Role for Ceramide in Cell Cycle Arrest (*)
- Supriya Jayadev,
- Bin Liu,
- Alicja E. Bielawska,
- Joanna Y. Lee,
- Fausta Nazaire,
- Marina Yu. Pushkareva,
- Lina M. Obeid and
- Yusuf A. Hannun(§)
- From the Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710
- §Mallinckrodt Scholar. To whom correspondence should be addressed: Dept. of Medicine, Duke University Medical Center, Box 3355, Durham, NC 27710. Tel.: 919-684-2449; Fax: 919-681-8253.
Abstract
The dependence of some cell types on serum factors for growth may represent a powerful, but poorly studied, model for antimitogenic pathways. In this study, we examine ceramide as a candidate intracellular mediator of serum factor dependence. In Molt-4 leukemia cells, serum withdrawal caused a significant arrest in cell cycle progression (80% of cells in G0/G1), accompanied by a modest apoptotic cell death (12%). Serum deprivation of these cells resulted in significant sphingomyelin hydrolysis (72%; corresponding to hydrolysis of 47 pmol/nmol phosphate), which was accompanied by a profound and progressive elevation (up to 10-15-fold) in endogenous levels of ceramide. Withdrawal of serum caused the activation of a distinct, particulate, and magnesium-dependent sphingomyelinase. The addition of exogenous C6-ceramide induced a dramatic arrest in the G0/G1 phase of the cell cycle comparable to the effects observed with serum withdrawal, albeit occurring much sooner. Unlike serum withdrawal, however, the addition of C6-ceramide resulted in more pronounced apoptosis. Because of the previously noted ability of exogenously added phorbol esters to inhibit ceramide-mediated apoptosis, we investigated the hypothesis that endogenous activation of the diacylglycerol/protein kinase C pathway may modulate the response to serum withdrawal. Indeed, serum withdrawal resulted in 3-4-fold elevation in endogenous diacylglycerol levels. The addition of exogenous diacylglycerols resulted in selective attenuation of ceramide's effects on apoptosis but not on cell cycle arrest. Thus, the combination of ceramide and diacylglycerol recapitulated the complex effects of serum withdrawal on cell cycle arrest and apoptosis. These studies identify a novel role for ceramide in cell cycle regulation, and they may provide the first evidence for an intracellular signal transduction pathway in mammalian cells mediating cell cycle arrest. These studies also underscore the importance of lipid second messengers and the significance of the interplay between glycerolipid-derived and sphingolipid-derived lipid mediators.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant GM43825. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- DAG
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diacylglycerol
- PCD
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programmed cell death
- diC8
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dioctanoylglycerol
- FCS
-
fetal calf serum
- PBS
-
phosphate-buffered saline
- FACS
-
fluorescence-activated cell sorting.
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↵2In U937 cells, 30 μM diC8 and 100 nM phorbol 12-myristate 13-acetate were both able to reverse DNA fragmentation induced by 10 μM C2-ceramide treatment in a dose-dependent manner. In U937 viability assays, C6-ceramide decreased viability by 50%; 10 μM diC8 restored viability to 70% of control, and 30 μM diC8 completely reversed C6-ceramide's effects on viability (L. Karolak, P. Jeancake, L. M. Obeid, and Y. A. Hannun, unpublished observations).
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↵3G. Dbaibo, M. Pushkareva, S. Jayadev, J. K. Schwartz, J. M. Horowitz, L. M. Obeid, and Y. A. Hannun, manuscript in preparation.
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- Received September 1, 1994.
- Revision received November 21, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
