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Identification of hypoxia regulated genes unaffected by pH

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Abstract:

Background.

Genes upregulated by low oxygen have been suggested as endogenous markers for tumour hypoxia. Yet, most of the genes investigated have shown inconsistent results, which have led to concerns about their ability to be true hypoxia makers. Our previous studies have demonstrated that gene expression can be affected by factors other than oxygen concentration, e.g. extracellular pH (pHe). The current study is an in vitro approach, where we have used two different cell lines (SiHa and FaDuDD) under different conditions, and analysed gene expression with microarray to identify genes that are upregulated by hypoxia, independent of pHe. Methods.

Human tumor cell lines [uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma (FaDuDD)] were used. Hypoxia was induced by gassing exponential growing cells in air tight chambers with various oxygen concentrations (21%, 5%, 1%, 0.1%, 0%) for 24 hours. Low pH media, titrated to pH 6.3 with a Tris-MES buffer system, was applied to some of the cells during the hypoxia treatment. RNA was extracted and gene expression was analysed using the Affymetrix – Human Genome U133 Plus 2.0 Array. Results.

Using a multiclass SAM analysis combining both cell lines using four groups: normal oxygen – normal pH (21% and 5% oxygen at pH 7.4), low oxygen – normal pH (0.1% and 0% oxygen at pH 7.4), normal oxygen – low pH (21% and 5% oxygen at pH 6.3), low oxygen – low pH (0.1% and 0% oxygen at pH 6.3), we selected 1077 probesets (false discovery rate ∼1%) that distinguish between the four groups. Amongst those probesets we could differentiate between genes induced at low pH, independent of oxygen concentration, genes induced at low oxygen only at normal pH, and genes induced at low oxygen at both normal and low pH. As our previous studies have shown, genes as Ca9 were found amongst the genes that was upregulated at low oxygen only at normal pH. 139 probesets, representing 96 different genes were found to be upregulated at low oxygen, independent of pH. Included in these 96 genes was Lysyl Oxidase, LOX, a recent suggested and promising hypoxia marker.

Document Type: Abstract

DOI: http://dx.doi.org/10.1111/j.1600-0463.2008.001165_32.x

Publication date: May 1, 2008

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