Attenuation of ceramide-induced apoptosis by diglyceride in human myeloid leukemia cells.

Prior studies demonstrated that increased intracellular availability of ceramide induces apoptotic DNA degradation and cell death in the human leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77). The present findings show that diglyceride opposes ceramide-related apoptosis in HL-60 and U937 cells. Acute (6-12-h) exposure to sphingomyelinase (100 milliunits/ml) or synthetic ceramide (10 microM) promoted apoptotic degradation of genomic DNA as indicated by (a) the appearance of both approximately 50-kilobase pair (kbp) DNA fragments and approximately 0.2-1.2-kbp DNA fragment ladders on agarose gels, (b) formation and release of small double-stranded DNA fragments, and (c) loss of integrity of bulk DNA. DNA damage was associated with reduced clonogenicity and expression of apoptotic morphology. In contrast, exposure to phospholipase C (0.001-100 milliunits/ml) or synthetic diglyceride (10 microM) failed to promote apoptosis and abolished the lethal actions of ceramide as defined by each of the indices outlined above. Ceramide-related apoptosis was also reduced by acute (6-h) exposure to tumor promoters such as phorbol dibutyrate and mezerein and the non-tumor-promoting agent bryostatin 1; conversely, chronic (24-h) pretreatment with these agents failed to modify ceramide-mediated cytotoxicity, but abolished the protective actions of diglyceride. These findings demonstrate that diglyceride and pharmacological protein kinase C activators reduce or abolish ceramide-mediated apoptosis in human leukemia cells and support the concept of a cytoprotective function for protein kinase C in the regulation of leukemic cell survival. In addition, the capacity of diglyceride to prevent very early genomic lesions (e.g. generation of 50-kbp DNA fragments) suggests that acute activation of protein kinase C arrests apoptosis at an initial stage.