Sp1 is involved in 8-chloro-adenosine-upregulated death receptor 5 expression in human hepatoma cells.
8-Chloro-adenosine (8-Cl-Ado) is an adenosine derivative, which inhibits proliferation and induces apoptosis in various tumor cells. Subtoxic concentration of 8-Cl-Ado sensitizes human hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-triggered apoptosis. However, the molecular mechanism by which TRAIL cytotoxicity is amplified by 8-Cl-Ado is unknown. In the present study, we demonstrated by Western blot and real-time PCR that 8-Cl-Ado selectively up-regulated death receptor 5 (DR5), but not death receptor 4 (DR4), at both protein and RNA levels in human hepatoma cell line BEL-7402. Analysis of the transcriptional regulation of DR5 expression by using Dual-Luciferase reporter assay system demonstrated that the 5'-flanking fragment -207 to -145 upstream to the ATG site within the DR5 promoter region was responsible for the 8-Cl-Ado-upregulated DR5 expression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) confirmed that 8-Cl-Ado treatment facilitated transcription factor Sp1 binding to its cis-element -198/-189 in the DR5 promoter, suggesting that Sp1 is at least one of the 8-Cl-Ado-responsive transcription factors. However, we observed that nuclear factor kappaB (NF-kappaB) activity remained invariable in the cells treated with 8-Cl-Ado. These data allowed us to draw a conclusion that 8-Cl-Ado-enhanced DR5 expression is regulated by Sp1 binding to the -198/-189 cis-element in DR5 promoter without affecting NF-kappaB activity in the hepatoma cells. This study may shed light on further screening the regulators of DR5 expression and developing novel therapeutic drugs for liver cancer.
Sun
Minmin
M
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, PR China.
Zhang
Jinchun
J
Liu
Shilian
S
Liu
Yanxin
Y
Zheng
Dexian
D
D000241:Adenosine / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007136:Immunoglobulins / Q000187:drug effects / Q000378:metabolism*; D047468:Immunoprecipitation; D008113:Liver Neoplasms / Q000378:metabolism*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000187:drug effects; D015854:Up-Regulation
D000970:Antineoplastic Agents; D007136:Immunoglobulins; D012333:RNA, Messenger; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; C090698:SP1 antigen; D000241:Adenosine