©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role for Ceramide in Cell Cycle Arrest (*)

(Received for publication, September 1, 1994; and in revised form, November 21, 1994)

Supriya Jayadev Bin Liu Alicja E. Bielawska Joanna Y. Lee Fausta Nazaire Marina Yu. Pushkareva Lina M. Obeid Yusuf A. Hannun (§)

From the Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 G(0)/G(1)), 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 C(6)-ceramide induced a dramatic arrest in the G(0)/G(1) phase of the cell cycle comparable to the effects observed with serum withdrawal, albeit occurring much sooner. Unlike serum withdrawal, however, the addition of C(6)-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.


INTRODUCTION

Serum deprivation is a powerful mechanism by which cells can be arrested in the cell cycle and induced to undergo programmed cell death (1, 2, 3) . As such, the serum deprivation model has been used extensively to study both of these events. However, a mechanistic basis for this growth arrest and apoptosis has yet to be found. With the discovery of the sphingomyelin cycle, sphingolipid-derived molecules have gained prominence as critical antimitogenic molecules of cells(4, 5) . Ceramide, a product of regulated sphingomyelin hydrolysis, has been shown to be released within minutes to hours following stimulation of cells with agents such as 1alpha,25-dihydroxyvitamin D(3)(6, 7) , tumor necrosis factor alpha(8, 9) , interleukin-1beta(10, 11) , and -interferon(8) . Agonist-induced mobilization of ceramide has been found to precede the antimitogenic effects of the agonists, and exogenous addition of short chain membrane-permeable ceramides has been shown to reproduce the antiproliferative and differentiative effects of these agonists(7, 12) . Furthermore, in the case of tumor necrosis factor alpha, ceramide has been implicated as a mediator of programmed cell death(13) . These studies suggested that the antiproliferative effects of ceramide may be a consequence of apoptosis; however, an effect of ceramide on cell cycle progression has not been examined. Prompted by these findings and considerations, we investigated a role for ceramide in mediating the growth suppressive effects of serum deprivation.

In this study, we show that ceramide levels respond progressively to serum withdrawal through activation of a magnesium-dependent, membrane-associated, neutral sphingomyelinase. Ceramide is found to induce a significant block in cell cycle progression accompanied by apoptosis. Interestingly, diacylglycerol (DAG)(^1), which also increases with serum withdrawal, counters the effects of ceramide on apoptosis but not on cell cycle, suggesting a protective role for DAG. More importantly, the combination of ceramide and DAG recapitulates the effects of serum withdrawal on cell cycle arrest. The implications of these studies on the role of ceramide in cell cycle arrest are discussed.


EXPERIMENTAL PROCEDURES

Materials

Molt-4 human leukemia cells and Wi38 human fibroblasts were purchased from ATCC (Rockville, MD). RPMI 1640 and fetal calf serum were purchased from Life Technologies, Inc. TLC plates were purchased from Fisher. All other reagents were purchased from Sigma.

Methods

Cell Culture

Molt-4 cells were maintained at 37 °C in a 5% CO(2) incubator. For general maintenance, Molt-4 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum and Wi38 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For treatment, Molt-4 cells in log phase growth were washed once with PBS and seeded at 5 times 10^5/ml in 2% FCS-supplemented RPMI 1640 or serum-free RPMI 1640. For treatment, Wi38 cells were seeded and allowed to grow for 3 days. Cells were then re-fed with normal growth media and treated. Time-matched controls were always run concurrently.

Cell Cycle and Apoptosis Studies

Following the indicated times of treatment, 2 times 10^6 cells were harvested and prepared as described(14) . Briefly, cells were resuspended in 1 ml of PBS fixed in 4 ml of absolute ethanol and stored at -20 °C. On the day of analysis, the fixation buffer was removed and the cells were resuspended in 1 ml of PBS. To remove RNA prior to staining, cells were treated with 100 µl of 200 µg/ml DNase-free RNase A at 37 °C for 30 min. Cells were stained by treating with 100 µl of 1 mg/ml propidium iodide for 5-10 min and analyzed using a FACStar flow cytometer (Becton Dickinson, San Jose, CA). Flow cytometric analyses were performed using an argon laser at 488 nm. Apoptotic cells were evident as cells containing less than 2 N DNA.

