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Ceramide Reduction and Transcriptional Up-Regulation of Glucosylceramide Synthase through Doxorubicin-Activated Sp1 in Drug-Resistant HL-60/ADR Cells

¹ Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto, Japan; ² Frontier Research Program, Institute of Chemical and Physical Research, RIKEN, Saitama, Japan; and ³ Department of Dermatology, School of Medicine, University of California and Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, San Francisco, California

Treatment with doxorubicin (DOX) induced apoptosis with an increase of ceramide content in drug-sensitive HL-60 cells, but not in drug-resistant HL-60/ADR cells. In HL-60/ADR cells (but not in HL-60 cells), the levels of mRNA, protein, and activity in glucosylceramide synthase (GCS), which converts ceramide to glucosylceramide, were up-regulated in response to DOX. Thus, abrogation of apoptosis in HL-60/ADR cells might be involved in ceramide reduction through DOX-induced up-regulation of GCS function. Because we reported that a GC-rich/Sp1 promoter binding region was of importance in the regulation of GCS expression, the role of Sp1 in DOX-induced up-regulation of GCS and apoptosis was investigated. DOX induced Sp1 activation in HL-60/ADR cells, as assessed by Sp1 gel shift and promoter-luciferase reporter assays, whereas transfection of double-stranded oligodeoxynucleotides (ODNs) containing a GC-rich/Sp1 region (Sp1 decoy ODNs) inhibited DOX-induced Sp1 activation. In addition, DOX-increased mRNA and enzyme activity in GCS were inhibited by Sp1 decoy, in conjunction with corresponding elevations of ceramide content. Moreover, DOX-induced apoptotic cell death was significantly increased in Sp1 decoy ODN-transfected HL-60/ADR cells over mock-transfected HL-60/ADR cells. Together, the results suggest that transcriptional up-regulation of GCS through DOX-induced activation of Sp1 is one potential mechanism to regulate ceramide increase and apoptosis in HL-60/ADR cells.

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