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Papers In Press, published online ahead of print January 1, 2007
J. Lipid Res., doi:10.1194/jlr.M600399-JLR200

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Journal of Lipid Research, Vol. 48, 66-76, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Effects of ceramide-1-phosphate on cultured cells: dependence on dodecane in the vehicle

Loïc Tauzin^*, Christine Graf^*, Mei Sun, Philipp Rovina^*, Nicolas Bouveyron^*, Markus Jaritz^*, Anthony Winiski^*, Nicole Hartmann, Frank Staedtler, Andreas Billich^*, Thomas Baumruker^*, Mei Zhang^** and Frédéric Bornancin^1,*

^* Novartis Institutes for BioMedical Research, A-1235 Vienna, Austria
Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Novartis Institutes for BioMedical Research, CH-4057 Basel, Switzerland
^** Synta Pharmaceuticals Corp., Lexington, MA

The online version of this article (available at http://www.jlr.org) contains supplemental data in the form of 2 figures and 1 table.

Published, JLR Papers in Press, October 3, 2006.

¹ To whom correspondence should be addressed. e-mail: frederic.bornancin{at}novartis.com

Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A₂. Much of the present knowledge has relied on the use of C1P of various acyl chain lengths, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here, we show that addition of C1P in ethanol-dodecane to culture medium leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation, and cell death. Not only the toxicity of C1P, but also its ability to trigger prostaglandin E₂ release, is fully dependent upon addition of a premade C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and, in turn, help in unraveling C1P signaling, which is still poorly understood.

Supplementary key words ceramide kinase • sphingolipid • mitochondria • viability • phosphatidic acid • phospholipase A₂ • prostaglandin E₂

Abbreviations: ATCC, American Type Culture Collection; C1P, ceramide-1-phosphate; cPLA₂, cytosolic phospholipase A₂; ER, endoplasmic reticulum; LAMP2, lysosome-associated membrane protein 2; NBD-Cer, nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide; PA, phosphatidic acid; PG, phosphatidylglycerol; PGE₂, prostaglandin E₂; YFP, yellow fluorescent protein

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