Synthesis of C(6)-ceramide and Dihydro-C(6)-ceramide

(2S,3R)-N-Hexanoylsphingosine (D-erythro-C(6)-ceramide) was synthesized by acylation of (2S,3R)-sphingosine with either hexanoyl chloride (15) or N-succinimidylhexanoate(16) . (2S,3R)-Sphingosine was obtained via stereoselective synthesis using L-serine. Ceramide was purified via flash chromatography (EM-Science silica gel; 40-63 µ) using a methylene chloride-methanol system with increasing polarity from 100:0 to 96:4. The product from this was crystallized from methanol-water (1:1) to obtain pure product (yield 68%) as assessed by TLC and NMR analyses. TLC was done on Merck precoated Silica Gel 60 F-254 plates and methylene chloride-methanol (93:7) was used as solvent to develop plates. Detection was by: 1) UV, 2) iodine vapor, and 3) 5% potassium permanganate in 1 N potassium hydroxide.

^1H NMR spectra were obtained on a G.E. 500-MH(2) Omega spectrometer. Chemical shifts () were indicated as parts/million relative to a TMS internal standard. Mass spectra were obtained using a Hewlett-Packard 5988 GO/MS/DS system. The samples were analyzed using chemical ionization mass spectrometry. MS is m/z, 398 (M); ^1H NMR (CD(3)OD) is as follows: 0.84 (6H, dt, CH(3)-C(17), CH(3)-C(6`)), 1.25 (26H, sbr, CH(2)), 1.38-1.42 (2H, m, CH(2)), 1.52-1.62 (2H, m, CH(2)), 2.00-2.08 (2H, m, CH(2)-C(6)), 2.14-2.22 (2H, t, CH(2)-C(2`)), 3.67 (2H, d, H(2)C(1)), 3.83-3.87 (1H, m, HC(2)), 4.04 (1H, t, HC(3)), 5.42-5.50 (1H, m, HC(4), 5.62-5.72 (1H, m, HC(5)), 7.66-7.72 (1H, m, NH).

DL-Erythrodihydro-C(6)-ceramide was prepared via a similar procedure using DL-erythrodihydrosphingosine (Sigma D7033).

Ceramide and Diacylglycerol Measurements

Cells grown in serum-free media for the indicated times were harvested, and the lipids were extracted via the Bligh and Dyer method(17) . Lipids, dried under nitrogen, were resuspended in 100 µl of chloroform, 25 µl was set aside for phosphate measurements(18) , and 25 µl was used in the diacylglycerol kinase assay(19, 20) . The phosphorylated lipids were extracted and run on TLC using chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1) as solvent. The ceramide-phosphate and phosphatidic acid spots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Both ceramide and diacylglycerol were quantitated using external standards and were normalized to phosphate.

Sphingomyelin Measurements

Molt-4 cells serum-starved for the indicated times were harvested and the lipids extracted via the Bligh and Dyer method(17) . Lipids dried under nitrogen were resuspended in 100 µl of chloroform, 10-µl duplicates were set aside for phosphate measurements(18) , and 45 µl were run on TLC using chloroform/methanol/acetic acid/water (50:30:8:5) as the solvent. The sphingomyelin spots were visualized via iodine staining, scraped, and measured for phosphate. Sphingomyelin was quantitated using an external standard and was normalized to total phosphate. Sphingomyelin mass is represented as pmol/nmol phosphate.

Sphingomyelinase Assay

Molt-4 cells, serum-starved for the indicated times, were washed with PBS and resuspended in cold lysis buffer (25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1 mM ATP, 20 µg/ml chymostatin, 20 µg/ml leupeptin, 20 µg/ml antipain, 20 µg/ml pepstatin, 1 mM phenylmethlysulfonyl fluoride) to attain a final concentration of 5 times 10^7 cells/ml. Cells were lysed via nitrogen cavitation (20 min at 350 p.s.i., 4 °C), and post-nuclear supernatant was spun at 200,000 times g for 30 min. The resultant supernatant was designated the cytosol fraction and the pellet, resuspended in the original volume of lysis buffer, was designated the membrane fraction. Sphingomyelinase activity was assayed in all fractions using ^14C-labeled sphingomyelin as described(21) .


