DNA intrastrand cross-link at the 5′-GA-3′ sequence formed by busulfan and its role in the cytotoxic effect
DNA intrastrand cross-link at the 5′-GA-3′ sequence formed by busulfan and its role in the cytotoxic effect
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DOI：
10.1111/j.1349-7006.2004.tb03231.x
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发表时间：
2004-05-01
期刊：
影响因子：
5.7
通讯作者：
Kawanishi, S
中科院分区：
文献类型：
--
作者：
Iwanoto, T;Hiraku, Y;Kawanishi, S
白消安（1,4-丁二醇二甲磺酸酯）已广泛用于治疗慢性粒细胞白血病患者。白消安具有双功能，因此可以有效诱导 DNA 损伤，这可能在细胞毒性中发挥重要作用。在本研究中，我们比较了双功能白消安与单功能甲磺酸乙酯 (EMS) 对人早幼粒细胞白血病 HL-60 细胞的细胞毒性。白消安对细胞生长具有显着的抑制作用，而细胞在 EMS 存在下生长。为了阐明白消安的细胞毒性机制，我们使用从人 p16 肿瘤抑制基因获得的 P-32-5' 末端标记的 DNA 片段研究了白消安诱导的 DNA 损伤。白消安引起的 DNA 损伤呈剂量依赖性，而 EMS 引起的 DNA 损伤很小。使用哌啶和 3-甲基腺嘌呤 DNA 糖基化酶进行的 DNA 测序实验表明，白消安主要在 5'-GA-3' 序列处引起双碱基损伤，在较小程度上也在 5'-GG-3' 序列处引起双碱基损伤。飞行时间质谱分析证实，除了单烷基化之外，白消安还在 5'-GA-3' 序列处形成链内交联。讨论了 5'-GA-3' 序列交联与白消安诱导的细胞毒性相关的机制和作用。
Busulfan (1,4-butanediol dimethanesulfonate) has been used widely for the treatment of patients with chronic myelogenous leukemia. Busulfan is bifunctional and thus may effectively induce DNA damage, which may play an important role in the cytotoxicity. In this study, we compared the cytotoxicity of bifunctional busulfan with that of monofunctional ethyl methanesulfonate (EMS) in human promyelocytic leukemia HL-60 cells. Busulfan showed a significant inhibitory effect on cell growth, whereas the cells grew in the presence of EMS. To clarify the mechanism of cytotoxicity of busulfan, we investigated DNA damage induced by busulfan using P-32-5'-end-labeled DNA fragments obtained from the human p16 tumor suppressor gene. Busulfan induced DNA damage dose-dependently, whereas EMS caused little DNA damage. DNA-sequencing experiments using piperidine and 3-methyladenine DNA glycosylase indicated that busulfan caused double-base lesions mainly at 5'-GA-3' and, to a lesser extent, at 5'-GG-3' sequences. Time of flight mass spectrometry confirmed that busulfan forms an intrastrand cross-link at the 5'-GA-3' sequence, in addition to mono-alkylation. The mechanism and the role of cross-linking at the 5'-GA-3' sequence are discussed in relation to the cytotoxicity induced by busulfan.