title: The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine.
abstract: OBJECTIVE
  Clear cell renal carcinoma (ccRCC) is strongly associated with loss of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene is functionally lost through hypermethylation in up to 19% of sporadic ccRCC cases. We theorized that re-expressing VHL silenced by methylation in ccRCC cells, using a hypo-methylating agent, may be an approach to treatment in patients with this type of cancer. We test the ability of two hypo-methylating agents to re-express VHL in cell culture and in mice bearing human ccRCC and evaluate the effects of re-expressed VHL in these models.
  
  
  METHODS
  Real-time reverse transcription-PCR was used to evaluate the ability of zebularine and 5-aza-2'-deoxycytidine (5-aza-dCyd) to re-express VHL in four ccRCC cell lines with documented VHL gene silencing through hypermethylation. We evaluated if the VHL re-expressed after hypo-methylating agent treatment could recreate similar phenotypic changes in ccRCC cells observed when the VHL gene is re-expressed via transfection in cell culture and in a xenograft mouse model. Finally we evaluate global gene expression changes occurring in our cells, using microarray analysis.
  
  
  RESULTS
  5-Aza-dCyd was able to re-express VHL in our cell lines both in culture and in xenografted murine tumors. Well described phenotypic changes of VHL expression including decreased invasiveness into Matrigel, and decreased vascular endothelial growth factor and glucose transporter-1 expression were observed in the treated lines. VHL methylated ccRCC xenografted tumors were significantly reduced in size in mice treated with 5-aza-dCyd. Mice bearing nonmethylated but VHL-mutated tumors showed no tumor shrinkage with 5-aza-dCyd treatment.
  
  
  CONCLUSIONS
  Hypo-methylating agents may be useful in the treatment of patients having ccRCC tumors consisting of cells with methylated VHL.
authors.affiliation: Howard Hughes Medical Institute, Chevy Chase, MD, USA.
authors.forename: Wade G
authors.initials: WG
authors.lastname: Alleman
authors.forename: Ray L
authors.initials: RL
authors.lastname: Tabios
authors.forename: Gadisetti V R
authors.initials: GV
authors.lastname: Chandramouli
authors.forename: Olga N
authors.initials: ON
authors.lastname: Aprelikova
authors.forename: Carlos
authors.initials: C
authors.lastname: Torres-Cabala
authors.forename: Arnulfo
authors.initials: A
authors.lastname: Mendoza
authors.forename: Craig
authors.initials: C
authors.lastname: Rogers
authors.forename: Craig
authors.initials: C
authors.lastname: Rodgers
authors.forename: Nikolai A
authors.initials: NA
authors.lastname: Sopko
authors.forename: W Marston
authors.initials: WM
authors.lastname: Linehan
authors.forename: James R
authors.initials: JR
authors.lastname: Vasselli
mesh_terms: D018262:Adenocarcinoma, Clear Cell / Q000235:genetics* / Q000473:pathology; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003562:Cytidine / Q000031:analogs & derivatives; D019175:DNA Methylation; D000077209:Decitabine; D004195:Disease Models, Animal; D020868:Gene Silencing; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000473:pathology; D051379:Mice; D011741:Pyrimidine Nucleosides / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein
chemical_list: D000964:Antimetabolites, Antineoplastic; D011741:Pyrimidine Nucleosides; D025521:Tumor Suppressor Proteins; D003562:Cytidine; D000077209:Decitabine; C009131:pyrimidin-2-one beta-ribofuranoside; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D001374:Azacitidine