大部分细胞毒性是由于鸟嘌呤O-6位的DNA烷基化。与DNA鸟嘌呤的O-6氯乙基化进展为链间交联的概念一致，彗星试验通过测量链断裂引起的DNA迁移减少来测量DNA交联，结果表明氯雷他嗪和90CE，但不是仅具有氨基甲酰化作用的试剂1,2-双（甲基磺酰基）-1-[(甲氨基)羰基]肼（101MDCE），会产生DNA交联，并且在等摩尔浓度下，氯雷他嗪比90CE引起更多的DNA交联。细胞周期分析表明，90CE和101MDCE分别在5和80 μmol/L的浓度下产生相似程度的G2-M期阻滞。90CE在过夜孵育后对DNA合成产生选择性抑制，而101MDCE导致对RNA、DNA和蛋白质合成的快速且非选择性抑制。90CE和101MDCE均诱导组蛋白H2AX磷酸化，尽管动力学不同。这些结果表明：（a）肿瘤细胞和宿主细胞中O6-烷基鸟嘌呤-DNA烷基转移酶的差异表达似乎是氯雷他嗪发挥肿瘤选择性的原因；（b）101MDCE增强DNA交联活性；（c）90CE在低于引起细胞周期和大分子合成改变的浓度下诱导细胞死亡。