RESULTS AND DISCUSSION

Molt-4 T leukemia cells were found to require serum factors for growth in tissue culture; thus, withdrawal of serum factors led to both an arrest of cells in the G(0)/G(1) phase of the cell cycle and programmed cell death (PCD) (Fig. 1). Cell cycle arrest was observed as early as 12 h following serum withdrawal. By 26 h, cell cycle arrest was pronounced, with almost 80% of cells in the G(0)/G(1) stage as opposed to 61% in control cultures. By 48 h of serum starvation, very few cells could be found in either the S (2%) or the G(2)/M (2%) phases, and nearly all cells had arrested in G(0)/G(1). Some cells also underwent PCD, which was observed by 26 h. By 48 h following serum deprivation, a significant proportion (12%) of cells appeared in a pre-G(0)/G(1), apoptotic peak (Fig. 1, A and C). Therefore the growth arrest observed with serum deprivation involves predominantly cell cycle arrest with a small component of apoptosis.


Figure 1: Effects of serum deprivation on cell cycle and PCD. Molt-4 cells were grown with or without 10% fetal calf serum (FCS) for the indicated periods of time. Cells were fixed, stained, and analyzed as described under ``Methods.'' A, representative tracings from FACS analyses; B and C, quantitative measure of FACS data using the sum of broadened rectangles (SOBR) model.



Since little insight is available concerning the mechanisms for the antimitogenic effects of serum factor withdrawal, we initially wondered whether ceramide, which has been implicated as an intracellular mediator of tumor necrosis factor alpha-induced apoptosis(13) , may play a role. Therefore, the response of endogenous ceramide to serum withdrawal was investigated. Serum starvation of Molt-4 cells led to a remarkable increase in endogenous ceramide (Fig. 2A). A 3-fold increase in ceramide levels could be observed as early as 24 h following the removal of serum. This initial elevation was comparable to agonist-induced ceramide elevations described in other cell systems. However, unlike other agonist-stimulated systems where ceramide levels returned to basal values(6, 7) , prolonged serum starvation produced further elevations in ceramide. Thus, by 48 h an 8-fold increase in ceramide could be observed, and by 96 h ceramide had increased further to 15-fold above basal. Since ceramide levels were normalized for total phospholipid levels, these results reflected specific increases in ceramide levels. These increases in ceramide coincided with both the cell cycle arrest and PCD elicited by serum deprivation.


Figure 2: Effects of serum deprivation on sphingomyelin metabolism. Molt-4 cells were serum-starved for the indicated time periods and analyzed as described under ``Methods.'' The results shown are representative of two to five separate experiments. A, time course of ceramide elevations; B, mass of sphingomyelin following 72 h of treatment; C, sphingomyelinase activity from cells treated for 72 h. Neutral sphingomyelinase activity in the presence of Mg is shown. No activity was observed in the absence of Mg.



Since the kinetics and magnitude of the ceramide response were sufficiently distinct and more pronounced than those previously observed with tumor necrosis factor alpha and other extracellular cytokines, it became important to determine the source of ceramide as well as the mechanisms involved in generating ceramide. The elevation in ceramide levels was paralleled by a concomitant decrease (from 64 to 17 pmol/nmol phosphate) in the mass of sphingomyelin (Fig. 2B). This loss in sphingomyelin (47 pmol/nmol phosphate at 72 h) accounts for most of the generated ceramide at the same time point (38 pmol/nmol phosphate). However, investigation of the mechanism of hydrolysis of sphingomyelin revealed that serum withdrawal did not induce any substantial activity in the cytosolic/magnesium-independent, neutral sphingomyelinase (Fig. 2C), which has been implicated previously in the mechanism of action of 1alpha,25-dihydroxyvitamin D(3)(22) . On the other hand, withdrawal of serum factors resulted in substantial activation of a particulate, magnesium-dependent, neutral sphingomyelinase with an increase from 10,000 to 24,000 cpm/mg protein (Fig. 2C). Thus, serum withdrawal appears to stimulate a signaling cascade distinct from agonist stimulation of the sphingomyelin cycle(6, 7, 8, 9, 10, 11) . However, the outcome of both pathways is the induction of sphingomyelin hydrolysis and ceramide generation, although the magnitude and persistence of the ceramide response upon serum withdrawal reaches levels and durations not seen with agonist stimulation.

