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J. Biol. Chem., Vol. 282, Issue 23, 16718-16728, June 8, 2007

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Involvement of Dihydroceramide Desaturase in Cell Cycle Progression in Human Neuroblastoma Cells^*

Jacqueline M. Kraveka¹, Li Li, Zdzislaw M. Szulc, Jacek Bielawski, Besim Ogretmen, Yusuf A. Hannun, Lina M. Obeid^¶||, and Alicja Bielawska

From the Division of Hematology/Oncology, Department of Pediatrics, and the Departments of Biochemistry and Molecular Biology and ^¶Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and the ^||Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401

The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12'-(1''-pyridinium)-dodecanoyl]-4,5-dihydrosphingosine bromide (C₁₂-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12'-(1''-pyridinium)dodecanoyl]sphingosine bromide (C₁₂-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C₈-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C₁₂-dhCCPS to C₁₂-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G₀/G₁. This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.

Received for publication, January 23, 2007

Note Added in Proof—While these studies were in progress, another paper by Zheng et al. (Biochim. Biophys. Acta (2006) 1758, 1864–1884) has reported that 4-HPR inhibits the desaturase and elevates dihydroceramides.

^* 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 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Div. of Hematology/Oncology, Dept. of Pediatrics, Medical University of South Carolina, 135 Rutledge Ave., P. O. Box 250558, Charleston, SC 29425. Tel.: 843-792-2957; Fax: 843-792-8912; E-mail: kravekjm{at}musc.edu.

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