The potential involvement of ceramiderelated signaling processes in the induction of apoptosis by tumor necrosis factor a was assessed by multiple biochemical strategies in the human leukemic cell lines HL-60 and U937 and the murine fibrosarcoma cell lines L929/LM and WEHI 164/13. Exposure of these cells to tumor necrosis factor a resulted in internucleosomal cleavage of genomic DNA, yielding laddered patterns of oligonucleosomal fragments characteristic of apoptosis when resolved by agarose gel electrophoresis; similar responses were observed after exposure to exogenous sphingomyelinase or synthetic ceramides. Quantitative spectrofluorophotometry demonstrated that these treatments promoted time-and concentration-dependent degradation of DNA, resulting in the formation ofand eventual release ofsmall DNA fragments (<3.0 kb). Corresponding damage to bulk DNA was demonstrated by enhanced-fluorescence alkaline unwinding analysis. DNA fragmentation was not induced by phospholipase C or synthetic diglyceride; in fact, the effects of sphingomyelinase and ceramide were substantially reduced by coexposure to these agents, suggesting opposing roles for diglyceride-and ceramide-mediated signals in the regulation of apoptosis. Phospholipase A2 and arachidonic acid failed to promote DNA fragmentation, as did phospholipase D. Characterization of DNA strand breaks by alkaline and neutral elution analyses confirmed that ceramide action was restricted to breakage of mature, double-stranded DNA but not of nascent DNA. The induction of DNA damage was associated with appearance of apoptotic morphology and decreased clonogenicity. These results demonstrate that the ceramidedependent signaling system selectively induces apoptosis and raise the possibility that ceramide-activated enzymes represent important components in a signaling cascade involved in the regulation of programmed cell death.Programmed cell death, or apoptosis, is an active, energydependent process through which living cells participate in their own destruction and is initiated by a variety of physiological and pharmacological stimuli (1-4). A fundamental component of this response is the stereotypical degradation of genomic DNA to oligonucleosomal fragments (3, 4). The inflammatory cytokine tumor necrosis factor a (TNF-a) has been shown to initiate apoptotic cell death and DNA fragmentation in several mammalian cell lines, including the human leukemia cell lines HL-60 (5, 6) and U937 (6-8) and the murine fibrosarcoma cell lines L929 (9, 10) and WEHI (11,12). Two subtypes of TNF receptor, a high-affinity, 75-kDa (type A) form and a low-affinity, 55-kDa (type B) form, are expressed in comparable copy numbers in U937 and HL-60 cells (13); activation of the latter species has beenThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. implicated in the induction of apoptotic DNA degradation and cell death in bot...

). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0.001-10 M) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations ( 750 nM), however, sphingoid bases synergistically augmented the apoptotic capacity of ceramide (10 M), producing both a leftward shift in the ceramide concentration-response profile and a pronounced increase in the response to maximally effective levels of ceramide. Thus, sphingosine and sphinganine increased both the potency and efficacy of ceramide. The apoptotic capacity of bacterial sphingomyelinase (50 milliunits/ml) was similarly enhanced by either (a) acute co-exposure to highly selective pharmacological inhibitors of protein kinase C such as calphostin C and chelerythrine or (b) chronic pre-exposure to the nontumor-promoting protein kinase C activator bryostatin 1, which completely down-modulated total assayable protein kinase C activity. These findings demonstrate that inhibition of protein kinase C by physiological or pharmacological agents potentiates the lethal actions of ceramide in human leukemia cells, providing further support for the emerging concept of a cytoprotective function of the protein kinase C isoenzyme family in the regulation of leukemic cell survival.Recent investigation has examined the participation of sphingophospholipid-and glycerophospholipid-derived messengers in the regulation of leukemic cell survival. We (1, 2) and others (3) have demonstrated that increased intracellular availability of ceramide induces programmed cell death or apoptosis in the human myeloid leukemia cell lines HL-60 and U937. Ceramide interacts with at least two distinct intracellular target enzymes, ceramide-activated protein kinase (4 -6) and ceramide-activated protein phosphatase (7-9). A cytotoxic role for ceramide-activated protein phosphatase and ceramideactivated protein kinase in ceramide action has been inferred, although the relative contributions of these enzymes to the initiation of apoptosis is presently uncertain (10, 11). A contrasting cytoprotective function of diglyceride and, therefore, of one or more isoforms of protein kinase C (PKC) 1 is supported by several lines of evidence. Increased intracellular availability of diglyceride abrogates the initiation of apoptotic DNA damage by ceramide in both HL-60 and U937 cells (1, 2); this effect is mimicked by such diverse pharmacological PKC activators as the stage 1 tumor promoters phorbol dibutyrate (2) and phorbol myristate acetate (2, 3), the stag...

We have examined the in vivo radioprotective effects of the macrocyclic lactone protein kinase C (PK-C) activator, bryostatin 1, administered either alone or in conjunction with recombinant murine granulocyte- macrophage colony-stimulating factor (rmGM-CSF), in Balb/c and C3H/HeN mice subjected to lethal total body irradiation (TBI). When administered alone on a divided dose schedule (24 hours and 30 minutes before TBI), rmGM-CSF (20 micrograms/kg) was ineffective in increasing survival in either strain. However, in Balb/c mice, bryostatin 1 alone (1 microgram) permitted the long-term survival (60 days) of 70% of the animals following TBI, and 80% when administered in conjunction with rmGM-CSF. Bryostatin 1 administered alone according to this schedule exerted minimal radioprotective effects in C3H/HeN mice, but, when combined with a subeffective dose of rmGM-CSF, allowed 50% of the animals to survive. Treatment of Balb/c mice with bryostatin 1 administered as a single dose 4 hours before TBI resulted in a 20% survival rate, and 45% when administered with rmGM-CSF; corresponding values for the C3H/HeN strain were 60% and 40%, respectively. Lastly, the survival rates of Balb/c mice treated with bryostatin 1 administered as a single dose 4 hours following TBI was 20%, and 25% with rmGM-CSF; corresponding values were 50% and 25% for C3H/HeN mice. These findings indicate that the PK-C activator bryostatin 1 exhibits intrinsic in vivo radioprotective effects in lethally irradiated Balb/c and C3H/HeN mice, and may, under some circumstances, augment the radioprotective capacity of rmGM-CSF. They also underscore the critical role that strain differences and scheduling considerations play in determining the in vivo radioprotective capacity of bryostatin 1, as well as its interactions with rmGM-CSF.

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