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Investigation of Tumor Hypoxia Using A Askoxylakis et al. Radiation Oncology 2011, 6:35 http://www.ro-journal.com/content/6/1/35 RESEARCH Open Access Investigation of tumor hypoxia using a two- enzyme system for in vitro generation of oxygen deficiency Vasileios Askoxylakis1,5*, Gunda Millonig2, Ute Wirkner1,3, Christian Schwager1,3, Shoaib Rana4, Annette Altmann5, Uwe Haberkorn4,5, Jürgen Debus1, Sebastian Mueller2 and Peter E Huber1,3 Abstract Background: Oxygen deficiency in tumor tissue is associated with a malign phenotype, characterized by high invasiveness, increased metastatic potential and poor prognosis. Hypoxia chambers are the established standard model for in vitro studies on tumor hypoxia. An enzymatic hypoxia system (GOX/CAT) based on the use of glucose oxidase (GOX) and catalase (CAT) that allows induction of stable hypoxia for in vitro approaches more rapidly and with less operating expense has been introduced recently. Aim of this work is to compare the enzymatic system with the established technique of hypoxia chamber in respect of gene expression, glucose metabolism and radioresistance, prior to its application for in vitro investigation of oxygen deficiency. Methods: Human head and neck squamous cell carcinoma HNO97 cells were incubated under normoxic and hypoxic conditions using both hypoxia chamber and the enzymatic model. Gene expression was investigated using Agilent microarray chips and real time PCR analysis. 14C-fluoro-deoxy-glucose uptake experiments were performed in order to evaluate cellular metabolism. Cell proliferation after photon irradiation was investigated for evaluation of radioresistance under normoxia and hypoxia using both a hypoxia chamber and the enzymatic system. Results: The microarray analysis revealed a similar trend in the expression of known HIF-1 target genes between the two hypoxia systems for HNO97 cells. Quantitative RT-PCR demonstrated different kinetic patterns in the expression of carbonic anhydrase IX and lysyl oxidase, which might be due to the faster induction of hypoxia by the enzymatic system. 14C-fluoro-deoxy-glucose uptake assays showed a higher glucose metabolism under hypoxic conditions, especially for the enzymatic system. Proliferation experiments after photon irradiation revealed increased survival rates for the enzymatic model compared to hypoxia chamber and normoxia, indicating enhanced resistance to irradiation. While the GOX/CAT system allows independent investigation of hypoxia and oxidative stress, care must be taken to prevent acidification during longer incubation. Conclusion: The results of our study indicate that the enzymatic model can find application for in vitro investigation of tumor hypoxia, despite limitations that need to be considered in the experimental design. Background during angiogenesis are highly irregular and possess Reduced oxygen levels are measured in several solid incomplete endothelial linings and basement mem- tumors mainly as result of tumor outgrowing the exist- branes, as well as arteriovenous shunts, resulting in dis- ing vasculature but also as result of structural and func- turbances of blood flow and oxygen delivery [2]. Tumor tional disturbances of tumor vasculature [1]. In hypoxia is associated with a more aggressive neoplastic particular, tumor blood vessels that are newly formed phenotype, characterized by high invasiveness and increased metastatic potential. Genes with key-role in metastatic processes, such as lysyl oxidase (LOX), met * Correspondence: [email protected] proto-oncogene (MET) and c-X-c chemokine receptor 4 1Department of Radiooncology and Radiation Therapy, University of Heidelberg, Heidelberg, Germany (CXCR4) have been identified to be upregulated under Full list of author information is available at the end of the article © 2011 Askoxylakis et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Askoxylakis et al. Radiation Oncology 2011, 6:35 Page 2 of 12 http://www.ro-journal.com/content/6/1/35 hypoxic conditions [3,4]. In regard to therapy outcome volume, controlled oxygen depletion can be achieved and prognosis, hypoxic regions within a solid tumor are and also stably maintained for at least 12-24 h [10]. characterized by increased resistance towards che- Aim of the present work is to investigate the effects of motherapy or radiotherapy. In particular, oxygen defi- rapidly induced hypoxia on cellular processes using the ciency upregulates the expression of the multidrug enzymatic GOX/CAT system in comparison to the estab- resistance gene (MDR1), leading to efflux of chemother- lished method of hypoxia chamber. Since hypoxia is apeutic drugs [5]. In respect to radiation therapy both known to be a feature of human head and neck squa- chemical and biological mechanisms are found to be mous cell carcinoma [11], the HNSCC cell line HNO97 important for increased radioresistance. Oxygen defi- was chosen for investigation under normoxic and ciency disturbs the radiolysis of H2O leading to reduced