Ceramide reduction and transcriptional up-regulation of glucosylceramide synthase through doxorubicin-activated Sp1 in drug-resistant HL-60/ADR cells.
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.
Uchida
Yoshikazu
Y
Department of Hematology and Oncology, Clinical Sciences for Pathological Organs, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Itoh
Mitsuru
M
Taguchi
Yoshimitsu
Y
Yamaoka
Syohei
S
Umehara
Hisanori
H
Ichikawa
Shin-Ichi
S
Hirabayashi
Yoshio
Y
Holleran
Walter M
WM
Okazaki
Toshiro
T
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D015854:Up-Regulation
D000903:Antibiotics, Antineoplastic; D002518:Ceramides; D009841:Oligonucleotides; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D004317:Doxorubicin; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase