Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1189-94. doi: 10.1073/pnas.95.3.1189.

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.

MeSH terms

  • Antiviral Agents / therapeutic use*
  • Base Sequence
  • Cell Division
  • Cells, Cultured
  • Gene Expression
  • Genetic Engineering
  • Humans
  • Keratinocytes / virology*
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oncogene Proteins, Viral / antagonists & inhibitors
  • Oncogene Proteins, Viral / genetics*
  • Papillomaviridae
  • Papillomavirus E7 Proteins
  • Papillomavirus Infections / prevention & control*
  • Protein Biosynthesis
  • RNA, Catalytic / therapeutic use*
  • RNA, Messenger / metabolism*
  • Repressor Proteins*
  • Transfection
  • Tumor Virus Infections / prevention & control*

Substances

  • Antiviral Agents
  • E6 protein, Human papillomavirus type 16
  • Oncogene Proteins, Viral
  • Papillomavirus E7 Proteins
  • RNA, Catalytic
  • RNA, Messenger
  • Repressor Proteins
  • oncogene protein E7, Human papillomavirus type 16