Sørensen Brita Singers, Hao Jing, Overgaard Jens, Vorum Henrik, Honoré Bent, Alsner Jan, Horsman Michael R
Department of Experimental Clinical Oncology, Aarhus University Hospital, Denmark.
Radiother Oncol. 2005 Aug;76(2):187-93. doi: 10.1016/j.radonc.2005.06.037.
This study was designed to determine the oxygen dependency for expression of the endogenous hypoxic markers carbonic anhydrase IX (protein: CAIX/gene: CA9), glucose transporter 1 (GLUT1/GLUT1), osteopontin (OPN/OPN) and lactate dehydrogenase A (LDH-A/LDHA), and how this expression was influenced by extracellular pH (pHe).
Human cervix squamous cell carcinoma (SiHa) cells were used in all experiments. These cells were gassed in an enclosed environment under either anoxia (95% N2+5% CO2) for various times (0-30 h) or under different oxygen concentrations (0-21% O2) for 24 h at normal pHe (7.4) or low pHe (6.3). Response to radiation (7 Gy) was estimated using a clonogenic assay. Gene expression was determined by real-time PCR (normalized to the housekeeping gene, TFRC) and protein expression by Western blots.
Under normal pHe conditions, CA9, GLUT1 and LDHA gene expression was upregulated within 1-3h of anoxia, reaching near maximal values by 6h. OPN showed a slow increase over 24 h. At 24 h the relative increase was 135, 12, 90 and 5 times for CA9, GLUT1, OPN and LDHA, respectively. No induction was seen with the EGF receptor (EGFR). Gassing cells with differing oxygen concentrations for 24h resulted in a maximum level of expression for CA9 at 1% oxygen, whereas with GLUT1 and LDHA maximal expression occurred at 0.01% oxygen, but at 0% oxygen with OPN. The oxygen dependency for radiation response was identical to that seen for GLUT1 and LDHA. Expression of CA9, GLUT1, OPN and LDHA was inhibited under hypoxic conditions when pHe was reduced to 6.3. Expression of CAIX protein mimicked the CA9 gene expression patterns.
The expression of all the endogenous markers were upregulated by hypoxia, but the timing and oxygen dependencies were different and their expression was influenced by low pHe. This raises concerns about the generalised use of these agents as markers for hypoxia.
本研究旨在确定内源性缺氧标志物碳酸酐酶IX（蛋白质：CAIX/基因：CA9）、葡萄糖转运蛋白1（GLUT1/GLUT1）、骨桥蛋白（OPN/OPN）和乳酸脱氢酶A（LDH - A/LDHA）表达的氧依赖性，以及细胞外pH值（pHe）如何影响这种表达。
所有实验均使用人宫颈鳞状细胞癌（SiHa）细胞。这些细胞在封闭环境中，于正常pHe（7.4）或低pHe（6.3）条件下，分别在缺氧（95% N2 + 5% CO2）环境中处理不同时间（0 - 30小时），或在不同氧浓度（0 - 21% O2）下处理24小时。使用克隆形成试验评估对辐射（7 Gy）的反应。通过实时PCR（以管家基因TFRC标准化）测定基因表达，通过蛋白质印迹法测定蛋白质表达。
在正常pHe条件下，缺氧1 - 3小时内CA9、GLUT1和LDHA基因表达上调，6小时时达到接近最大值。OPN在24小时内缓慢增加。在24小时时，CA9、GLUT1、OPN和LDHA的相对增加倍数分别为135、12、90和5倍。表皮生长因子受体（EGFR）未见诱导。用不同氧浓度处理细胞24小时，CA9在1%氧浓度时达到最大表达水平，而GLUT1和LDHA在0.01%氧浓度时达到最大表达，但OPN在0%氧浓度时达到最大表达。辐射反应的氧依赖性与GLUT1和LDHA相同。当pHe降至6.3时，缺氧条件下CA9、GLUT1、OPN和LDHA的表达受到抑制。CAIX蛋白的表达模仿了CA9基因的表达模式。
所有内源性标志物的表达均因缺氧而上调，但时间和氧依赖性不同，且其表达受低pHe影响。这引发了对将这些因子普遍用作缺氧标志物的担忧。
