Abstract
Ceramide is a bioactive lipid involved in the induction of apoptosis and is the precursor to several sphingolipids, including sphingomyelin, the gangliosides, and sphingosine. Ceramide production is increased in response to stress and toxic agents. Because modulation of ceramide levels has been shown to affect sensitivity and/or resistance to therapeutic agents, it will be important to assess the activity of sphingolipid metabolic pathways when investigating the mode of action of antitumor drugs. This chapter summarizes protocols for quantitating the level of apoptosis, the activities of acidic sphingomyelinase, neutral sphingomyelinase, glycosylceramide synthase, sphingomyelin synthase, and ceramidase, and the amount of ceramide in tumor xenografts in nude mice.
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References
Jarvis, W. D. and Grant, S. (1998) The role of ceramide in the cellular response to cytotoxic agents. Curr. Opin. Oncol. 10, 552–559.
Liu, G., Kleine, L., and Hebert, R. L. (1999) Advances in the signal transduction of ceramide and related sphingolipids. Crit. Rev. Clin. Lab. Sci. 36, 511–573.
Pena, L. A., Fuks, Z., and Kolesnick, R. (1997) Stress-induced apoptosis and the sphingomyelin pathway. Biochem. Pharmacol. 53, 615–621.
Levade, T. and Jaffrezou, J. P. (1999) Signalling sphingomyelinases: which, where, how and why? Biochim. Biophys. Acta 1438, 1–17.
Liu, B., Obeid, L. M., and Hannun, Y. A. (1997) Sphingomyelinases in cell regulation. Semin. Cell Dev. Biol. 8, 311–322.
Cabot, M. C., Giuliano, A. E., Han, T. Y., and Liu, Y. Y. (1999) SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drugresistant cancer cells. Cancer Res. 59, 880–885.
Bose, R., Verheij, M., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995) Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82, 405–414.
Liu, Y. Y., Han, T. Y., Giuliano, A. E., and Cabot, M. C. (1999) Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells. J. Biol. Chem. 274, 1140–1146.
Senchenkov, A., Litvak, D. A., and Cabot, M. C. (2001) Targeting ceramide metabolism-a strategy for overcoming drug resistance. J. Natl. Cancer Inst. 93, 347–357.
Kolesnick, R. (2002) The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J. Clin. Invest. 110, 3–8.
Modrak, D. E., Rodriguez, M. D., Goldenberg, D.M., Lew, W., and Blumenthal, R. D. (2002) Sphingomyelin enhances chemotherapy efficacy and increases apoptosis in human colonic tumor xenografts. Int. J. Oncol. 20, 379–384.
Merrill, A. and Hannun, Y. (eds.) (2000) Sphingolipid Metabolism and Cell Signalling, Part A, Vol. 311. Academic, London.
Merrill, A. and Hannun, Y. (eds.) (2000) Sphingolipid Metabolism and Cell Signalling, Part B, Vol. 312. Academic, London.
Hansen, H. J., Goldenberg, D. M., Newman, E. S., Grebenau, R., and Sharkey, R. M. (1993) Characterization of second-generation monoclonal antibodies against carcinoembryonic antigen. Cancer 71, 3478–3485.
Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917.
Ames, B. N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Meth. Enzymol. 8, 115–118.
Bodennec, J., Brichon, G., Koul, O., El Babili, M., and Zwingelstein, G. (1997) A two-dimensional thin-layer chromatography procedure for simultaneous separation of ceramide and diacylglycerol species. J. Lipid Res. 38, 1702–1706.
Morgan, E. T., Nikolova-Karakashian, M., Chen, J. Q., and Merrill, A. H. Jr. (1996) Sphingolipid-dependent signaling in regulation of cytochrome P450 expression. Meth. Enzymol. 272, 381–388.
Watts, J. D., Gu, M., Polverino, A. J., Patterson, S. D., and Aebersold, R. (1997) Fas-induced apoptosis of T cells occurs independently of ceramide generation. Proc. Natl. Acad. Sci. USA 94, 7292–7296.
Tepper, A. D. and Van Blitterswijk, W. J. (2000) Ceramide mass analysis by normalphase high-performance liquid chromatography. Meth. Enzymol. 312, 16–22.
Liu, P. and Anderson, R. G. (1995) Compartmentalized production of ceramide at the cell surface. J. Biol. Chem. 270, 27,179–27,185.
Sillence, D. J., Raggers, R. J., and van Meer, G. (2000) Assays for transmembrane movement of sphingolipids. Meth. Enzymol. 312, 562–579.
Liu, B. and Hannun, Y. A. (2000) Sphingomyelinase assay using radiolabeled substrate. Meth. Enzymol. 311, 164–167.
Hertervig, E., Nilsson, A., Nyberg, L., and Duan, R. D. (1997) Alkaline sphingomyelinase activity is decreased in human colorectal carcinoma. Cancer 79, 448–453.
Shayman, J. A. and Abe, A. (2000) Glucosylceramide synthase: assay and properties. Meth. Enzymol. 311, 42–49.
Nikolova-Karakashian, M. (2000) Assays for the biosynthesis of sphingomyelin and ceramide phosphoethanolamine. Meth. Enzymol. 311, 31–42.
Nikolova-Karakashian, M. and Merrill, A. H. Jr. (2000) Ceramidases. Meth. Enzymol. 311, 194–201.
Perry, D. K. and Hannun, Y. A. (1999) The use of diglyceride kinase for quantifying ceramide. Trends Biochem. Sci. 24, 226–227.
Liu, B. and Hannun, Y. A. (1997)Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione. J. Biol. Chem. 272, 16,281–16,287.
Brenkert, A. and Radin, N. S. (1972) Synthesis of galactosyl ceramide and glucosyl ceramide by rat brain: assay procedures and changes with age. Brain Res. 36, 183–193.
Shukla, G. S. and Radin, N. S. (1990) Glucosyceramide synthase of mouse kidney: further characterization with an improved assay method. Arch. Biochem. Biophys. 283, 372–378.
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Modrak, D.E. (2005). Measurement of Ceramide and Sphingolipid Metabolism in Tumors. In: Blumenthal, R.D. (eds) Chemosensitivity: Volume II. Methods in Molecular Medicine™, vol 111. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-889-7:183
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DOI: https://doi.org/10.1385/1-59259-889-7:183
Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-59259-889-2
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