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

L M Alvarez-Salas1, A E Cullinan, A Siwkowski

  • 1Laboratory of Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 14, 1998
PubMed

Insights

Engineered ribozymes targeting Human Papillomavirus type 16 (HPV-16) E6 and E7 genes effectively inhibit cervical keratinocyte immortalization. This dual-action approach, combining cleavage and passive hybridization, shows promise for anti-cancer therapy.

Area of Science:

  • Molecular Biology
  • Virology
  • Biochemistry

Background:

  • Human Papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins are crucial for cervical keratinocyte immortalization.
  • These oncoproteins represent significant therapeutic targets for cervical cancer treatment.

Purpose of the Study:

  • To develop and evaluate engineered hairpin (HP) ribozymes for disrupting HPV-16 E6/E7 mRNA.
  • To assess the efficacy of a specific anti-E6 HP ribozyme (R434) in inhibiting HPV-16-mediated immortalization in vitro and in vivo.

Main Methods:

  • Production and biochemical characterization of engineered HP ribozymes targeting HPV-16 E6/E7 mRNA.
  • In vitro translation inhibition assays using the selected R434 ribozyme.
  • Cis-expression of R434 in normal human keratinocytes containing HPV-16 E6/E7 genes.
  • Reverse transcription-PCR (RT-PCR) analysis to quantify E6/E7 transcript cleavage and elimination.

Main Results:

  • The R434 ribozyme demonstrated efficient inhibition of E6 in vitro translation.
  • Cis-expression of R434 in keratinocytes reduced cell growth rate and prevented immortalization.
  • RT-PCR confirmed cleavage and substantial elimination of E6/E7 transcripts in transfected cells.
  • An inactive HP ribozyme also showed inhibitory effects, suggesting a role for passive hybridization.

Conclusions:

  • Engineered HP ribozymes are effective inhibitors of HPV-16 E6/E7 mRNA.
  • The combination of catalytic cleavage and passive hybridization by antisense RNA provides a potent strategy against HPV-16-induced immortalization.
  • This approach holds potential for developing novel anti-cancer therapies targeting HPV-driven cancers.

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