Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA
Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA
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DOI：
10.1016/s0165-1218(97)00037-2
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发表时间：
1997-06-13
影响因子：
1.9
通讯作者：
Poirier, MC
中科院分区：
文献类型：
--
作者：
Olivero, OA;Chang, PK;Poirier, MC
暴露于顺式二氨二氯铂（II）（顺铂）24小时的中国仓鼠卵巢（CHO）细胞中的DNA加合物水平已显示线粒体（mt）DNA比核（n）DNA高4至6倍（Olivero et al.，突变研究所，346（1995）221）。本研究的目的是了解如果优先顺铂在线粒体DNA中的结合是部分由于缺乏加合物去除线粒体。将中国仓鼠卵巢细胞暴露于50 μ M顺铂中6 h，然后在无顺铂培养基中孵育24和48 h。在30小时时间点（使用顺铂6小时，不使用顺铂24小时），从每个板收获一半的细胞，培养剩余细胞并在54小时收获（使用顺铂6小时，不使用顺铂48小时）。30和54小时时间点分别称为“T-30”和“T-54”。通过解离增强镧系元素荧光免疫测定法（DELFIA），一种灵敏的竞争性微量滴定法，利用针对顺铂修饰的DNA引起的抗血清，在细胞核和线粒体组分的DNA中测定顺铂-DNA加合物。在T-30时，与nDNA相比，在mtDNA中观察到初始更高水平的顺铂-DNA加合物。此外，在T-54的细胞中证明了mtDNA中的加合物缺乏去除。在采用[H-3]胸苷掺入的脉冲追踪实验中记录了DNA复制对DNA加合物的稀释。通过修复相关机制的加合物去除被认为包括总DNA加合物去除和与DNA复制相关的加合物去除之间的差异。最终结果表明，较高的初始结合和缺乏顺铂-DNA加合物的去除似乎有助于在CHO细胞中观察到的优先顺铂-mtDNA结合。
Levels of DNA adducts in Chinese hamster ovary (CHO) cells exposed to cis-diamminedichloroplatinum(II) (cisplatin) for 24 h, have been shown to be 4- to 6-fold higher in mitochondrial (mt) DNA as compared to nuclear (n) DNA (Olivero et al., Mutation Res., 346 (1995) 221). The aim of the present study was to understand if the preferential cisplatin binding in mtDNA is partially caused by lack of adduct removal in the mitochondria. Chinese hamster ovary cells were exposed for 6 h to 50 mu M cisplatin, followed by incubation for 24 and 48 h in cisplatin-free medium. At the 30-h time point (6 h with cisplatin, 24 h without cisplatin), half of the cells from each plate were harvested and the remainder were cultured and harvested at 54 h (6 h with cisplatin, 48 h without cisplatin). The 30- and 54-h time points are called 'T-30' and 'T-54', respectively. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An initial higher level of cisplatin-DNA adducts was observed in mtDNA when compared to nDNA, at T-30. In addition, a lack of removal of adducts in mtDNA was demonstrated in cells at T-54. Dilution of DNA adducts by DNA replication was documented in pulse-chase experiments that employed [H-3]thymidine incorporation. Adduct removal by repair-related mechanisms was considered to comprise the difference between total DNA adduct removal and adduct removal related to DNA replication. The final results demonstrated that both, higher initial binding and lack of removal of cisplatin-DNA adducts appear to contribute to the preferential cisplatin-mtDNA binding observed in CHO cells.