Influence of oxygen concentration and pH on expression of hypoxia induced genes.
OBJECTIVE
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).


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.


CONCLUSIONS
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.
Sørensen
Brita Singers
BS
Department of Experimental Clinical Oncology, Aarhus University Hospital, Denmark. bsin@oncology.dk
Hao
Jing
J
Overgaard
Jens
J
Vorum
Henrik
H
Honoré
Bent
B
Alsner
Jan
J
Horsman
Michael R
MR
D000951:Antigens, Neoplasm / Q000235:genetics; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000503:physiopathology; D015687:Cell Hypoxia / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D051272:Glucose Transporter Type 1 / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D000080864:Lactate Dehydrogenase 5; D053495:Osteopontin; D010100:Oxygen / Q000502:physiology*; D012795:Sialoglycoproteins / Q000235:genetics; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D051272:Glucose Transporter Type 1; D007527:Isoenzymes; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D010100:Oxygen