Mitochondrial ceramidase overexpression up-regulates Bcl-2 prot ein level in K562 cells probably through its metabolic product sphingosine-1-ph osphate

AIM:To investigate the role of mitochondri al ceramidase in mitochondrial functions,especially in the regulation of apopto sis.METHODS:pCDNA3.1/His-MtCDase plasmid,containing mitochondrial ceramidase cDNA sequence,was transfected into K562 cells by liposome,and G418 was used to screen the positive clones.A stable transfected K562 cell line was established and defined as ‘K562TC’.The differences between K562 an d K562TC cells in serum withdrawal resistance and Bcl-2 protein expression were evaluated by annexin V/PI test,flow cytometry and Western blotting,respectivel y.RESULTS:K562TC cells with elevated Bcl-2 protein expression lev el identified by FCM or Western blotting showed stronger resistance to apoptosis induced by serum withdrawal than their parental cells.Inhibition of mitochondr ial ceramidase expression in K562TC cells by its specific antisense oligodeoxynu cleotide was correlated with a decrease in Bcl-2 protein level.N,N'-dimethylsp hingosine (DMS),a sphingosine kinase inhibitor,depleted intracellular sphingos ine-1-phosphate (SPP) production,also abrogated Bcl-2 protein expression in K56 2TC cells,while exogenous sphingosine-1-phosphate up-regulated Bcl-2 protein le vel in K562 cells.CONCLUSION:Mitochondrial ceramidase overexpression in K562 cell s leads to markedly elevated level of Bcl-2 protein a nd results in more resistance to serum withdrawal.This effect is initiated no t by sphingosine,the direct metabolite of mitochondrial ceramidase,but via sph ingosine-1-phosphate,its phosphorylated form,indicating that mitochondrial cer amidase,through its sphingoid metabolite sphingosine-1-phosphate,up-regulates Bcl-2 protein expression in K562 cells.