Dorai T, Olsson C A, Katz A E, Buttyan R
Department of Urology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Prostate. 1997 Sep 1;32(4):246-58. doi: 10.1002/(sici)1097-0045(19970901)32:4<246::aid-pros4>3.0.co;2-h.
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.
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.
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.
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.
bcl-2癌蛋白可抑制细胞凋亡，在前列腺癌细胞中过度表达时，会使这些细胞对多种治疗药物产生抗性，包括激素消融疗法。因此，bcl-2为开发用于治疗人类前列腺癌的基因敲除疗法提供了一个战略靶点。为此，我们基于核酶技术合成了一种抗bcl-2基因治疗试剂，并在体外和体内测试了其对bcl-2 mRNA的有效性。
通过将两个催化RNA结构域重组到人类bcl-2 mRNA编码区的反义片段中，构建了一种二价锤头状核酶。还构建了一种缺乏催化活性的失活核酶作为实验的对照试剂。测试了核酶对合成的和天然的bcl-2 mRNA的内切核酸酶活性。然后采用简单的转染程序将核酶导入培养的前列腺癌细胞（LNCaP衍生物）。我们测量了核酶对这些细胞中bcl-2 mRNA和蛋白质内源性表达的影响以及它们诱导细胞凋亡的能力。
有功能的而非失活的核酶能够在体外迅速降解bcl-2 mRNA，无需任何其他细胞蛋白或因子。直接转染到LNCaP细胞变体中时，它在处理后18小时内显著降低了bcl-2 mRNA和蛋白质水平。这种活性足以在低bcl-2表达的LNCaP变体中诱导细胞凋亡，但在高bcl-2表达의LNCaP细胞系中则不然。然而，对于高bcl-2表达变体，它确实恢复了对第二种凋亡剂佛波酯产生基因反应的能力，这可通过佛波酯在这些细胞中诱导NGF1A mRNA的新能力得到证明。
本研究支持抗bcl-2核酶试剂在降低或消除激素难治性前列腺癌细胞中bcl-2表达以及杀死前列腺癌细胞方面的潜在效用。因此，这是朝着有效治疗激素难治性人类前列腺癌的基因疗法迈出의第一步。
