E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes.
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.
Kim
Yoonah
Y
Department of Medicine, St Vincent's Hospital Clinical School, University of New South Wales, Sydney 2010, Australia. lsun2@medau.jnj.com
Cairns
Murray J
MJ
Marouga
Rita
R
Sun
Lun-Quan
LQ
D017209:Apoptosis / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D015536:Down-Regulation; D020869:Gene Expression Profiling; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein
C494772:BAK1 protein, human; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; D016337:RNA, Catalytic; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; C427204:hammerhead ribozyme; D016685:Mitomycin; D044767:Ubiquitin-Protein Ligases