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J. Biol. Chem., Vol. 282, Issue 15, 10922-10934, April 13, 2007

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Alterations of Ceramide/Sphingosine 1-Phosphate Rheostat Involved in the Regulation of Resistance to Imatinib-induced Apoptosis in K562 Human Chronic Myeloid Leukemia Cells^*

Yusuf Baran¹², Arelis Salas¹³, Can E. Senkal, Ufuk Gunduz, Jacek Bielawski, Lina M. Obeid^¶, and Besim Ogretmen⁴

From the Department of Biochemistry and Molecular Biology, and Hollings Cancer Center and the ^¶Division of General Internal Medicine, Ralph H. Johnson Veterans Administration Hospital, and Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and the Department of Biology, Middle East Technical University, Ankara, Turkey

In this study, mechanisms of resistance to imatinib-induced apoptosis in human K562 cells were examined. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of K562/IMA-0.2 and -1 cells, which expressed 2.3- and 19-fold resistance, respectively. Measurement of endogenous ceramides by high performance liquid chromatography/mass spectroscopy showed that treatment with imatinib increased the generation of ceramide, mainly C₁₈-ceramide, which is generated by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Inhibition of hLASS1 by small interfering RNA partially prevented imatinib-induced cell death in sensitive cells. In reciprocal experiments, overexpression of hLASS1, and not hLASS6, in drug-resistant cells caused a marked increase in imatinib-induced C₁₈-ceramide generation, and enhanced apoptosis. Interestingly, there were no defects in the levels of mRNA and enzyme activity levels of hLASS1 for ceramide generation in K562/IMA-1 cells. However, expression levels of sphingosine kinase-1 (SK1) and generation of sphingosine 1-phosphate (S1P) were increased significantly in K562/IMA-1 cells, channeling sphingoid bases to the sphingosine kinase pathway. The partial inhibition of SK1 expression by small interference RNA modulated S1P levels and increased sensitivity to imatinib-induced apoptosis in resistant cells. On the other hand, forced expression of SK1 in K562 cells increased the ratio between total S1P/C₁₈-ceramide levels 6-fold and prevented apoptosis significantly in response to imatinib. Additional data indicated a role for SK1/S1P signaling in the up-regulation of the Bcr-Abl expression at the post-transcriptional level, which suggested a possible mechanism for resistance to imatinib-mediated apoptosis. In conclusion, these data suggest a role for endogenous C₁₈-ceramide synthesis mainly via hLASS1 in imatinib-induced apoptosis in sensitive cells, whereas in resistant cells, alterations of the balance between the levels of ceramide and S1P by overexpression of SK1 result in resistance to imatinib-induced apoptosis.

Received for publication, October 30, 2006 , and in revised form, February 12, 2007.

^* This work was supported in part by National Institutes of Health (NIH) Grants CA88932, DE01657, and CA097132, Dept. of Defense Program Project Phase 7, through Hollings Cancer Center, National Science Foundation/Experimental Program to Stimulate Competitive Research (EPSCoR) Grant EPS-0132573, and the Middle East Technical University Research Project (Grant BAP-2004-07-02-00-20 to U. G.). The Lipidomics Core and Flow Cytometry Facility at Medical University of South Carolina are partly supported by grants from NIH. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ Both authors contributed equally to this work.

² Sponsored by a fellowship from The Scientific and Technological Research Council of Turkey through NATO-A2 research grant.

³ Recipient of the minority student research award from the Comprehensive Minority Biomedical Branch of National Institutes of Health.

⁴ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Hollings Cancer Center, Charleston, SC 29425. Tel.: 843-792-0940; Fax: 843-792-8568; E-mail: ogretmen{at}musc.edu.

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