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La,25-Dihydroxyvitamin D3 Inhibits Programmed Cell Death in HL-60 Cells by Activation of Sphingosine Kinase1

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La,25-Dihydroxyvitamin D3 Inhibits Programmed Cell Death in HL-60 Cells by Activation of Sphingosine Kinase1 (CANCER RESEARCH 58. 1817-1824, May 1. I99K] la,25-Dihydroxyvitamin D3 Inhibits Programmed Cell Death in HL-60 Cells by Activation of Sphingosine Kinase1 Burkhard Kleuser, Olivier Cuvillier, and Sarah Spiegel2 Department of Biochemistn1 ariti Molecular Biology. Georgetown University Medical Center, Washington, DC 20007 (OH)-,D3 (8) is accompanied by activation of Mg2+-independent, ABSTRACT neutral cytosolic SMase, resulting in hydrolysis of sphingomyelin to Sphingolipid breakdown products [ceramide, sphingosine, and sphin- ceramide and phosphorylcholine (9, 10). Moreover, cell-permeable gosine-1-phosphate (SPP)] are emerging as a new class of bioactive mol ecules. In agreement with previous studies, treatment of human promy- synthetic ceramide analogues or treatment with bacterial SMase in hibited growth and induced differentiation of HL-60 cells, suggesting elocytic leukemia HL-60 cells with l-a,25-dihydroxyvitamin l>, [1,25- an important role for ceramide in 1,25-(OH)2D,-mediated differenti (OII),I),1 induced a transient increase of ceramide levels within 2 h, which ation (9-11). However, it has also been well documented that ceram then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h ide is an important regulatory component of programmed cell death of exposure, leading to a concomitant increase in SPP level. Unlike treat (apoptosis) induced by various stress stimuli, including members of ments with cell-permeable ceramide analogues or sphingomyelinase, the TNF superfamily, serum withdrawal, and ionizing radiation (re which induce apoptosis, 1,25-(OH)2D, did not induce apoptosis, despite viewed in Refs. 12-14). Moreover, increased cellular levels of cer the early formation of ceramide. Moreover, prolonged treatment of HL-60 amide by treatment with cell-permeable ceramide analogues or SMase cells with l.25-(OIIi,I), suppressed ceramide-induced apoptosis. There enhance apoptosis (15-17). Surprisingly, treatment of HL-60 cells was a correlation between the time course and dose response of the with 1,25-(OH)2D3, which elevates ceramide levels, made them re activation of sphingosine kinase by I,25-(OII),I>, and the protection against apoptosis. In contrast, treatment with all-/ronv-retinoic acid nei sistant to apoptotic cell death induced by calcium ionophores and cancer chemotherapeutic drugs (18-20). Antisense inhibition of VDR ther stimulated sphingosine kinase activity nor protected cells from cer amide-induced apoptosis. Treatment with SPP protected HL-60 cells from expression induces apoptosis in U937 cells, suggesting that cell sur ceramide-induced apoptosis, and /VyV-dimethylsphingosine (DMS), a com vival is mediated via binding of 1,25-(OH)2D3 to its nuclear receptor petitive inhibitor of sphingosine kinase, prevented the survival effect of (21). 1,2S-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting Recently, we have shown that SPP, which is a metabolite of that SPP is a critical component of the cytoprotective effect of 1,25- ceramide, prevents the appearance of the hallmarks of apoptosis (OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, mark induced by ceramide (22). Moreover, we have shown that the disso edly reduced sphingosine kinase activity and the apoptosis-sparing effect ciation of stress-induced apoptosis from growth factor-mediated pro of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecano- liferation is a consequence of formation of distinct sphingolipid- ylphorbol-13-acetate not only suppressed ceramide-induced apoptosis but derived second messengers. Because the relative balance of the also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collec sphingolipid metabolites, ceramide, and SPP has recently been shown tively, our observations indicate that the cytoprotective effect of 1,25- to influence opposing pathways of apoptosis and cell survival, it was (OH)2D3 is mediated by SPP, which is formed as a consequence of acti of interest to determine whether SPP production might play a role in vation of sphingosine kinase. the survival effect of 1,25-(OH)2D3. In this study, we found that, although 1,25-(OH)2D3 induces a rapid increase in ceramide, this is INTRODUCTION followed by a delayed increase in sphingosine kinase activity leading 1,25-(OH)2D3,3 a member of the steroid hormone family, controls to increased levels of SPP, which, in turn, may be an important component of the cytoprotective effect of 1,25-(OH)2D3. Moreover, calcium homeostasis (1). It also has been shown to inhibit growth of our study demonstrates that agonist-induced activation of sphingosine malignant cells, including breast and colon cancer cells (2, 3), and to kinase can suppress concomitant ceramide-mediated apoptosis. induce differentiation of immature hematopoietic cells (4). These observations have stimulated interest in the therapeutic use of this secosteroid in cancer, acute promyelocytic leukemia, and psoriasis MATERIALS AND METHODS (1). The effects of 1,25-(OH)2D3 are mediated mainly via interaction Materials. 