INDUCTION OF APOPTOSIS AND POTENTIATION OF TNF-MEDIATED AND FAS-MEDIATED APOPTOSIS IN U937 CELLS BY THE XANTHOGENATE COMPOUND D609

Apoptosis induced by ligation of either the tumor necrosis factor (TNF ) p55 receptor or the Fas/APO-1/CD95 receptor has been suggested to re quire ceramide as a signaling molecule. Ceramide is formed as a result ; of sphingomyelinase (SMase) activation in the sphingomyelin cycle, a nd ligation of TNF and Fas receptors has been reported to stimulate SM ase activity. We have studied the effects of D609, a xanthogenate comp ound with antitumoral properties, on TNF- or Fas-induced apoptosis of monocytic U937 cells, First, the effects of D609 on SMase activity wer e assessed using in vitro assays for neutral and acidic SMase, and the results suggested that D609 caused a modest stimulation of the activi ty of both SMases in U937 cells. Exposure of U937 cells for 6 h to TNF or anti-Fas mAb induced apoptosis in 40-45% of the cells, as measured by fluorescent staining of nuclear chromatin, Cotreatment with D609 p otentiated TNF- as well as Fas-mediated apoptosis up to 70 and 90%, re spectively. Further-more, in incubations with D609 alone, 60% of the c ells became apoptotic within 16 h. Since D609 has been reported to inh ibit. protein kinase C (PKC) activity, the effect of phorbol 12-myrist ate 13-acetate (PMA) on D609-induced apoptosis was investigated. PMA w as found to inhibit D609-induced apoptosis in U937 cells as well as ce ll death induced by TNF and anti-Fas mAb. Thus, PKC inactivation may p lay an important role in the regulation of apoptosis in U937 cells. in summary, the present results show that D609 stimulates SMase activity , potentiates TNF- and Fas-induced apoptosis, and induces apoptosis on its own in U937 cells. (C) 1997 Academic Press.