Synthetic ceramides induce apoptotic death of Jurkat and HL60 leukaemia cell lines. By contrast we show here that ceramide induces non-apoptotic killing of malignant cells from patients with B-chronic lymphocytic leukaemia (B-CLL) and of normal B lymphocytes. The protein phosphatase inhibitor okadaic acid readily induces apoptosis of B-CLL cells, indicating that this death pathway is fully functional in these cells. The ability of ceramide to activate the apoptotic protease caspase 3 in HL60 cells but not in B-CLL cells, as well as the lack of correlation of ceramidemediated killing of di erent B-CLL isolates with expression of the apoptosis-regulating proteins bcl-2 and bax reinforce the conclusion that ceramide killing of B-CLL cells is by a non-apoptotic mechanism. Fludarabine treatment or g-irradiation of B-CLL cells resulted in ceramide elevation and in killing by both apoptotic and non-apoptotic mechanisms, suggesting that a ceramide-triggered non-apoptotic mechanism may play a role in the killing of these cells. Therefore, the results here show that ceramide can induce either apoptotic or non-apoptotic death, depending on the cellular context. The inability of synthetic dihydroceramide to kill B-CLL cells or normal B lymphocytes suggests that non-apoptotic killing by ceramide is via interaction with a speci®c, but unidenti®ed, cellular target.

The bcl-2 protein suppresses apoptosis and the bax protein opposes the cytoprotective effect of bcl-2. A decrease in bcl-2 levels has been implicated in the induction of apoptosis during the terminal differentiation of HL60 myeloid leukaemia cells. We show here that bax protein also declined with a time course similar to the downregulation of bcl-2 following treatment of HL60 with phorbol myristate acetate (PMA), dimethyl sulphoxide (DMSO) or retinoic acid (RA). Decreased bcl-2 protein expression in induced cells was associated with downregulation of its mRNA. By contrast, the decrease in bax occurred by a post-transcriptional mechanism. Co-ordinate downregulation of bcl-2 and bax proteins may fine-tune the induction of apoptosis during cellular differentiation.

Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents. Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation. The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or g-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells. The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-X L anti-apoptotic proteins. In contrast, herbimycin protected Philadelphianegative HL60 cells from apoptosis induction by etoposide and did not a ect killing of NC37 and CEM cells. The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage. Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.

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