title: siRNA-mediated silencing of Cockayne Cyndrome group B gene potentiates radiation-induced apoptosis and antiproliferative effect in HeLa cells.
abstract: BACKGROUND
  Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.
  
  
  METHODS
  The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.
  
  
  RESULTS
  Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation.
  
  
  CONCLUSIONS
  siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.
authors.affiliation: Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Zi-jian
authors.initials: ZJ
authors.lastname: Yu
authors.forename: Jian-li
authors.initials: JL
authors.lastname: Sui
authors.forename: Bei
authors.initials: B
authors.lastname: Bai
authors.forename: Ping-kun
authors.initials: PK
authors.lastname: Zhou
mesh_terms: D017209:Apoptosis / Q000528:radiation effects*; D002453:Cell Cycle / Q000528:radiation effects; D049109:Cell Proliferation / Q000528:radiation effects; D002945:Cisplatin / Q000494:pharmacology; D003057:Cockayne Syndrome / Q000235:genetics*; D020868:Gene Silencing; D015316:Genetic Therapy; D006367:HeLa Cells / Q000528:radiation effects*; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics*; D011836:Radiation Tolerance; D014466:Ultraviolet Rays
chemical_list: D034741:RNA, Small Interfering; D002945:Cisplatin