title: Adenosine promotes GATA-2-regulated p53 gene transcription to induce HepG2 cell apoptosis.
abstract: OBJECTIVE
  In our earlier study, adenosine induced apoptosis in HepG2 human hepatoma cells by tuning of apoptosis-mediator gene transcription. The present study aimed at understanding the regulatory mechanism underlying the apoptosis-mediator gene transcription under the control of adenosine.
  
  
  METHODS
  For HepG2 cells with and without knocking-down p53 or GATA-2, cell viability, mitochondrial membrane potentials, caspase activity, and transcriptional activity were monitored, and Western blotting, RT-PCR, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were carried out.
  
  
  RESULTS
  Extracellular adenosine upregulated expression of the p53 mRNA and protein in HepG2 human hepatoma cells. Adenosine induced apoptosis, disrupted mitochondrial membrane potentials, and activated caspase-3, -8 and -9 in HepG2 cells, and those effects were inhibited by silencing the p53-targetd gene. In the assay of transcriptional activity using full-length p53 gene promoter and 5' deletion mutants combined with the luciferase reporter vector, adenosine enhanced transcriptional activity for full-length p53 gene promoter, that was prevented by deleting from -240 to -146 bp on the promoter. In the EMSA using a (32)P-labeled DNA probe to detect binding to the putative GATA-2 biding site on the p53 gene promoter, adenosine produced (32)P-positive signals in nuclear extracts from HepG2 cells. In the Western blot analysis, adenosine increased presence of GATA-2 in nuclear extracts. In the ChIP assay, adenosine increased PCR products for the p53 gene promoter in chromosomal extracts from HepG2 cells, immunoprecipitated using an anti-GATA-2 antibody. Adenosine-induced upregulation of the p53 mRNA expression was suppressed by knocking-down GATA-2.
  
  
  CONCLUSIONS
  The results of the present study show that p53 is a transcriptional target of GATA-2 and that adenosine upregulates GATA-2-regulated p53 expression, thereby activating caspase-3, -8, and -9 to induce HepG2 cell apoptosis.
authors.affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yaguchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakano
authors.forename: Akinobu
authors.initials: A
authors.lastname: Gotoh
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nishizaki
mesh_terms: D000241:Adenosine / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D050989:GATA2 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D050989:GATA2 Transcription Factor; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D000241:Adenosine