线粒体神经酰胺酶上调K562细胞Bcl-2蛋白表达水平

最近，克隆了一种新的选择性定位于线粒体的神经酰胺酶，提示线粒体存在着神经酰胺代谢途径，可能对线粒体的功能，特别对凋亡产生影响。本研究将线粒体神经酰胺酶基因转染到K562细胞，以此了解线粒体神经酰胺酶的细胞生物学效应。以脂质体介导将含有线粒体神经酰胺酶cDNA的pcDNA3．1／His—CDase质粒转染到K562细胞，G418筛选阳性克隆，建立稳定表达线粒体神经酰胺酶的K562TC细胞株。用MTT法、annexin V／PI法、FCM及Westernblot分别检测K562与K562TC细胞在细胞毒药物抗性、血清剥夺耐受性及Bcl-2蛋白表达水平方面的差异。结果表明：虽然K562TC与K562细胞对阿霉素、足叶乙甙及亚砷酸的敏感性没有差异，但是K562TC细胞Bcl-2蛋白水平明显升高，抗血清剥夺能力明显增强。以硫代反义寡核苷酸特异性封闭K562TC细胞线粒体神经酰胺酶，下调Bcl-2蛋白表达水平；二甲基鞘氨醇(鞘氨醇激酶抑制剂，降低细胞内1-磷酸鞘氨醇水平)同样下调K562TC细胞Bcl-2蛋白表达水平，而1-磷酸鞘氨醇明显上调K562细胞Bcl-2表达水平。结论：线粒体神经酰胺酶将线粒体神经酰胺代谢为鞘氨醇，进而在鞘氨醇激酶作用下生成1-磷酸鞘氨醇，此代谢途径上调K562细胞Bcl-2蛋白表达水平。

Mitochondrial Ceramidase Overexpression Up-regulates Bcl-2 Protein Level in K562 Cells,Probably Through Its Metabolite Sphingosine-1-phosphate

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.