We next investigated what role ceramide may play in mediating the effects of serum deprivation. A short chain synthetic ceramide, C(6)-ceramide, was employed to treat Molt-4 cells in the presence of serum. Addition of C(6)-ceramide not only caused PCD (Fig. 3), it also induced significant cell cycle arrest. Ceramide-induced cell cycle arrest occurred rapidly (within 4 h) and doses as low as 15 µM C(6)-ceramide (approximately 4 nmol/10^6 cells; comparable to doses achieved upon serum deprivation) were able to reproduce effects reminiscent of prolonged serum deprivation: almost complete G(0)/G(1) arrest following 14 h of treatment. Higher doses (20-40 µM) were more rapidly effective (within 4 h) and lower doses (10 µM) exhibited decreased efficacy, illustrating that ceramide's effects on both PCD and cell cycle exhibited time and dose dependence (Fig. 3, B and C). Furthermore the effects of ceramide on cell cycle arrest was found to be a more generalized phenomenon, not restricted to the transformed Molt-4 cell line. In a number of non-transformed cells, including the human Wi38 human diploid fibroblast line, 15 µM C(6)-ceramide was able to produce substantial cell cycle arrest in a time-dependent fashion (Fig. 3D).


Figure 3: Effects of C(6)-ceramide and dihydro-C(6)-ceramide on cell cycle and PCD. Molt-4 cells and Wi38 cells were grown as indicated in ``Methods.'' Cells were treated with C(6)-ceramide, dihydro-C(6)-ceramide, or ethanol vehicle for the indicated times. Cells were prepared and analyzed as described under ``Methods.'' A, representative FACS tracings at 14 h; B-F, quantitative measure of FACS data using the SOBR model (B and C, Molt-4 cells treated with C(6)-ceramide; D, Wi38 cells treated with 15 µM C(6)-ceramide; E and F, dihydro-C(6)-ceramide treatments).



Next, it became important to establish the specificity of ceramide action. Thus, Molt-4 cells were treated with the closely related lipid molecule, dihydroceramide, a biosynthetic precursor for ceramide. Dihydroceramide differs structurally from ceramide in only one respect; it lacks the double bond between carbons 4 and 5 of the sphingoid backbone. In Molt-4 cells, dihydro-C(6)-ceramide treatment, at concentrations and times comparable to those that were effective with C(6)-ceramide, proved to be ineffective at producing either cell cycle arrest or PCD (Fig. 3, E and F), although both molecules show similar kinetics of uptake and minimal metabolism(23) . Thus, the differences in biologic activity are not a result of differential delivery, but probably reflect specific interaction of ceramide with an intracellular target that does not interact with dihydroceramide. A possible candidate is ceramide-activated protein phosphatase, which is specifically activated in vitro by C(6)-ceramide and not by dihydro-C(6)-ceramide(24) . Furthermore, the specificity of action of ceramide was also supported by the lack of effect of diacylglycerol (a structurally related molecule) on cell cycle progression (see below).

Thus, ceramide appears to regulate specifically the biological effects of serum deprivation. However, one critical difference between the biology elicited by ceramide and that elicited upon serum starvation related to the more pronounced effects of ceramide at stimulating PCD (compare Fig. 1C and Fig. 3C). This observation raised the possibility that other factors may operate in modulating cell viability following serum deprivation. In U937, HL60, L929/LM, and WEHI 164/13 cells, phorbol esters (activators of protein kinase C) have been noted to oppose the effects of ceramide on PCD(13, 25) . We therefore investigated whether diacylglycerols (endogenous activators of protein kinase C) may play a role in the response to serum deprivation.

During serum deprivation of Molt-4 cells, DAG levels were found to become elevated (Fig. 4A). By 24 h following serum starvation, DAG levels increased 1.5-fold (whereas ceramide levels had tripled). By 96 h, DAG levels increased by a total of 4-fold (Fig. 4A), demonstrating a progressive build-up of DAG levels, albeit to a level significantly less than that seen with ceramide (4-fold versus 15-fold at 96 h; compare Fig. 2A and Fig. 4A).


Figure 4: Effects of serum deprivation on endogenous diacylglycerol levels and effects of diacylglycerol on C(6)-ceramide-induced PCD and cell cycle arrest. A, Molt-4 cells were serum-starved for the indicated time periods. Cells were harvested, and the lipids were analyzed as described under ``Methods.'' The results shown are representative of five separate experiments. B, Molt-4 cells, grown in 2% FCS, were treated with dioctanoylglycerol plus C(6)-ceramide or ethanol for the indicated times. Cells were prepared and analyzed as described under ``Methods.''



