title: 5-Aza-2''-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene.
abstract: OBJECTIVE
  To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase (DNMT) inhibitor, on the growth and survival of the Chinese retinoblastoma (RB) cell line HXO-RB44.
  
  
  METHODS
  The DNA methylation status of the Ras association domain family (RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction (MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining, respectively, when cells were treated with 5.0µmol/L of 5-Aza-CdR. The effect of 5.0µmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry.
  
  
  RESULTS
  5-Aza-CdR efficiently induced cell cycle arrest at G0/G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0µmol/L 5-Aza-CdR. Accordingly, RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA, with a plateau on day four. 5-Aza-CdR at 5.0µmol/L completely demethylated the RASSF1A promoter in HXO-RB44 cells on day four, and as a result, RASSF1A expression increased significantly from day 4 to day 7.
  
  
  CONCLUSIONS
  5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China ; Department of Ophthalmology, the First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan Province, China ; Institute of Translational Medicine, the First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan Province, China.
authors.forename: Ru
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
authors.forename: Xiao-Huan
authors.initials: XH
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Wang
authors.affiliation: Institute of Translational Medicine, the First People's Hospital of Chenzhou City, Chenzhou 423000, Hunan Province, China.
authors.forename: Di-Xian
authors.initials: DX
authors.lastname: Luo
authors.affiliation: Department of Ophthalmology, the Second Xiangya Hospital Central South University, Changsha 410011, Hunan Province, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Gao
keywords: 5-Aza-2′-deoxycytidine; Ras association domain family; apoptosis; methylation; retinoblastoma