title: Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.
abstract: BACKGROUND
  Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130.
  
  
  METHODS
  We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated.
  
  
  RESULTS
  We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected.
  
  
  CONCLUSIONS
  HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.
authors.affiliation: Experimental Oncology, "Regina Elena" National Cancer Institute, IRCCS, Via Elio Chianesi, 53, 00144, Rome, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Mileo
authors.affiliation: Department of Molecular Biology, University of Geneva, 1211, Geneva, Switzerland.
authors.forename: Stefano
authors.initials: S
authors.lastname: Mattarocci
authors.affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Matarrese
authors.affiliation: National AIDS Center, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Anticoli
authors.affiliation: Experimental Oncology, "Regina Elena" National Cancer Institute, IRCCS, Via Elio Chianesi, 53, 00144, Rome, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Abbruzzese
authors.affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Catone
authors.affiliation: Gastroenterology and Metabolic Diseases, Department of Gastroenterology, 56124 Pisa University Hospital, Pisa, Italy.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Sacco
authors.affiliation: Experimental Oncology, "Regina Elena" National Cancer Institute, IRCCS, Via Elio Chianesi, 53, 00144, Rome, Italy. paggi@ifo.it.
authors.forename: Marco G
authors.initials: MG
authors.lastname: Paggi
authors.affiliation: Department of Veterinary Public Health & Food Safety, Istituto Superiore di Sanità, 00161, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Ruggieri
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015870:Gene Expression; D005786:Gene Expression Regulation; D020868:Gene Silencing; D018936:Hepatitis C Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130 / Q000235:genetics* / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018936:Hepatitis C Antigens; D016160:Retinoblastoma Protein; D050717:Retinoblastoma-Like Protein p130; D014758:Viral Core Proteins; C499352:core protein p22, Hepatitis C virus