Gross Johann, Fuchs Julia, Machulik Astrid, Jahnke Volker, Kietzmann Thomas, Bockmühl Ulrike
Department of Otorhinolaryngology, Charité Hospital, Humboldt University, Berlin, Germany.
Int J Oncol. 2005 Sep;27(3):807-14.
The objective of this study was to examine the mode of cell death and the hypoxia inducible factor-1 (HIF-1) expression of human head and neck squamous cell carcinoma (HNSCC) exposed to hypoxia in vitro. Apoptosis and necrosis rates were examined using flow cytometry. The findings suggest that HNSCC cells show a considerable heterogeneity in cell size and in response to hypoxia. A small-cell population showed a high spontaneous apoptosis and necrosis rate which was in-sensitive to hypoxia. A large-cell population responded to hypoxia by increase of apoptosis rate in parallel to recruitment of HIF-1. Hypoxia led to increased HIF-1alpha protein levels in nuclear extract using ELISA-binding activity. In all cells, accumulation of HIF-1 in the nuclei during hypoxia and a rapid degradation of HIF-1 in the post-hypoxic period were observed immunocytochemically. The HIF-1alpha mRNA level showed an expression of 10-40 pg/microg total RNA and remained unchanged in one cell line, while slightly decreasing in the other. Remarkably, no increased luciferase activity response was found on the reporter gene level using pGL3 reporter gene with three erythropoietin hypoxia responsive elements, either by hypoxia or by application of lactacystin, desferrioxamine or CoCl2. These findings suggest that, in HNSCC cells, hypoxia induces HIF-1alpha to stabilize and accumulate in the cell nuclei but have a cell-specific transcriptional complex.
本研究的目的是检测体外暴露于低氧环境下的人头颈部鳞状细胞癌（HNSCC）的细胞死亡模式及低氧诱导因子-1（HIF-1）的表达。采用流式细胞术检测凋亡率和坏死率。研究结果表明，HNSCC细胞在细胞大小及对低氧的反应方面表现出显著的异质性。一小部分细胞群体显示出较高的自发凋亡和坏死率，且对低氧不敏感。一大细胞群体对低氧的反应是凋亡率增加，同时伴有HIF-1的募集。使用ELISA结合活性检测发现，低氧导致核提取物中HIF-1α蛋白水平升高。免疫细胞化学检测显示，在所有细胞中，低氧期间细胞核内HIF-1积聚，低氧后阶段HIF-1迅速降解。HIF-1α mRNA水平显示为每微克总RNA表达10 - 40 pg，在一个细胞系中保持不变，而在另一个细胞系中略有下降。值得注意的是，使用带有三个促红细胞生成素低氧反应元件的pGL3报告基因，无论是低氧处理还是应用乳酸链球菌素、去铁胺或氯化钴，在报告基因水平均未发现荧光素酶活性增加的反应。这些研究结果表明，在HNSCC细胞中，低氧诱导HIF-1α在细胞核内稳定并积聚，但具有细胞特异性转录复合体。
