Induction of Apoptosis and Potentiation of Ceramide-mediated Cytotoxicity by Sphingoid Bases in Human Myeloid Leukemia Cells

Abstract

). 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...

Activation of caspase-3-like proteases in apoptosis induced by sphingosine and other long-chain bases in Hep3B hepatoma cells

“…Previous studies have demonstrated that sphingosine is an endogenous modulator that mediates apoptotic signals induced by TNFα in neutrophils and cardic myocytes [44,45]. It has also been reported that sphingosine can markedly potentiate the induction of apoptosis by ceramide when present at submicromolar levels, which may also have physiological significance [46]. Additionally, it should be noted that sphingosine was added exogenously in our study, and thus the amount of sphingosine taken up by the Hep3B cells was not known.…”

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells

“…Cells were exposed or not exposed to 1 Gy radiation and after 24 h or 48 h media was aspirated and cells ®xed. Terminal Uridyl-Nucleotide End Labelling (TUNEL) was performed on the ®xed cells as described (Jarvis et al, 1996). Data shown are from a representative experiment (n=3) of the mean number of staining cells from randomly selected ®elds of ®xed cells (n=5 per slide+s.e.m.)…”

“…Terminal uridyl-nucleotide end labeling and propidium iodide staining for A431 cells A431 cells were grown in 2 well glass chamber slides as described, treated with or without varying concentrations of PD98059 DMSO control 30 min prior to irradiation, irradiated, and cells ®xed after either 24 h or 48 h. Terminal Uridyl Nucleotide End Labeling (TUNEL) was performed on these cells as described previously (Jarvis et al, 1996). Randomly selected ®elds of ®xed cells (n=5 per slide) were counted initially using propidium iodide counter stain, followed by examination and counting of TUNEL positive staining cells of the same ®eld under FITC/¯orescence light.…”

The roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway; a potential route to radio- and chemo-sensitization of tumor cells resulting in the induction of apoptosis and loss of clonogenicity

“…Studies from our laboratories have demonstrated that sphingosine, which has been shown to promote apoptosis, 92 also induces a rapid inactivation of the MAP kinase pathway in U937 cells. 93 We found that a similar inactivation of the MAP kinase pathway can be obtained by exposing cells to a relatively specific inhibitor of MEK1 (PD98059, amino methoxyflavone).…”

“…97,100 More recently, bryostatin 1 and the PKC inhibitor safingol have been shown to block Ara-Cmediated MAP kinase activation in association with potentiation of leukemic cell apoptosis, effects which have found to be mimicked by the MEK1 inhibitor PD98059. 92,93,101 Bryostatin 1 and PD98059 have also been found to promote taxolinduced apoptosis in leukemic cells. 102 Collectively, these findings raise the possibility that interruption of the PKC/MAP kinase pathway facilitates drug-induced apoptosis, perhaps by preventing one or more cytoprotective responses.…”