TOPOISOMERASE INHIBITORS ENHANCE THE CYTOCIDAL EFFECT OF AAV-HSVTK/GANCICLOVIR ON CANCER CELLS

腺相关病毒(AAV)是一种单链DNA基因组的非致病性病毒。AAV载体具有一些独特的特性，适合于基因治疗应用。然而，转基因应用的一个障碍是转基因表达效率低，这主要是由于有限的第二链合成。在本研究中，我们研究了拓扑异构酶抑制剂(依托泊苷和喜树碱)是否能增强AAV载体介导的转基因表达以及AAVtk/GCV系统的杀伤效应。在人喉癌细胞(Hep-2细胞)和HeLa细胞中，转基因表达呈浓度依赖性增强。Southern分析证实，依托泊苷能促进Hep-2细胞和HeLa细胞中AAV载体基因组的双链合成。AAVtk/GCV系统如预期的那样有效地杀伤了细胞。更重要的是，依托泊苷和喜树碱均能增强AAVtk/GCV系统的杀伤作用。在体外实验的基础上，我们检测了AAV载体在裸鼠皮下接种Hep-2后的转基因表达和抗肿瘤活性。与AAVLacZ/GCV、AAVtk/PBS等对照相比，AAVtk/GCV系统抑制了移植瘤的生长。这些发现表明，AAV介导的自杀基因治疗与拓扑异构酶抑制剂治疗相结合，在癌症治疗中可能具有协同治疗作用。

Adeno-associated virus(AAV) is a nonpathogenic virus with a single-strand DNA genome. AAV vectors have several unique properties suited for gene therapy applications. However, an obstacle to their application is a low efficiency of transgene expression, mainly due to a limited second-strand synthesis. In the present study, we investigated whether topoisomerase inhibitors (etoposide and camptothecin) enhance the AAV vector-mediated transgene expression and the killing effect by AAVtk/GCV system. The enhancement of transgene expression was observed in a concentration-dependent manner on human laryngeal carcinoma cells (HEp-2 cells) and HeLa cells. Southern analysis confirmed that etoposide enhanced the double-strand synthesis of the AAV vector genome in HEp-2 cells and HeLa cells. The cells were efficiently killed by AAVtk/GCV system, as expected. More importantly, both etoposide and camptothecin augmented the cytocidal effect of the AAVtk/GCV system. Following the in vitro experiments, we evaluated the transgene expression and the antitumor activity of the AAV vectors in nude mice inoculated with HEp-2 subcutaneously. The LacZ expression was observed in the xenografted tumors and the AAVtk/GCV system suppressed the tumors growth compared with any other control such as AAVLacZ/GCV, AAVtk/PBS. These findings suggest that the combination of AAV-mediated suicide gene therapy and treatment with topoisomerase inhibitors may have synergistic therapeutic effects in the treatment of cancers.