Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer

Prostate. 1997 Sep 1;32(4):246-58. doi: 10.1002/(sici)1097-0045(19970901)32:4<246::aid-pros4>;2-h.


Background: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo.

Methods: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis.

Results: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells.

Conclusions: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • DNA-Binding Proteins / biosynthesis*
  • Early Growth Response Protein 1
  • Genes, bcl-2*
  • Genetic Therapy*
  • Humans
  • Immediate-Early Proteins*
  • Male
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Prostatic Neoplasms / therapy*
  • Proto-Oncogene Proteins c-bcl-2 / antagonists & inhibitors
  • Proto-Oncogene Proteins c-bcl-2 / biosynthesis
  • RNA, Catalytic / biosynthesis*
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism
  • RNA, Messenger / chemistry
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Tetradecanoylphorbol Acetate
  • Transcription Factors / biosynthesis*
  • Transfection / methods*
  • Tumor Cells, Cultured
  • Zinc Fingers


  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Immediate-Early Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Catalytic
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Tetradecanoylphorbol Acetate