Abstract
Prior studies demonstrated that ceramide promotes apoptotic cell death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77), and that this lethal process is potently suppressed by diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L., Gewirtz, D. A., Kolesnick, R. N., and Grant, S. (1994) J. Biol. Chem. 269, 31685-31692). 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 microM) 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 microM), 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 non-tumor-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.
Highlights
§ Recipient of National Research Service Award CA-09380 from NCI, National Institutes of Health
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
Sphingoid bases represent a versatile class of endogenous inhibitory effectors of the protein kinase C (PKC) isoenzyme family [13, 14], and have been found to suppress or attenuate numerous PKCdependent aspects of leukemic cell survival
Summary
Synthetic preparations of D-erythro-sphingosine and D-erythrosphinganine were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Pelleted cells were resuspended in PBS and lysed by addition of 10 mM Tris-HCl, 15 mM EGTA, 15 mM EDTA, 0.1% Nonidet P-40, pH 7.4, yielding a final concentration of 4 ϫ 107 cells/ml; the lysates were treated with proteinase-K (200 g/ml; Sigma) at 55 °C for 24 h. Aliquots of final lysate preparations (corresponding to 2 ϫ 106 cells) were loaded into 2.25% agarose gels (Metaphor; FMC) impregnated with ethidium bromide (0.5 g/ml); low molecular weight DNA fragments were resolved by electrophoresis at 6 V/cm for 90 –180 min in 1 ϫ Tris acetate/ EGTA buffer at 10 °C. The pellets were discarded, and the presence of non-sedimenting DNA fragments in the supernatant from lysate and medium extracts was determined by dilution in modified Tris-sodium/EGTA buffer (3 mM NaCl, 10 mM Tris-HCl, 1 mM EGTA, pH 8.0) containing 1.0 g/ml bis-benzimide trihydrochloride (Hoechst 33258; Sigma), and monitoring net fluorescence in each sample (ex ϭ 365, em ϭ 460). Membranes were sequentially incubated in (a) rabbit anti-human polyclonal antibody (1:5000; Santa Cruz) for 1 h and (b) goat anti-rabbit polyclonal antibody horseradish peroxidase conjugate (1:5000; Calbiochem) for 1 h; immunoreactive cPKC␣ was visualized by enhanced chemiluminescence
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