Inhibition of HPV-16 E6/E7 Immortalization of Normal Keratinocytes by Hairpin Ribozymes

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1998-02, Vol.95 (3), p.1189-1194
Main Authors: Alvarez-Salas, Luis M., Cullinan, Amy E., Siwkowski, Andrew, Hampel, Arnold, DiPaolo, Joseph A.
Format: Article
Language:English
Subjects:
Antiviral Agents - therapeutic use
Antivirals
Base Sequence
Biological Sciences
Cancer
Cell Division
Cell growth
Cells
Cells, Cultured
Cellular biology
DNA
Gene Expression
Genes
Genetic Engineering
Genetic vectors
Human papillomavirus 16
Humans
Keratinocytes - virology
Messenger RNA
Molecular Sequence Data
Nucleic Acid Conformation
Nucleotides
Oncogene Proteins, Viral - antagonists & inhibitors
Oncogene Proteins, Viral - genetics
Papillomaviridae
Papillomavirus E7 Proteins
Papillomavirus Infections - prevention & control
Plasmids
Protein Biosynthesis
Repressor Proteins
RNA
RNA, Catalytic - therapeutic use
RNA, Messenger - metabolism
Transfection
Tumor Virus Infections - prevention & control
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Summary: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 anti-sense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.95.3.1189