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J. Biol. Chem., Vol. 279, Issue 22, 23238-23249, May 28, 2004

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De Novo Ceramide Accumulation Due to Inhibition of Its Conversion to Complex Sphingolipids in Apoptotic Photosensitized Cells^*

Vladislav Dolgachev, M. Sharjeel Farooqui, Olga I. Kulaeva, Michael A. Tainsky, Biserka Nagy¶, Kentaro Hanada||, and Duska Separovic**

From the Occupational and Environmental Health Sciences, The Department of Fundamental and Applied Sciences, Eugene Applebaum College of Pharmacy and Health Sciences and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, the ^¶Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia, and the ^||Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [¹⁴C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [¹⁴C]ceramide with a concomitant decrease in [¹⁴C]sphingomyelin was detected. The labeling of [¹⁴C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.

Received for publication, October 31, 2003 , and in revised form, February 13, 2004.

^* This work was supported by United States Public Health Service Grant R29 CA77475 from the NCI, National Institutes of Health. 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.

^** To whom correspondence should be addressed: Occupational and Environmental Health Sciences, Rm. 5142, Eugene Applebaum College of Pharmacy and Health Sciences, 259 Mack Ave., Detroit, MI 48201. Tel.: 313-577-8065; Fax: 313-577-0097; E-mail: dseparovic{at}wayne.edu.

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