Brand K, Löser P, Arnold W, Bartels T, Strauss M
Institut für Biologie, Humboldt-Universität Berlin, Germany.
Gene Ther. 1998 Oct;5(10):1363-71. doi: 10.1038/sj.gt.3300728.
Treatment of colorectal liver metastases with the HSVtk/GCV approach and adenoviral vectors is highly toxic. We present a nontoxic alternative using the cell type-specific CEA promoter instead of the widely used hCMV immediate-early promoter to drive tk gene expression in the context of a recombinant adenovirus. Analysis of CEA promoter-dependent tk gene expression showed significant activity of this promoter in several human and rat tumor-derived cell lines but not in rat primary hepatocytes and in mouse liver, whereas the CMV promoter was highly active in all cell types and tissues investigated. CEA promoter-dependent tk gene expression was sufficient to kill 100% of cancer cells in vitro, even if less than 10% were infected by the adenoviral vector, indicating a significant bystander effect. Moreover, treatment of subcutaneous tumors in SCID mice with Ad.CEA-tk led to a several-fold reduction of tumor growth, and tail vein injection of a high dose of Ad.CEA-tk caused no side-effects in the liver. The CMV promoter was more potent than the CEA promoter in mediating GCV sensitivity to cancer cells in vitro and in vivo, but even a 20-fold reduction of the dose of Ad.CMV-tk did not prevent its liver cell toxicity after systemic application to mice and still resulted in the death of all animals within 4 days after the start of GCV treatment. These results indicate that restriction of tk gene expression to tumor cells in the liver prevents systemic toxicity. Moreover, the CEA promoter is a safe and efficient tool for tumor cell-specific expression of suicide genes in the liver.
采用单纯疱疹病毒胸苷激酶（HSVtk）/丙氧鸟苷（GCV）方法和腺病毒载体治疗结直肠癌肝转移具有高毒性。我们提出了一种无毒替代方案，使用细胞类型特异性癌胚抗原（CEA）启动子而非广泛使用的人巨细胞病毒（hCMV）立即早期启动子，以在重组腺病毒的背景下驱动胸苷激酶（tk）基因表达。对CEA启动子依赖性tk基因表达的分析表明，该启动子在几种人和大鼠肿瘤来源的细胞系中具有显著活性，但在大鼠原代肝细胞和小鼠肝脏中无活性，而CMV启动子在所有研究的细胞类型和组织中均具有高活性。CEA启动子依赖性tk基因表达足以在体外杀死100%的癌细胞，即使感染腺病毒载体的细胞少于10%，这表明存在显著的旁观者效应。此外，用Ad.CEA - tk治疗SCID小鼠的皮下肿瘤导致肿瘤生长减少数倍，尾静脉注射高剂量的Ad.CEA - tk对肝脏无副作用。在体外和体内介导GCV对癌细胞的敏感性方面，CMV启动子比CEA启动子更有效，但即使将Ad.CMV - tk的剂量降低20倍，在对小鼠全身应用后仍不能防止其肝细胞毒性，并且在开始GCV治疗后4天内所有动物仍会死亡。这些结果表明，将tk基因表达限制在肝脏中的肿瘤细胞可防止全身毒性。此外，CEA启动子是在肝脏中肿瘤细胞特异性表达自杀基因的安全有效工具。
