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

腺相关病毒（Adeno-associated virus，AAV）是一种非致病性的单链DNA病毒。AAV载体具有适合于基因治疗应用的若干独特性质。然而，它们应用的障碍是转基因表达效率低，主要是由于第二链合成有限。在本研究中，我们研究拓扑异构酶抑制剂（依托泊苷和喜树碱）是否增强AAV载体介导的转基因表达和AAVtk/GCV系统的杀伤效果。在人喉癌细胞（HEp-2细胞）和HeLa细胞上观察到转基因表达以浓度依赖性方式增强。Southern分析证实依托泊苷增强了HEp-2细胞和HeLa细胞中AAV载体基因组的双链合成。AAVtk/GCV系统能有效地杀伤细胞。更重要的是，依托泊苷和喜树碱增强了AAVtk/GCV系统的细胞杀伤作用。在体外实验之后，我们评估了AAV载体在皮下接种HEp-2的裸鼠中的转基因表达和抗肿瘤活性。结果表明，AAVtk/GCV系统能有效抑制肿瘤生长，且与其他对照组（如AAVLacZ/GCV，AAVtk/PBS）相比，肿瘤生长受到明显抑制。这些发现表明，AAV介导的自杀基因治疗和拓扑异构酶抑制剂治疗的组合可能在癌症治疗中具有协同治疗作用。

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.