Wirth Thomas, Zender Lars, Schulte Bernd, Mundt Bettina, Plentz Ruben, Rudolph Karl Lenhard, Manns Michael, Kubicka Stefan, Kühnel Florian
Department of Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, 30625 Hannover, Germany.
Cancer Res. 2003 Jun 15;63(12):3181-8.
The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional up-regulation of hTERT. Several studies demonstrated that the hTERT promoter can be used to restrict gene expression of E1-deleted replication defective adenoviral vectors to telomerase-positive cancer cells. In this study, a conditionally replicating adenovirus (hTERT-Ad) expressing E1A genes under control of a 255-bp hTERT-promoter was constructed. Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was comparable with that of adenovirus wild type and significantly higher compared with ONYX-015, regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors. These in vivo experiments also revealed that hTERT-Ad-mediated oncolysis was more efficient than ONYX-015 treatment. These results demonstrate that expression of E1A under transcriptional control of the hTERT promoter is sufficient for effective telomerase-dependent adenovirus replication as a promising perspective for the treatment of the majority of epithelial tumors.
人端粒酶逆转录酶（hTERT）的催化成分在大多数原代人体体细胞中不表达，而大多数癌细胞通过hTERT的转录上调重新激活端粒酶。多项研究表明，hTERT启动子可用于将E1缺失的复制缺陷型腺病毒载体的基因表达限制在端粒酶阳性癌细胞中。在本研究中，构建了一种在255 bp的hTERT启动子控制下表达E1A基因的条件性复制腺病毒（hTERT-Ad）。此外，在E1B基因座下游插入了一个内部核糖体进入位点增强型绿色荧光蛋白盒，以监测体内病毒复制。在所有研究的端粒酶阳性肿瘤细胞系中均可观察到hTERT-Ad的腺病毒复制及增强型绿色荧光蛋白表达的增强。相比之下，hTERT-Ad感染端粒酶阴性的原代人肝细胞并未导致显著复制。hTERT-Ad在肿瘤细胞中诱导细胞病变效应的能力与腺病毒野生型相当，且与ONYX-015相比显著更高，无论肿瘤细胞的p53状态如何。低剂量hTERT-Ad单次应用于肿瘤异种移植可显著抑制肿瘤生长，证实了条件性复制腺病毒载体的潜在治疗价值。这些体内实验还表明，hTERT-Ad介导的溶瘤作用比ONYX-015治疗更有效。这些结果表明，在hTERT启动子的转录控制下E1A的表达足以实现有效的端粒酶依赖性腺病毒复制，这为大多数上皮肿瘤的治疗提供了一个有前景的方向。
