Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer
✍ Scribed by Dorai, Thambi; Olsson, Carl A.; Katz, Aaron E.; Buttyan, Ralph
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 503 KB
- Volume
- 32
- Category
- Article
- ISSN
- 0270-4137
No coin nor oath required. For personal study only.
✦ Synopsis
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.