BACKGROUND AND PURPOSE: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND AND PURPOSE: 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). MATERIALS AND METHODS: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. RESULTS: 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. CONCLUSION: 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.
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