Hypoxia down-regulates CCAAT/enhancer binding protein-alpha expression in breast cancer cells.
The transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBP alpha mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBP alpha mRNA by approximately 30%. C/EBP alpha gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1 alpha, as essential for down-regulation of C/EBP alpha transcription in hypoxia. Immunocytochemical analysis showed that C/EBP alpha was localized in the nucleus at 21% O(2), but was mostly cytoplasmic under 1% O(2). Knockdown of HIF-1 alpha by RNAi restored C/EBP alpha to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBP alpha and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBP alpha expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBP alpha in hypoxia is mediated by HIF-1.
Seifeddine
Ramzi
R
INSERM U612, Institut Curie, Orsay, France.
Dreiem
Anne
A
Blanc
Etienne
E
Fulchignoni-Lataud
Marie-Claude
MC
Le Frère Belda
Marie-Aude
MA
Lecuru
Fabrice
F
Mayi
Thérèse Hervèe
TH
Mazure
Nathalie
N
Favaudon
Vincent
V
Massaad
Charbel
C
Barouki
Robert
R
Massaad-Massade
Liliane
L
D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D048708:Cell Growth Processes / Q000502:physiology; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D022763:CCAAT-Enhancer-Binding Protein-alpha; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003676:Deferoxamine