Bhatt Rupal S, Landis Daniel M, Zimmer Michael, Torregrossa Joelle, Chen Shaoyong, Sukhatme Vikas P, Iliopoulos Othon, Balk Steve, Bubley Glenn J
Division of Cancer Biology, Beth Israel Deaconess Medical Center, Boston, MA, USA.
BJU Int. 2008 Aug;102(3):358-63. doi: 10.1111/j.1464-410X.2008.07558.x. Epub 2008 Apr 3.
To determine the role of hypoxia-inducible factor-2alpha (HIF2alpha) on the sensitivity of renal cell carcinoma (RCC) cell lines to ionizing radiation and to determine if the mTOR antagonist, rapamycin, could decrease HIF2alpha protein levels.
Cell lines expressing stable short-hairpin RNAs (shRNAs) encoding HIF2alpha shRNAs or an empty vector were transfected with a hypoxia responsive element (HRE)-driven firefly luciferase reporter gene. Two separate paired cell lines were assayed for their response to increasing doses of ionizing radiation. Proliferation and cell cycle kinetics were compared for cell lines expressing HIF2alpha shRNAs and empty vectors. The effect of an mTOR antagonist, rapamycin on HIF1alpha and HIF2alpha proteins levels was also assessed.
We confirmed that the 786-O RCC lines with stably integrated shRNAs against HIF2alpha had decreased activation of a plasmid with a HRE-driven firefly luciferase reporter gene. Lines from two separate cell clones with decreased HIF2alpha levels showed a significant increase in radiation sensitivity and an increase in G2 cell cycle arrest. Rapamycin, while effective in decreasing HIF1alpha protein levels, did not affect HIF2alpha levels in either of the RCC cell lines.
These results show that decreasing levels of HIF2alpha leads to an increased sensitivity to ionizing radiation. This finding may explain in part, the known resistance of RCC to radiation therapy. Although mTOR antagonists are approved for the treatment of RCC, these agents do not decrease HIF2alpha levels and therefore might not be effective in enhancing the radio-sensitivity of these tumours.
确定缺氧诱导因子-2α（HIF2α）在肾细胞癌（RCC）细胞系对电离辐射敏感性中的作用，并确定mTOR拮抗剂雷帕霉素是否能降低HIF2α蛋白水平。
将表达编码HIF2α短发夹RNA（shRNA）或空载体的稳定短发夹RNA的细胞系用缺氧反应元件（HRE）驱动的萤火虫荧光素酶报告基因进行转染。对两个单独的配对细胞系进行检测，以观察它们对递增剂量电离辐射的反应。比较表达HIF2α shRNA的细胞系和空载体细胞系的增殖及细胞周期动力学。还评估了mTOR拮抗剂雷帕霉素对HIF1α和HIF2α蛋白水平的影响。
我们证实，稳定整合有针对HIF2α的shRNA的786-O RCC细胞系，其带有HRE驱动的萤火虫荧光素酶报告基因的质粒激活降低。来自两个HIF2α水平降低的单独细胞克隆的细胞系，其辐射敏感性显著增加，且G2期细胞周期阻滞增加。雷帕霉素虽然能有效降低HIF1α蛋白水平，但对两种RCC细胞系中的HIF2α水平均无影响。
这些结果表明，降低HIF2α水平会导致对电离辐射的敏感性增加。这一发现可能部分解释了RCC对放射治疗已知的抗性。尽管mTOR拮抗剂已被批准用于治疗RCC，但这些药物不会降低HIF2α水平，因此可能无法有效提高这些肿瘤的放射敏感性。
