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E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

Cancer Gene Therapy volume 10, pages 707–716 (2003)Cite this article

Abstract

E6AP was originally identified as the ubiquitin–protein ligase involved in human papillomavirus (HPV) E6-mediated p53 degradation and has since been shown to act as an E3 ubiquitin–protein ligase in the ubiquitination of several other protein substrates. To further define E6AP function at the molecular and cellular levels, a ribozyme-based gene inactivation approach was adopted. A library of hammerhead ribozymes, with randomized arm sequences, was used to screen active molecules along the entire E6AP transcript for ribozyme-cleavable sites. Ligation-anchored PCR was adapted to detect cleavage products, and ribozymes designed to the selected sites were characterized both in vitro and in vivo. Ribozyme-mediated reduction in E6AP expression was found to enhance the apoptotic response of HeLa cells to mitomycin C-induced DNA damage. These findings suggest that E6AP has potential as a drug target, as its suppression can potentiate apoptosis in HPV-positive cells treated with a cytotoxic drug.

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Acknowledgements

We thank Dr JM Huibregtse for the gift of the pGEM150-120 E6AP cDNA, Dr PR Daniels for the anti-E6AP polyclonal antibody and Dr WL Gerlach for critical review of the manuscript.

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Authors and Affiliations

  1. Department of Medicine, St Vincent's Hospital Clinical School, University of New South Wales, Sydney, 2010, Australia

    Yoonah Kim & Lun-Quan Sun

  2. Johnson and Johnson Research Laboratories, Level 4, 1 Central Ave., Eveleigh, Sydney, 1430, Australia

    Murray J Cairns, Rita Marouga & Lun-Quan Sun

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  1. Yoonah Kim
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  2. Murray J Cairns
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  4. Lun-Quan Sun
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Correspondence to Lun-Quan Sun.

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Kim, Y., Cairns, M., Marouga, R. et al. E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes. Cancer Gene Ther 10, 707–716 (2003). https://doi.org/10.1038/sj.cgt.7700623

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