title: Attenuation of ceramide-induced apoptosis by diglyceride in human myeloid leukemia cells.
abstract: 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.
authors.affiliation: Department of Medicine, Medical College of Virginia, Richmond 23298.
authors.forename: W D
authors.initials: WD
authors.lastname: Jarvis
authors.forename: F A
authors.initials: FA
authors.lastname: Fornari
authors.forename: J L
authors.initials: JL
authors.lastname: Browning
authors.forename: D A
authors.initials: DA
authors.lastname: Gewirtz
authors.forename: R N
authors.initials: RN
authors.lastname: Kolesnick
authors.forename: S
authors.initials: S
authors.lastname: Grant
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002518:Ceramides / Q000494:pharmacology*; D004249:DNA Damage; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007951:Leukemia, Myeloid / Q000473:pathology*; D011493:Protein Kinase C / Q000502:physiology; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000494:pharmacology
chemical_list: D002518:Ceramides; D004075:Diglycerides; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase