Overexpression of the human aldehyde dehydrogenase class I results in increased resistance to 4-hydroperoxycyclophosphamide.
Overexpression of the human aldehyde dehydrogenase class I results in increased resistance to 4-hydroperoxycyclophosphamide.
复制标题
DOI：
--
复制
发表时间：
1996
影响因子：
6.4
通讯作者：
J. Moreb;M. Schweder;A. Suresh;J. Zucali
中科院分区：
文献类型：
--
作者：
J. Moreb;M. Schweder;A. Suresh;J. Zucali
乙醛脱氢酶的过度表达与广泛使用的抗癌药物恶氮磷的耐药性之间存在相关性。为探讨人醛脱氢酶1类(ALDH-1)在其中一种抗药性中的直接作用，将环磷酰胺的活性代谢物4-羟基环磷酰胺(4-HC)、带有野生型ALDH-1正义互补DNA的新霉素可选择质粒或逆转录病毒载体导入K562白血病细胞系。聚合酶链式反应(PCR)分析证实载体DNA在稳定表达的K562细胞中存在。逆转录酶聚合酶链式反应和Northern和Western印迹分析显示，ALDH-1转染组有正义方向的ALDH-1mRNA和蛋白表达，而单独转染组ALDH-1mRNA和蛋白不表达。用分光光度法检测ALDH-1的活性。用不同剂量的4-HC处理稳定表达的K562细胞，用克隆形成细胞半固体培养法检测细胞存活率。结果表明，正义方向转导ALDH-1的K562细胞与野生型或载体转染型K562细胞相比，对4-HC的抗性增强。此外，加入ALDH-1的特异性抑制剂二乙氨基苯甲醛，可恢复表达ALDH-1的K562细胞对4-HC的敏感性。因此，这些数据明确了ALDH-1在保护4-HC细胞毒性中的直接作用。
A correlation between overexpression of aldehyde dehydrogenase and resistance to oxazaphosphorines, widely used anticancer agents, has been shown. To investigate the direct role of the human aldehyde dehydrogenase class 1 (ALDH-1) in the resistance to one of these agents, 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide, neomycin-selectable plasmid or retroviral constructs harboring the wild-type ALDH-1 complementary DNA in the sense orientation were transfected into K562 leukemic cell lines. Polymerase chain reaction (PCR) analysis confirmed the presence of vector DNA in the stably transfected K562 cells. Reverse transcriptase PCR and Northern and Western blot analysis showed expression of ALDH-1 mRNA and protein in the cells transfected with ALDH-1 in the sense orientation but not in cells transfected with vector alone. The activity of the expressed ALDH-1 was demonstrated using spectrophotometric assay. Stably transfected K562 cells were subjected to various doses of 4-HC, and cell viability was assayed using clonogenic cell culture in semisolid medium. Results demonstrate that K562 cells transfected with ALDH-1 in the sense orientation display increased resistance to 4-HC compared with wild-type or vector-transfected K562 cells. Furthermore, the addition of diethylaminobenzaldehyde, a specific inhibitor for ALDH-1, restored the sensitivity of the ALDH-1-expressing K562 cells to 4-HC. Thus, the data pinpoint the direct role of ALDH-1 in the protection against 4-HC cytotoxicity.