1,25-(OH)2D, was a generous gift from Dr. Lise Binderup with an intracellular receptor (VDR), which heterodimerizes with the (Leo-Pharmaceutical Products. Ballerup, Denmark). RA was purchased from retinoic acid receptor (RXR), enhancing gene activity (5, 6). How Fluka (Ronkomkoma. NY). SPP. sphingosine. and chelerythrine chloride were ever, some of its rapid effects (such as increased cytosolic free from Biomol (Plymouth Meeting. PA). [mefAv/-3H]Thymidine (70-90 Ci/ calcium) have been suggested to be independent of VDR (7). Previ mmol), [y-32P]ATP (3000 Ci/mmol), ['H]acetic anhydride (50 mCi/mmol), ously, it has been demonstrated that monocytic differentiation of and ['Hjenhancer spray were from NEN (Boston, MA). SMase (from Strep- human promyelocytic leukemia HL-60 cells induced by 1,25- tomyces sp. and Slaphylococcus áureas), ceramides (from bovine brain, type III), and diethelenetriaminepentaacetic acid were from Sigma Chemical Co. (St. Louis. MO). The various standard phospholipids and cardiolipin were from Received 10/6/97; accepted 3/4/98. Avanti Polar Lipids (Birmingham, AL). /V-Acetyl sphingosine (C2-Cer) was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with from Matreya (Pleasant Gap. PA). Escherichia coli DAG kinase and octyl-ß- 18 U.S.C. Section 1734 solely to indicate this fact. D-glucopyranoside were purchased from Calbiochem (La Jolla, CA). Insulin 1This work was supported by NIH Research Grants CM 43880 and CA61774. 2 To whom requests for reprints should be addressed, at Department of Biochemistry and transferrin were from Collaborative Research (Lexington, MA). PCS was and Molecular Biology, Georgetown University Medical Center, 353 Basic Science purchased from PAA Laboratories (Newport Beach. CA). Silica gel G60 plates Building, 3900 Reservoir Road NW, Washington, DC 20007. Phone: (202)687-1432; were from EM Sciences (Cherry Hill. NJ). and high-performance TLC plates Fax: (2021687-0260. were from Whatman International Ltd. (Maidstone. England). 'The abbreviations used are: 1,25-(OH)2D„ l-a.25-dihydroxyvitamin D,; SMase, Cell Culture. Human promyelocytic leukemia HL-60 cells were from sphingomyelinase; TNF, tumor necrosis factor; SPP, sphingosine-1-phosphate; RA, all- rrani-retinoic acid; DAG, diacylglycerol; DMS, ¿V.A'-dimethylsphingosine: PKC. protein American Type Culture Collection. Cells were cultured in RPMI 1640 with kinase C; TP A, 12-O-tetradecanoylphorbol-13-acetate. 10% PCS at 37°Cin 5% CO,. Cells were washed twice with PBS and then 1817 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1998 American Association for Cancer Research. l-a.25-DIHYDROXYVITAMIN D, AND SPHINGOSINE-] -PHOSPHATE resuspended at a cell density of 0.3-0.5 x IO6 cells/ml in serum-free medium acetic acid, and water (80:20:10:20, v/v/v/v) as solvent. The radioactive spots supplemented with transferrin (5 fig/liter), insulin (5 /ig/liter), and BSA corresponding to authentic SPP were visualized by autoradiography, scraped (0.5%). from the plates, and counted in a scintillation counter (25). Mass Measurement of Ceramide. Cells were treated with the indicated Quantitation of SPP. SPP levels were determined by minor modifications concentrations of 1,25-(OH)2D3 for various incubation periods. Lipids were ex of a method previously described by Yatomi et al. (26). Briefly, after treatment tracted and the mass amounts of ceramide in cellular extracts were measured by with 1,25-(OH)2D3 for various times, cells were washed two times with PBS; the use of the DAG kinase enzymatic method (23). Briefly, 1 x IO7 cells were suspended in 3.5 ml of methanol, chloroform, and PBS (2:1:0.5, v/v/v); and centrifuged, and 3 ml of methanol, chloroform, and water (1:1:1, v/v/v) were sonicated on ice. Alkaline extraction was performed by addition of 4.1 ml of added to the cell pellet. Two hundred ¿tlofthe chloroform phase were dried under chloroform, l M KC1, and concentrated NH4OH (2:2:0.1, v/v/v). The aqueous a nitrogen stream; the lipids or standard bovine brain type III ceramides were phase containing SPP was extracted with 3.2 ml of chloroform-concentrated suspended in 40 fi\ of 7.5% (w/v) octyl-ß-glycopyranoside-5 mM cardiolipin in 1 HC1 (3:0.2, v/v), and the organic phases were then evaporated under N, and mM diethelenetriaminepentaacetic acid-10 mM imidazole (pH 6.6) and then solu- solubilized in 20 ¿Uof0.008 N NaOH-methanol and 20 ¿ilof 10 mM [3H]acetic bilized by freeze-thawing and subsequent sonication. The enzymatic reaction was anhydride (10 (nCi). Acetylation reactions proceeded at 37°Cfor 2 h. Acety- started by the addition of 20 ftl of DTT (20 mM). 20 ¿UofE.
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