title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes.
abstract: HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.
authors.affiliation: Laboratory of Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Alvarez-Salas
authors.forename: A E
authors.initials: AE
authors.lastname: Cullinan
authors.forename: A
authors.initials: A
authors.lastname: Siwkowski
authors.forename: A
authors.initials: A
authors.lastname: Hampel
authors.forename: J A
authors.initials: JA
authors.lastname: DiPaolo
mesh_terms: D000998:Antiviral Agents / Q000627:therapeutic use*; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D015603:Keratinocytes / Q000821:virology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009856:Oncogene Proteins, Viral / Q000037:antagonists & inhibitors / Q000235:genetics*; D027383:Papillomaviridae; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000517:prevention & control*; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism*; D012097:Repressor Proteins; D014162:Transfection; D014412:Tumor Virus Infections / Q000517:prevention & control*
chemical_list: D000998:Antiviral Agents; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16