Post D E, Van Meir E G
Neurosurgery Department, Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.
Gene Ther. 2001 Dec;8(23):1801-7. doi: 10.1038/sj.gt.3301605.
Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.
缺氧在所有生物体中都会引发适应性生理反应，并在多种人类疾病的发病机制中发挥作用。缺氧/缺氧诱导因子-1（HIF-1）转录因子通过与靶基因内存在的顺式作用缺氧反应元件（HRE）结合来介导对缺氧的转录反应。利用HIF-1/HRE基因调控系统可作为一种机制，将治疗性基因的表达靶向缺氧细胞或因细胞转化而具有组成性活性HIF-1/HRE途径的细胞。鉴于肿瘤对单一治疗策略的快速耐药性，需要开发能够提供多模式治疗的新载体系统。在此我们表明，HREs作为经典增强子元件发挥作用，并双向发挥功能以共同调节两个基因的表达。我们使用源自人血管内皮生长因子（VEGF）和促红细胞生成素（EPO）基因的HREs设计了一系列新型双向缺氧/HIF反应性表达载体。我们通过在三种人胶质瘤细胞系（LN229、U251MG和U138MG）中进行瞬时转染，测量了这些构建体在常氧（21% O₂）与缺氧（1%、3%、5%和10% O₂）条件下双向表达荧光素酶和LacZ/β-半乳糖苷酶（β-gal）报告基因的能力。鉴定出九个构建体，它们在1% O₂时表现出中度至高诱导性，同时在常氧条件下保持严格调控。此外，激活水平是O₂浓度的函数，并且在O₂水平低于5%时呈指数关系。这些载体将成为针对HIF-1/HRE途径病理激活的各种基因治疗应用中的有价值工具。
