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 Baran,Arelis Salas,Can E Senkal,Ufuk Gunduz,Jacek Bielawski,Lina M Obeid,Besim Ogretmen

doi:10.1074/jbc.m610157200

Abstract

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 approximately 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 approximately 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.

Highlights

sphingosine 1-phosphate (S1P)/C18-ceramide levels ϳ6-fold and prevented apoptosis significantly in response to imatinib
Additional data indicated a role for sphingosine kinase-1 (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
The error bars represent the standard deviations, and when not seen, they are smaller than the thickness of the lines on the graphs. These results further suggest that defects in the generation, or accumulation/metabolism of C18ceramide, might result in increased resistance to imatinib-induced cell death in K562 cells

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions—The Ph chromosomepositive K562 human CML cells were obtained from the German Collection of Microorganisms and Cell Cultures and maintained in RPMI 1640 growth medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen) at 37 °C in 5% CO2. Detection of the Loss of Mitochondrial Membrane Potential— The collapse of an electrochemical gradient across the mitochondrial membrane during apoptosis was measured using a JC-1 mitochondrial membrane potential detection kit (Cell Technology) by flow cytometry as described previously [32]. 1 ␮l of the reverse transcriptase reaction was amplified using these primers by PCR for 35 cycles (94 °C for 1 min, 55– 68 °C for 2.5 min, and 72 °C for 2 min), and their levels were normalized to that of ␤-actin as described previously [31]. Quantitative real-time PCR for the measurement of mRNA levels of human SK1, Abl, LASS1, LASS2, LASS4, LASS5, LASS6, and rRNA were performed using TaqMan௡ gene expression kit and gene-specific real-time-PCR TaqMan primer/probe mix (Applied Biosystems) with ABI 7300 Q-PCR system as described by the manufacturer. Statistical significance was examined using analysis of variance or Student’s t test analysis, and p Ͻ 0.05 was considered significant

RESULTS

DISCUSSION

Obeid and Besim Ogretmen