Sørensen Brita Singers, Alsner Jan, Overgaard Jens, Horsman Michael R
Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark.
Radiother Oncol. 2007 Jun;83(3):362-6. doi: 10.1016/j.radonc.2007.04.028. Epub 2007 May 18.
Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression.
Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu(DD)]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR.
In both SiHa and FaDu(DD) cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu(DD) cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu(DD). Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu(DD) cells.
As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers.
碳酸酐酶IX（Ca9）、葡萄糖转运蛋白1（Glut1）、乳酸脱氢酶A（LDH-A）、骨桥蛋白（OPN）和赖氨酰氧化酶（LOX）等基因已被认为是缺氧标志物，但结果不一致表明除氧气外的其他因素会影响它们的表达。本研究使用两种人类细胞系在6.3至7.5的一系列pH值下进行详细研究，以确定缺氧诱导基因表达的pH依赖性。
使用人类肿瘤细胞系（子宫颈鳞状细胞癌（SiHa）和咽鳞状细胞癌[FaDu(DD)]）。通过在密闭腔室中用不同氧气浓度（21%、1%、0.1%、0.01%和0%）对细胞通气长达24小时来诱导缺氧。将培养基滴定到一系列pH值（7.5、7.0、6.7、6.5和6.3）。通过实时PCR测定基因表达。
在SiHa和FaDu(DD)细胞中，Ca9和LOX在1%氧气时达到最高表达水平。在FaDu(DD)细胞中，pH 6.5对缺氧诱导的Ca9表达有中等抑制作用。pH 6.3导致SiHa和FaDu(DD)中Ca9和LOX的表达严重受抑。Glut1和LDH-A在两种细胞系中的表达模式相似，在0.01%氧气时表达最高。对于这些基因，pH 6.5和6.3改变了SiHa细胞中的表达模式。OPN在SiHa细胞中低氧时上调，但在FaDu(DD)细胞中未被缺氧诱导。
由于肿瘤缺氧发生在营养缺乏的微环境中，其他环境因素，例如低pH，可能与低氧浓度对基因表达的影响相互作用。本研究表明，两种细胞系中的pH对某些内源性缺氧标志物的氧依赖性诱导有深远影响。
