Expression of the TRAIL Gene for Cancer Therapy

由于它们能够在多种肿瘤细胞中引起细胞死亡，而在正常细胞中则不会，因此tnfalpha相关凋亡诱导配体（TRAIL）的重组蛋白似乎是治疗癌症患者的有希望的药物。然而，最近发现重组TRAIL蛋白在原代正常人肝细胞中诱导细胞凋亡，这引起了人们对TRAIL蛋白全身给药可能产生严重毒性的警告。我们的初步研究也表明，全长TRAIL蛋白在原代人肝细胞中的表达导致了这些细胞的大量凋亡。因此，肿瘤中基因的局部表达可能是减少全身毒性作用的好方法，同时在癌症部位提供恒定的治疗效果。本提案的目标是开发能够靶向TRAIL基因对癌细胞的药物作用的技术。我们假设，在85%的原发性人类癌症中活跃的人类端粒酶逆转录酶（hTERT）启动子将能够引发TRAIL基因的肿瘤特异性表达，从而可以用来避免对易感正常细胞的毒性。在一项初步研究中，我们发现TRAIL基因的体外转移在结肠癌DLD-1细胞中引发了细胞凋亡和旁观者效应，而在正常成纤维细胞中则没有。肿瘤内递送TRAIL基因可有效抑制DLD-1异种移植物的肿瘤生长。我们还证明了hTERT启动子可以在癌细胞中诱导高水平的转基因表达，而不是在体外和体内的正常细胞中。我们的初步结果表明，hTERT启动子可以在体外和体内阻止GFP-TRAIL融合基因在肝细胞中的表达。为了实现我们的目标，我们将评估从hTERT启动子表达的TRAIL基因的治疗价值。本文拟对hTERT启动子表达的GFP/TRAIL基因通过腺病毒载体传递后的基因转移效率、转基因表达水平、诱导细胞凋亡、旁观者效应及其可能机制、抗肿瘤活性、抗转移活性和可能毒性进行体外和体内研究。完成拟议的研究将使我们能够开发针对TRAIL基因对癌症的药物作用的技术，从而使治疗具有最大的治疗效果，但毒性最小。我们获得的关于hTERT启动子驱动的肿瘤特异性表达和hTERT- trail构建体的治疗/毒性谱的临床前文件可能为该方法在人类中的未来临床应用提供科学依据。

Because of their ability to elicit cell death in a variety of tumor cells but not in normal cells, recombinant proteins of the TNFalpha-related-apoptosis-inducing ligand (TRAIL) appear to be promising medicine for cancer patients. However, a recent finding of apoptosis induction by recombinant TRAIL protein in primary normal human hepatocytes raised the alarm about the possibility of serious toxicity when the TRAIL proteins are administered systemically. Our preliminary study also showed that expression of the full-length TRAIL protein in primary human hepatocytes caused massive apoptosis in these cells. Consequently, localized expression of the gene in tumors may be a good way to reduce systemic toxic effects while providing a constant therapeutic effect at the cancer site. The goal of this proposal is to develop technology that can target pharmaceutical effects of the TRAIL gene to cancer cells. We hypothesize that the promoter for human telomerase reverse transcriptase (hTERT), active in 85 percent of primary human cancers, will be able to elicit tumor-specific expression of the TRAIL gene and thus can be used to avoid toxicity in susceptible normal cells. In a preliminary study, we found that in vitro transfer of the TRAIL gene elicited apoptosis and bystander effects in colon cancer DLD-1 cells but not in normal fibroblasts. Intratumoral delivery of the TRAIL gene effectively suppressed tumor growth of DLD-1 xenograft. We also demonstrated that the hTERT promoter could induce high levels of transgene expression in cancer cells but not in normal cells in vitro and in vivo. Our preliminary results showed that the hTERT promoter can be used to prevent GFP-TRAIL fusion gene expression in liver cells in vitro and in vivo. To achieve our goals, we will assess therapeutic value of the TRAIL gene expressed from the hTERT promoter. In vitro and in vivo studies are proposed to evaluate the efficiency of gene transfer, levels of transgene expression, apoptosis induction, the bystander effect and its possible mechanism, antitumor activity, anti-metastasis activity and possible toxicity of the GFP/TRAIL gene expressed from the hTERT promoter and delivered by adenoviral vector. Completion of the proposed studies will allow us to develop technologies for targeting the pharmaceutical effects of the TRAIL gene to cancers so that the treatment will have maximum therapeutic effects but minimum toxicity. The preclinical documentation we obtain on tumor-specific expression driven by the hTERT promoter and on the therapeutic/toxic profiles of hTERT-TRAIL constructs may provide a scientific basis for future clinical application of this approach in humans.