title: Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma.
abstract: OBJECTIVE
  Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells.
  
  
  METHODS
  Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA.
  
  
  RESULTS
  The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter.
  
  
  CONCLUSIONS
  Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.
authors.affiliation: Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kishi
authors.forename: Tadahiko
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Hironori
authors.initials: H
authors.lastname: Tanaka
authors.forename: Takahiro
authors.initials: T
authors.lastname: Tanaka
authors.forename: Tetsu
authors.initials: T
authors.lastname: Tomonari
authors.forename: Koichi
authors.initials: K
authors.lastname: Okamoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sogabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyamoto
authors.forename: Toshiya
authors.initials: T
authors.lastname: Okahisa
authors.forename: Naoki
authors.initials: N
authors.lastname: Muguruma
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kajimoto
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagawa
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Takayama
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D006801:Humans; D007124:Immunoenzyme Techniques; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D013058:Mass Spectrometry; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D015415:Biomarkers; D014408:Biomarkers, Tumor; D011498:Protein Precursors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C008208:acarboxyprothrombin; D011516:Prothrombin; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D000469:Alkaline Phosphatase
keywords: Biomarkers; Des-gamma-carboxy prothrombin; Hepatocellular carcinoma; Poly-(ADP-ribose) polymerase-1; Prothrombin