Values reflect mean SEM of quadruplicate determinations.DNA into the reasch Riboflavin medium, an aspect of the response attributable to physical dissolution of cells undergoing apoptosis.Values reflect the mean SEM of quadruplicate determinations.Both sphingosine and noctanoic acid were without effect, however, indicating that the bioactivity of ceramide was not mediated by deacylation catabolites.Exposure to ceramide and dihydroceramide for hr resulted in comparable DNA fragmentation in HL cells. A number of lipid messenger systems have been implicated in the mechanism of action of TNF in different cell types, including the generation of diglyceride by PLC and of arachidonic acid by PLA.Treatment of HL cells with PLC but did not modify ceramide levels.PLC failed to promote DNA fragmentation in to hr incubations, however, and reduced the basal level of DNA fragments. In related studies, treatment of HL cells with PLA increased levels of free arachidonic acid and lysophosphatidylcholine by; nonetheless, a hr treatment of these cells with PLA was without effect on DNA fragmentation, as was exposure to exogenous arachidonic acid. The induction of DNA fragmentation was invariably associated with an increased number of cells exhibiting cytoarchitectural features of apoptosis in both U cells, with spontaneous expression of apoptotic traits discernible in of the cells scored.Although gradual physical dissolution of apoptotic cells was noted at longer intervals, there was no FIG.U cells were exposed to saline for hr.We further suggest that these findings may have potentially more generalized implications for current efforts to understand the mechanisms underlying pharmacological induction of DNA damage by other agents, including antineoplastic drugs.The present findings confirm the ability of TNF to induce apoptosis in both hematopoietic and nonhematopoietic cell lines.Biochemical characterization of apoptotic DNA damage revealed that ceramide promotes the internucleosomal degradation of DNA, resulting in the formation and eventual release of oligonucleosomal DNA fragments; this damage appears to be restricted to breakage of mature, doublestranded DNA, but not of nascent DNA, and is closely correlated with impaired clonogenicity and manifestation of apoptotic cytoarchitecture.Moreover, the induction of apoptosis was selectively associated with the action of ceramide and could not be elicited by other lipid messengers such as diglyceride, arachidonic acid, or sphingosine.Taken together, the present results indicate that the sphingomyelin pathway selectively mediates the induction of apoptotic fragmentation of genomic DNA by activation of the type B TNF receptor.In addition, these findings suggest an involvement of ceramideactivated protein kinase, rather than of protein kinase C, in the induction of apoptosis, although the extent to which these enzymes participate in the regulation of programmed cell death remains to be determined.The specificity of the responses to these agents is further underscored by the observations that neither PLC nor diC promoted DNA damage.In summary, pharmacological manipulations that increase the availability of free ceramide selectively induce apoptotic DNA damage and cell death in mammalian cell lines of both hematopoietic and nonhematopoietic origin, indicating that, rebmetpe Snotseugybded aonlwoD. Friedberg, E. C. Hannawalt, P. C. DekMunger, C, Ellis, A, Woods, K, Randolph, J, Yanovich, S. Yang, Z, Costanzo, M, Golde, D. W. Kolesnick, R. N. J. Biol. Chem. Gewirtz, D. A, Vol, pp. Kanter, P. M. Schwartz, H. S. Mol. Pharmacol.