We therefore investigated if the simultaneous increase in both ceramide and DAG could explain the decreased effect of serum deprivation on PCD. To test this hypothesis, a cell-permeable diacylglycerol, dioctanoylglycerol (diC(8)), was employed, both alone and in combination with ceramide for impact on cell cycle and PCD. Alone, diC(8), at concentrations of 10 to 100 µM, was ineffective at causing either cell cycle arrest or PCD during 5-30 h (data not shown). However, in combination with C(6)-ceramide diC(8) reversed the apoptotic effects of ceramide by more than 50% (Fig. 4B). In a quantitative analysis, diC(8) and phorbol esters also inhibited ceramide-induced DNA fragmentation and loss of viability. (^2)In contrast, diC(8) exhibited minimal effects in reversing ceramide-induced cell cycle arrest (at best, a 10-15% reversal was observed). These data suggest that the increase in endogenous DAG may account for the decreased PCD observed with serum deprivation compared to that found upon ceramide stimulation. Importantly, ceramide and diacylglycerol, together, faithfully recapitulated the effects of serum withdrawal on growth and viability.

These studies identify a novel role for ceramide in regulating cell cycle progression whereby ceramide induces a significant G(0)/G(1) arrest. This observation carries several implications. First, ceramide may emerge as an endogenous mediator of cell cycle arrest. As opposed to the myriad of candidate intracellular messengers and mediators of cell cycle progression, little is known on the operation of intracellular mediators of cell cycle arrest. Thus, these studies identify an important candidate for this role. This function for ceramide is likely in the context of serum withdrawal where endogenous ceramide levels accumulate to very high levels, and where exogenous ceramide induces very early effects on cell cycle progression. Further examination of this hypothesis awaits the development of specific inhibitors of ceramide generation and/or action. Second, these studies raise the tantalizing possibility that ceramide modulates the endogenous machinery regulating cell cycle progression. Indeed, in ongoing studies, support for such a role is provided by the ability of ceramide to induce dephosphorylation of the retinoblastoma gene product (^3)with specificity, potency, and kinetics that match the ability of ceramide to induce cell cycle arrest. Third, the ability of diacylglycerol to oppose ceramide's effects on PCD preferentially over cell cycle arrest distinguishes these two outcomes of ceramide action as products of distinctly-regulated mechanisms. This raises the possibility that ceramide may function as a proximal sensor and transducer of cell deprivation/insult/injury with the ability to launch distinct programs of cell suppression (growth arrest and apoptosis), the outcome of which may be dependent on whether other modulatory signals (such as diacylglycerol) are also activated. Finally, this latter finding suggests that sphingolipid and glycerophospholipid signaling may be coupled or interrelated, with the opposing effects of ceramide and DAG of greater relevance in defining the outcome on growth and viability. A perturbation of this ratio, whether by changes in ceramide, DAG, or both, could therefore lead to changes in cell growth and viability.

Previous studies have identified ceramide and DAG as rapidly mobilized second messenger molecules in the sphingomyelin and phosphatidylinositol cycles, respectively(6, 7, 8, 9, 11, 26, 27, 28, 29) . With DAG, evidence is accumulating on more sustained DAG signals generated from either the phospholipase D pathway (30) or through de novo biosynthesis(31) . The current study shows that the role of ceramide (and DAG) in cell cycle arrest and PCD not only exhibits slower kinetics, it occurs over a prolonged period resulting in very high levels of accumulation. Moreover, the mechanism involved in this long term generation of ceramide (magnesium-dependent, particulate sphingomyelinase) is distinct from that operating in cytokine activated sphingomyelin cycle (magnesium-independent, cytosolic sphingomyelinase) and may represent the counterpart of phospholipase D in long term DAG signaling. This description of ceramide and DAG as long term effector molecules suggests a different perspective on lipid mediators. According to this hypothesis, sustained changes in ceramide and DAG levels (probably by distinct mechanisms, as shown here for ceramide) may induce long term, and perhaps permanent, reprogramming of cell function through the regulation of apoptotic and cell cycle machinery.


FOOTNOTES

*
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.

§
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.

(^1)
The abbreviations used are: DAG, diacylglycerol; PCD, programmed cell death; diC(8), dioctanoylglycerol; FCS, fetal calf serum; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting.

(^2)
In U937 cells, 30 µM diC(8) and 100 nM phorbol 12-myristate 13-acetate were both able to reverse DNA fragmentation induced by 10 µM C(2)-ceramide treatment in a dose-dependent manner. In U937 viability assays, C(6)-ceramide decreased viability by 50%; 10 µM diC(8) restored viability to 70% of control, and 30 µM diC(8) completely reversed C(6)-ceramide's effects on viability (L. Karolak, P. Jeancake, L. M. Obeid, and Y. A. Hannun, unpublished observations).

