RESUMO
CCAAT/enhancer binding protein-alpha (C/EBPalpha) is involved in the control of cell differentiation and proliferation. It has been previously shown that hypoxia (H) down-regulates C/EBPalpha in breast cancer cells; here the effect of estrogen (E(2)) during H on C/EBPalpha in T-47D cell line was examined. By quantitative RT-PCR the C/EBPalpha mRNA stability was analyzed at 21% O(2) and 1% O(2) under E(2). The H-E(2) combination but not E(2) alone reduced the half-life of the C/EBPalpha mRNA. C/EBPalpha promoter activity studies covering -576 bp do not show any effect of E(2); a significant decrease of C/EBPalpha transcriptional activity was found in the C/EBPalpha promoter between -576/416 bp as previously observed when cells were treated with hypoxia alone. By immunocytochemistry, H-E(2) combination alters the cellular distribution of C/EBPalpha in T-47D cells and locates this protein mainly at the cytosol. Therefore, the observed down-regulation of C/EBPalpha by the H-E(2) combination in T-47D cells is mainly due to the hypoxia effect.
Assuntos
Neoplasias da Mama/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hipóxia/genética , Neoplasias da Mama/patologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação para Baixo , Feminino , Meia-Vida , Humanos , Técnicas Imunoenzimáticas , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Sequência de Bases , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Hipóxia Celular/genética , Linhagem Celular Tumoral , Desferroxamina/farmacologia , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
Experimental and clinical studies have shown that both estrogen (E2) and hypoxia (H) are involved in tumor development and progression. A study was undertaken to determine whether these factors could interact to modulate gene expression using a microarray approach. We screened the transcript levels of over 8000 genes in the estrogen receptor (ERalpha) positive T-47D human breast cancer cell lines maintained at 21% O2 or 1% O2 with or without E2 co-treatment. Treatment by E2 or hypoxia alone altered the expression of 26 and 9 genes, respectively, whilst the expression of 31 genes was modulated by the H-E2 combination. The majority (21/31 genes) underwent a down-regulation. Microarray data was validated for 19 by quantitative real-time PCR and a good correlation noted (r2=0.8). Five out of these 19 genes were assayed for protein expression by Western blot. A correlation was also found between mRNA and protein levels. Statistical analysis showed that the gene expression modulation by the combined H and E2 treatment was additive in most cases, but for RasGRP2 and transferrin (TF) an antagonistic interaction was noted. The results demonstrate that hypoxic conditions and estrogen exposure interact to modulate the expression of a limited number of genes involved in cell growth and differentiation, angiogenesis, protein transport, metabolism and apoptosis.