Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter.
Histone deacetylase (HDAC) inhibitors are known to arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1). However, several studies have suggested the existence of a p21(WAF1/Cip1)-independent molecular pathway. We report here that HDAC inhibitors activate a member of the INK4 family, the INK4d gene, causing G1 phase arrest, in the human T cell leukemia cell line, Jurkat. One of the major Trichostatin A (TSA)-responsive elements is a specific Sp1 binding site in the INK4d promoter. Electrophoretic mobility-shift assay revealed that Sp1 and Sp3 can specifically interact with this Sp1 binding site. Furthermore, using chromatin immunoprecipitation assay, we demonstrated that HDAC2 was present in the INK4d proximal promoter region in the absence, but not the presence, of TSA. Taken together, these results suggest that treatment with TSA transcriptionally activates INK4d by releasing HDAC2 from the histone-DNA complex at the INK4d promoter. Using a p21(WAF1/Cip1)-deleted human colorectal carcinoma cell line, HCT116 p21 (-/-), we show that upregulation of p19(INK4d) by TSA is associated with inhibition of cell proliferation. Moreover, mouse embryo fibroblasts lacking Ink4d were resistant to the growth inhibitory effects of TSA as compared to their wild-type counterpart. Our findings suggest that p19(INK4d) in addition to p21(WAF1/Cip1) is an important molecular target of HDAC inhibitors inducing growth arrest.
Yokota
Tomoya
T
Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
Matsuzaki
Youichirou
Y
Miyazawa
Kazuhiro
K
Zindy
Frederique
F
Roussel
Martine F
MF
Sakai
Toshiyuki
T
D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D050764:Cyclin-Dependent Kinase Inhibitor p19; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D016193:G1 Phase; D017353:Gene Deletion; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
C493956:CDKN1A protein, human; C493974:CDKN2D protein, human; C493957:Cdkn1a protein, mouse; C493975:Cdkn2d protein, mouse; D018797:Cell Cycle Proteins; D002843:Chromatin; D050764:Cyclin-Dependent Kinase Inhibitor p19; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D012333:RNA, Messenger; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; C012589:trichostatin A; D008156:Luciferases