(^3)
G. Dbaibo, M. Pushkareva, S. Jayadev, J. K. Schwartz, J. M. Horowitz, L. M. Obeid, and Y. A. Hannun, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Gilbert Radcliff for assistance with the flow cytometer.


REFERENCES

  1. Howard, M. K., Burke, L. C., Mailhos, C., Pizzey, A., Gilbert, C. S., Lawson, W. D., Collins, M. K. L., Thomas, N. S. B., and Latchman, D. S. (1993) J. Neurochem. 60, 1783-1791 [Medline] [Order article via Infotrieve]
  2. Dean, M., Levine, R. A., Ran, W., Kindy, M. S., Sonenshein, G. E., and Campisi, J. (1986) J. Biol. Chem. 261, 9161-9166 [Abstract/Free Full Text]
  3. Shichiri, M., Hanson, K. D., and Sedivy, J. M. (1993) Cell Growth & Diff. 4, 93-104
  4. Hannun, Y. A., and Linardic, C. M. (1993) Biochim. Biophys. Acta 1154, 223-236 [Medline] [Order article via Infotrieve]
  5. Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125-3128 [Free Full Text]
  6. Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080 [Abstract/Free Full Text]
  7. Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. A. (1990) J. Biol. Chem. 265, 15823-15831 [Abstract/Free Full Text]
  8. Kim, M., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266, 484-489 [Abstract/Free Full Text]
  9. Dressler, K. A., Mathias, S., and Kolesnick, R. N. (1992) Science 255, 1715-1718 [Medline] [Order article via Infotrieve]
  10. Ballou, L. R., Chao, C. P., Holness, M. A., Barker, S. C., and Raghow, R. (1992) J. Biol. Chem. 267, 20044-20050 [Abstract/Free Full Text]
  11. Mathias, S., Younes, A., Kan, C., Orlow, I., Joseph, C., and Kolesnick, R. N. (1993) Science 259, 519-522 [Medline] [Order article via Infotrieve]
  12. Bielawska, A., Linardic, C. M., and Hannun, Y. H. (1992) FEBS Lett. 307, 211-214 [CrossRef][Medline] [Order article via Infotrieve]
  13. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771 [Medline] [Order article via Infotrieve]
  14. Walker, P. R., Kwast-Welfeld, J., Gourdeau, H., Leblanc, J., Neugebauer, W., and Sikorska, M. (1993) Exp. Cell Res. 207, 142-151 [CrossRef][Medline] [Order article via Infotrieve]
  15. Herold, P. (1988) Helv. Chim. Acta 71, 354-362
  16. Bielawska, A., Linardic, C. M., and Hannun, Y. A. (1992) J. Biol. Chem. 267, 18493-18497 [Abstract/Free Full Text]
  17. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  18. Ames, B. N., and Dubin, D. T. (1960) J. Biol. Chem. 235, 769-775 [Medline] [Order article via Infotrieve]
  19. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600 [Abstract/Free Full Text]
  20. Van Veldhoven, P. P., Bishop, W. R., and Bell, R. M. (1989) Anal. Biochem. 183, 177-189 [Medline] [Order article via Infotrieve]
  21. Jayadev, S., Linardic, C. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269, 5757-5763 [Abstract/Free Full Text]
  22. Okazaki, T., Bielawska, A., Domae, N., Bell, R. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269, 4070-4077 [Abstract/Free Full Text]
  23. Bielawska, A., Crane, H. M., Liotta, D., Obeid, L. M., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 26226-26232 [Abstract/Free Full Text]
  24. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530 [Abstract/Free Full Text]
  25. Jarvis, W. D., Kolesnick, R. N., Forani, F. A., Taylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77 [Abstract]
  26. Bell, R. M. (1986) Cell 45, 631-632 [Medline] [Order article via Infotrieve]
  27. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  28. Liscovitch, M. (1992) Trends Biochem. Sci. 17, 393-399 [CrossRef][Medline] [Order article via Infotrieve]
  29. Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267, 12393-12396 [Free Full Text]
  30. Exton, J. H. (1994) Biochim. Biophys. Acta 1212, 26-42 [Medline] [Order article via Infotrieve]
  31. Ishizuka, T., Hoffman, J., Cooper, D. R., Watson, J. E., Pushkin, D. B., and Farese, R. V. (1989) FEBS Lett. 249, 234-238 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.