Bernt Kathrin M, Steinwaerder Dirk S, Ni Shaoheng, Li Zong-Yi, Roffler Steve R, Lieber André
Division of Medical Genetics, Department of Medicine, University of Washington, Seattle 98195, USA.
Cancer Res. 2002 Nov 1;62(21):6089-98.
Adenoviruses (Ads) that selectively replicate in tumor cells have shown promising preliminary results in clinical trials, especially in combination with chemotherapy. Here, we describe a system that combines the antitumor synergy of Ads and chemotherapeutic agents with the benefits of enzyme-activated prodrug therapy. In this system, a functional transgene expression cassette is created by homologous recombination during adenoviral DNA replication. Transgene expression is strictly dependent on viral DNA replication, which in turn is tumor specific. We constructed replication-activated Ad vectors to express a secreted form of beta-glucuronidase and a cytosine deaminase/uracil phosphoribosyltransferase, which activate the prodrugs 9-aminocamptothecin glucuronide to 9-aminocamptothecin and 5-fluorocytosine to 5-fluorouracil (5-FU) and further to 5-fluoro-UMP, respectively. We demonstrated replication-dependent transgene expression, prodrug activation, and induction of tumor cell toxicity by secreted beta-glucuronidase and cytosine deaminase/uracil phosphoribosyltransferase. Furthermore, exposure of cells to activated prodrug or drug at subtoxic concentrations enhanced viral DNA replication. Characteristically, these agents induced changes in the cell cycle status of exposed cells (G(2) arrest), which closely resembled the effect of wild-type Ad infection, and are thought to be favorable for viral replication. We tested a number of cytostatic drugs (camptothecin, etoposide, daunorubicin, cisplatin, 5-fluorouracil, hydroxyurea, Taxol, and actinomycin D) for their effect on viral DNA replication and found considerable differences between individual agents. Finally, we show that the combination of viral and prodrug therapy enhances viral replication and spread in liver metastases derived from human colon carcinoma or cervical carcinoma in a mouse model. Our data indicate that specific vector/drug combinations tailored to be synergistic may have the potential to improve the potency of either therapeutic approach. These data also provide a new rationale for expressing prodrug-activating enzymes from conditionally replicating Ads.
在肿瘤细胞中选择性复制的腺病毒（Ads）在临床试验中已显示出有前景的初步结果，尤其是与化疗联合使用时。在此，我们描述了一种系统，该系统将腺病毒与化疗药物的抗肿瘤协同作用与酶激活前药疗法的优势相结合。在这个系统中，一个功能性转基因表达盒在腺病毒DNA复制过程中通过同源重组产生。转基因表达严格依赖于病毒DNA复制，而病毒DNA复制又具有肿瘤特异性。我们构建了复制激活型腺病毒载体，以表达分泌形式的β-葡萄糖醛酸酶和胞嘧啶脱氨酶/尿嘧啶磷酸核糖转移酶，它们分别将前药9-氨基喜树碱葡糖苷酸激活为9-氨基喜树碱，将5-氟胞嘧啶激活为5-氟尿嘧啶（5-FU），并进一步激活为5-氟尿苷一磷酸。我们证明了分泌的β-葡萄糖醛酸酶和胞嘧啶脱氨酶/尿嘧啶磷酸核糖转移酶的复制依赖性转基因表达、前药激活以及对肿瘤细胞毒性的诱导。此外，将细胞暴露于亚毒性浓度的激活前药或药物可增强病毒DNA复制。其特点是，这些药物诱导暴露细胞的细胞周期状态发生变化（G2期阻滞），这与野生型腺病毒感染的效果非常相似，并且被认为有利于病毒复制。我们测试了多种细胞生长抑制剂（喜树碱、依托泊苷、柔红霉素、顺铂、5-氟尿嘧啶、羟基脲、紫杉醇和放线菌素D）对病毒DNA复制的影响，发现不同药物之间存在显著差异。最后，我们表明在小鼠模型中，病毒疗法和前药疗法的联合可增强病毒在源自人结肠癌或宫颈癌的肝转移灶中的复制和扩散。我们的数据表明，量身定制以实现协同作用的特定载体/药物组合可能有潜力提高任何一种治疗方法的效力。这些数据还为从条件性复制的腺病毒中表达前药激活酶提供了新的理论依据。
