RESTORATION OF TNF-ALPHA-INDUCED CERAMIDE GENERATION AND APOPTOSIS INRESISTANT HUMAN LEUKEMIA KG1A CELLS BY THE P-GLYCOPROTEIN BLOCKER PSC833

Tumor necrosis factor (TNF-alpha) is a cytokine with antitumor activit y against several cellular models, TNF-alpha-induced apoptosis seems t o be mediated by a signaling pathway termed 'sphingomyelin-ceramide' p athway, which consists of the hydrolysis of sphingomyelin and the prod uction of its breakdown product ceramide, Our study shows that KG1a ce lls, which are inherently resistant to TNF-alpha and do not produce ce ramide upon cytokine stimulation, can be sensitized by the use of the P-glycoprotein inhibitor PSC833, Coincubation with 1 mu M of this cycl osporin derivative restored the apoptotic potential of 10 ng/ml TNF-al pha, This effect was associated with the restoration of ceramide gener ation (315%) and activation of neutral, but not acid sphingomyelinase activity (143%), Furthermore, we demonstrate that treatment of KG1a ce lls with 1 mu M PSC833 led to a threefold increase in inner plasma mem brane sphingomyelin content and basal neutral sphingomyelinase activit y, These results support the hypothesis whereby resistance to TNF-alph a-mediated apoptosis of certain leukemic cells is linked to the dispos ability of the sphingomyelin pool, These data also suggest a role for P-glycoprotein in sphingomyelin transverse plasma membrane asymmetry.