Raspaglio Giuseppina, Filippetti Flavia, Prislei Silvia, Penci Roberta, De Maria Ilaria, Cicchillitti Lucia, Mozzetti Simona, Scambia Giovanni, Ferlini Cristiano
Department of Oncology, Catholic University of the Sacred Heart, Campobasso, Italy.
Gene. 2008 Feb 15;409(1-2):100-8. doi: 10.1016/j.gene.2007.11.015. Epub 2007 Dec 3.
Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
Ⅲ类β微管蛋白（TUBB3）过表达是对紫杉烷类和长春花生物碱等微管相互作用剂产生耐药性的主要机制。在此，我们测试了缺氧作为TUBB3可能诱导因素的情况。在A2780细胞、其耐紫杉醇对应细胞（TC1）和HeLa细胞中，从mRNA和蛋白质水平监测缺氧对TUBB3表达的影响。缺氧是A2780细胞中TUBB3的强诱导因素，但在TC1和HeLa细胞中并非如此。在A2780细胞中使用RNA干扰敲低HIF-1α，并在常氧和缺氧条件下评估TUBB3表达。这种沉默消除了缺氧依赖性的TUBB3增加，从而证明HIF-1α在缺氧状态下介导TUBB3的诱导。为了研究这一现象，将人TUBB3的5'侧翼区域克隆到绿色荧光蛋白（GFP）上游作为报告基因。该区域包含启动子基因，但报告基因的活性不受缺氧影响。因此，我们查看了3'侧翼区域，并且在距终止密码子+168个核苷酸处，使用一个构建体（其中该位点克隆到GFP下游作为报告基因）证明一个HIF-1α结合位点在缺氧状态下具有活性。构建体中该位点的缺失消除了缺氧时GFP的增强。染色质免疫沉淀显示缺氧时HIF-1α与该位点结合。对这个3'增强子的甲基化分析表明，在A2780细胞中70%的细胞中它没有甲基化，而在TC1和HeLa细胞中均不到16%，从而提示缺氧时TUBB3的增加通过3'增强子的甲基化而被消除。
