Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1alpha in human glioma in vitro and in vivo.
OBJECTIVE
To identify molecular markers of tumor hypoxia and potential therapeutic targets in glioblastoma (GBM), we investigated the hypoxia-related expression of osteopontin (OPN), carbonic anhydrase 9 (CA9), erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha (HIF-1alpha) in vitro in human GBM cell lines and in vivo in human tumor samples of GBM, compared to low-grade astrocytoma (LGA).


METHODS
Expression of the hypoxia-induced genes OPN, CA9, EPO, VEGF and HIF-1alpha was analyzed in three GBM cell lines, GaMG, U373 and U251, under in vitro hypoxia (1, 6 or 24h at 5%, 1% or 0.1% O(2)) and in tumor samples from two patient groups with LGA and GBM (n=15 each), at the mRNA level (semiquantitative RT-PCR). Selected conditions and representative tumor samples were also evaluated at the protein level by Western blot.


RESULTS
OPN and CA9 mRNA was most consistently upregulated in relation to severity and duration of in vitro hypoxia. In tumor samples, mean expression levels (LGA vs. GBM, normalized to mean expression in normal brain) were 1.71 vs. 4.57 (p<0.001) for OPN, 1.11 vs. 3.35 (p<0.001) for CA9, 2.79 vs. 5.28 (not significant, n.s.) for Epo, 1.13 vs. 2.0 (p=0.007) for VEGF and 0.97 vs. 0.97 (n.s.) for HIF-1alpha. In tumor samples, GBM showed a particularly strong protein expression of OPN.


CONCLUSIONS
Among a panel of known hypoxia-inducible genes, OPN and CA9 emerge as most consistently induced by in vitro hypoxia in human GBM cell lines and most specifically expressed in patient GBM tumor tissue, rendering these two genes attractive targets for hypoxia-directed treatment approaches.
Said
Harun M
HM
Department of Radiation Oncology, University of Würzburg, Germany. said_h@klinik.uni-wuerzburg.de
Hagemann
Carsten
C
Staab
Adrian
A
Stojic
Jelena
J
Kühnel
Siglinde
S
Vince
Giles H
GH
Flentje
Michael
M
Roosen
Klaus
K
Vordermark
Dirk
D
D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D015972:Gene Expression Regulation, Neoplastic; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D053495:Osteopontin; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases