Rapid detection of the hypoxia-regulated CA-IX and NDRG1 gene expression in different glioblastoma cells in vitro.
Hypoxia-inducible factor-1 (HIF-1) is a key regulator of tumor cell hypoxia. It regulates the expression of several genes related to oxygen homeostasis in response to hypoxic stress. Carbonic anhydrase IX (Ca-IX) has been found to be a stable marker of acute or chronic hypoxia. N-Myc down-regulated gene 1 (NDRG1) has been shown to possess more specific characteristics for clinical analysis and identification purposes. HIF-1 activates gene expression of the two genes and promotes tumor cell survival under hypoxic conditions. Herein, we modified a flow cytometry protocol to separate NDRG1- and CA-IX-negative and -positive cells in vitro to sort chronically hypoxic cells from glioblastoma tumors. The FITC-anti-CA-IX fluorescence differed between positive and negative cells by a factor of 60-160 in U373, U87-MG, U251 and GaMG, respectively. A clear effect of the O2 concentration on CA-IX expression was visible in GaMG and U251 cell lines whereas U373 showed a less differentiated pattern. NDRG1 expression was present in U373, U251 and GaMG with the lowest expression rate in GaMG. It was stable over 48 h of reoxygenation after 24 h of extreme hypoxia (0.1% O2). During reoxygenation NDRG1 was relatively stable in the four tumor cell lines with the lowest expression in GaMG. An oxygen- and time-dependent elevation of nuclear HIF-1alpha binding on HRE was displayed. FACS analysis of CA-IX and NDRG1 expression may be a new approach to determining the hypoxic state of tumor cells. However, an extensive analysis of other hypoxia-regulated genes in different tumors is required to identify additional markers for the detection of the oxygenation state in human tumors in order to tailor effective tumor-specific therapeutic strategies.
Said
Harun M
HM
Department of Radiation Oncology, University of Würzburg, 97080 Würzburg, Germany. said_h@klinik.uni-wuerzburg.de
Polat
Buelent
B
Staab
Adrian
A
Hagemann
Carsten
C
Stein
Susanne
S
Flentje
Michael
M
Theobald
Mathias
M
Katzer
Astrid
A
Vordermark
Dirk
D
D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000235:genetics* / Q000473:pathology; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D014407:Tumor Cells, Cultured; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D000951:Antigens, Neoplasm; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D018797:Cell Cycle Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047908:Intracellular Signaling Peptides and Proteins; D010100:Oxygen; D012333:RNA, Messenger; D000099259:N-myc Downstream-Regulated Gene 1 Protein; C497442:HIF1A protein, human; C089226:CA9 protein, human