title: Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells.
abstract: OBJECTIVE
  In cervical cancer, high-risk human papillomavirus (HPV) genes are expressed solely in cancerous cells and have been proposed to be the most important etiological factors for cervical cancer, thus making them suitable targets for gene therapy. In this study, we aim to inactivate the HPV16 E7 in CaSki cells and test the possibility of reducing the tumorigenicity of these cells.
  
  
  METHODS
  The full-length HPV16 E7 cDNA was cloned in the pBabe-puro or pWZL-Hygro retrovirus vector in reverse orientation and was stably transfected into CaSki cells by replication-defective retrovirus infection giving rise to CaSki-E7AS and CaSki-E7AS2X cells. Immunoprecipitation/Western analysis and real-time RT-PCR were performed to document the levels of HPV16 E7 gene product. Flow cytometry was performed to study changes in the cell cycle in response to reduced E7 protein. The expression of bcl-2, RB, and E2F-1 was studied using Western blot analysis. Tumorigenicity of CaSki, CaSki-E7AS, and CaSki-E7AS2X cells was assayed with subepidermal tumor growth in nude mice.
  
  
  RESULTS
  We have documented that the delivery of the antisense gene construct resulted in the reduction of HPV16 E7 protein expression and cell proliferation in CaSki cells. Furthermore, we demonstrated that these changes were accompanied by cell cycle arrest, up-regulation of RB, and down-regulation of E2F-1 and bcl-2 proteins. More importantly, dose-dependent transduction of the antisense HPV16E7 construct was able to inhibit and/or retard the tumorigenicity of CaSki cells in vivo.
  
  
  CONCLUSIONS
  Down-regulation of HPV16 E7 with antisense RNA is beneficial in reducing the tumorigenicity of CaSki cells and can potentially be useful for HPV-associated malignancy gene therapy.
authors.affiliation: Department of Medicine, Department of Pathology, Cancer Research Centre, Queen Mary Hospital, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. ckchoo@hkucc.hku.hk
authors.forename: C K
authors.initials: CK
authors.lastname: Choo
authors.forename: M T
authors.initials: MT
authors.lastname: Ling
authors.forename: C K
authors.initials: CK
authors.lastname: Suen
authors.forename: K W
authors.initials: KW
authors.lastname: Chan
authors.forename: Y L
authors.initials: YL
authors.lastname: Kwong
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002455:Cell Division; D004268:DNA-Binding Proteins; D015536:Down-Regulation; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000378:metabolism; D016372:RNA, Antisense / Q000008:administration & dosage* / Q000235:genetics; D016160:Retinoblastoma Protein / Q000096:biosynthesis / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000628:therapy / Q000821:virology*
chemical_list: C533431:Arid4a protein, mouse; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016372:RNA, Antisense; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; C059731:oncogene protein E7, Human papillomavirus type 16