hypoxicconditionsusingahypoxiachamberandthe production of reactive species that are cytotoxic [6]. enzymatic model. We focused on three aspects: gene Furthermore, hypoxia promotes the activation of the expression, glucose metabolism and radioresistance. hypoxia inducible factor-1 (HIF-1), a heterodimeric tran- Gene expression was investigated using Agilent microar- scription factor that upregulates the expression of genes ray chip analysis and real time PCR. Cellular glucose involved in angiogenesis and tumorigenesis [7]. metabolism was assessed with 14C-FDG uptake assays The fact that tumor hypoxia is associated with and proliferation experiments after photon irradiation increased therapy resistance and poor prognosis reveals were carried out for investigation of hypoxia induced the necessity for extensive and detailed investigation of radioresistance. The results of our study indicate that the biological mechanisms associated with oxygen defi- enzymatic GOX/CAT system is an attractive alternative ciency. The established method for in vitro investigation technique for in vitro investigation of tumor hypoxia. of tumor hypoxia is the exposure of cultured cells to defined, oxygen deficient gaseous environments. The Methods most common apparatus used for this purpose is the Cell culture hypoxia chamber. However this approach has critical The human head and neck squamous cell carcinoma limitations, mainly in regard to oxygen diffusion and cell line HNO97 [12] was cultivated in Dulbecco’s Mod- equilibration. In particular, within a hypoxia chamber ified Eagle’s Medium (DMEM containing 4.5 g/L glucose oxygen reaches the cell surface after a protracted pro- and 58 ng/L L-glutamine but no sodium pyruvate) sup- cess, including transport in the chamber, passing plemented with 10% (v/v) fetal calf serum (Gibco, Invi- through the material of the cell culture plate, solubility trogen Life Technologies) at 37°C in a 5% CO2 depended entering the culture medium at the gas- incubator. medium interface and diffusion through the medium to the cell surface. Oxygen transport kinetic studies in the In vitro enzymatic and non-enzymatic hypoxia induction past have revealed required time periods of about 30 Enzymatic hypoxia min for equilibration of pO2 between the gas inside and Hypoxia medium was prepared by diluting glucose oxi- outside of the culture plate and more than 3 h for equi- dase and catalase at a constant 1:10 ratio in cell culture libration of the pO2 between the medium inside the medium (both Sigma cat. No. C3155 and G0543). plate and the gas outside of it [8]. Enzyme activities of stock solutions were 3 mM/s for Recently an alternative way to generate in vitro GOX and 998 s-1 for CAT. To obtain a defined, stable oxygen-deficient conditions has been evaluated [9,10]. oxygen concentration of 2% on cell surface stock solu- This system is based on the use of the enzymes glucose tions were diluted by 1:10,000 for GOX and 1:1,000 for oxidase (GOX) and catalase (CAT). Addition of glucose CAT. The medium volumes used were 2.5 ml for 6-well oxidase into the cell culture medium removes oxygen by plates and 10.63 ml for 10 cm cell culture plates and oxidizing glucose. The reaction leads to generation of the cells were incubated at 37°C. Previous experiments hydrogen peroxide, which is then removed by catalase, using a computer-driven oxygen electrode Oxi 325-B in order to prevent cytotoxic effects due to accumula- (WTW, Weilheim, Germany) for oxygen measurement tion. This enzymatic system was found to induce rapid have revealed that at those conditions 2% hypoxia was depletion of oxygen within minutes at a defined rate. rapidly induced within 15 min and maintained over Oxygen concentration in the cultured medium is 24 h [10]. For incubation periods longer than 24 h med- reported to be dependent by two factors: the activity of ium was replaced by pre-equilibrated hypoxic medium glucose oxidase and the medium volume. GOX activity to maintain nutrients and substrates such as glucose. has an influence on the depletion rate of oxygen, while medium volume affects the diffusion distance of oxygen Hypoxia chamber from gas-medium interface to the cells. Experiments Cells cultivated in 6-well plates or 10 cm cell culture have revealed that at defined GOX activity and medium dishes under a layer of exactly 2.5 ml and 10.63 ml cell Askoxylakis et al. Radiation Oncology 2011, 6:35 Page 3 of 12 http://www.ro-journal.com/content/6/1/35 culture medium respectively were placed in a hypoxia #74104) and 1.65 μg labelled RNA was used for chemi- chamber. The chamber was flushed with 2% O2/5% cal fragmentation and hybridisation (Gene expression CO2/93% N2 gas mixture for 5 min, sealed and kept hybridization kit, Agilent #5188-5242). Assembly of the at 37°C. For longer incubation periods the chamber gasket/slide-sandwich in the hybridisation chamber was refilled after 24 h to ensure constant oxygen (Agilent, #G2534A) was performed according to manu- concentrations.
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