RESUMO
Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3' enhancers deregulate the Bcl2 expression in vitro. However, the effects of the Igh 3' enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3' enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I-hypersensitive regions from the murine Igh 3' region were integrated 3' of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3'E-bcl2 mice. B cells from Igh-3'E-bcl2 mice showed an extended survival in vitro compared with B cells from wild-type (Wt) mice. The Bcl2 promoter shift from P1 (the 5' promoter) to P2 (the 3' promoter) was observed in B cells from Igh-3'E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3'E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies show that the Igh 3' enhancers have an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo.
Assuntos
Regiões 3' não Traduzidas/genética , Linfócitos B/patologia , Elementos Facilitadores Genéticos/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Linfoma de Células B/etiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Southern Blotting , Western Blotting , Ciclo Celular , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
To better understand the mechanisms underlying the role of the immunoglobulin heavy-chain gene (IgH) 3' enhancers on bcl-2 transcriptional deregulation in t(14;18) lymphoma, we characterized the physical interactions of the IgH 3' enhancer region with the bcl-2 promoters. Using the chromosome conformation capture technique, we found that the IgH 3' enhancers physically interact with the bcl-2 promoter region over a 350 kb genomic region in t(14;18) lymphoma cells. No interactions of the bcl-2 promoter region with sequences distant to the IgH enhancers were observed. The physical interactions of the IgH enhancers with the bcl-2 5' region are functionally involved in the transcriptional control of bcl-2. The histone deacetylase inhibitor, trichostatin A, repressed bcl-2 transcription and decreased the IgH enhancer-bcl-2 promoter region interactions. We showed by chromatin immunoprecipitation assay and small interference RNA transfection studies that the POU2 family transcription factor Oct-2 and its cofactor Bob-1 have an important function in mediating the IgH enhancer-bcl-2 promoter region interactions. This study reveals a new aspect of the regulatory role of the IgH 3' enhancers on bcl-2 transcription in t(14;18) lymphomas.
Assuntos
Elementos Facilitadores Genéticos/genética , Genes bcl-2/genética , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/metabolismo , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 18/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Linfoma/metabolismo , Fator 2 de Transcrição de Octâmero/genética , Fator 2 de Transcrição de Octâmero/metabolismo , RNA Interferente Pequeno/genética , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Translocação GenéticaRESUMO
The expression of c-myc is deregulated in Burkitt's lymphoma by the translocation t(8;14). Most of the increased c-myc expression is from the P1 promoter, which is normally a minor promoter. How the P1 promoter is activated by the immunoglobulin heavy chain gene enhancers is not understood. We identified a YY1 site in the immunoglobulin heavy-chain gene HS3 enhancer, which increased c-myc P1 promoter activity, and a MARE site, which decreased c-myc P1 activity. Small Maf proteins bound to the MARE site both in vitro and in vivo, recruited histone deacetylase 2, and resulted in deacetylation of histones H3 and H4 at the c-myc promoter region. In contrast, YY1 recruited CBP and increased histone acetylation at the c-myc promoter. Rb interacts with YY1 to prevent DNA binding in normal B cells, but no significant interaction with YY1 was detected in Burkitt's cells, and binding of YY1 to the HS3 enhancer was observed by chromatin immunoprecipitaton. Increased expression of MafK and/or decreased expression of YY1 by silencing RNA downregulated endogenous c-myc mRNA levels and increased the sensitivity of the cells to doxorubicin. Mutation of the major active sites (nuclear factor-kappa B and YY1) in the enhancers prevented c-myc activation.
Assuntos
Linfoma de Burkitt/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Linfoma de Burkitt/imunologia , Linhagem Celular Tumoral , Cromatina/genética , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 8 , Genes Reporter , Humanos , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Translocação GenéticaRESUMO
In t(14;18) lymphomas, bcl-2 is juxtaposed to the immunoglobulin heavy-chain gene (IgH), resulting in increased bcl-2 transcription and resistance to apoptosis. Regulatory elements of both the bcl-2 promoter and the IgH enhancers are believed to play a role in the increased expression of bcl-2 in t(14;18) lymphoma cells. In addition, transcription of the translocated bcl-2 allele is deregulated with activation of the normally minor bcl-2 P2 promoter. The mechanisms involved in the promoter shift from P1 to P2 are not known. We found that the murine IgH 3' enhancers increased bcl-2 P2 promoter activity in an episomal model of the translocation, and IgH enhancer region HS12 had the greatest effect. Quantitative chromatin immunoprecipitation (ChIP) assays revealed that localized histone H3 hyperacetylation of the P2 promoter was observed on the translocated allele in t(14;18) DHL-4 cells and also on the stably transfected bcl-2 promoter-IgH enhancer episomal construct. Analysis of the HS12 enhancer region revealed that a previously identified nuclear factor-kappaB (NF-kappaB) site and a previously uncharacterized downstream Cdx site, both of which are conserved in the human and murine IgH enhancers, were important for its enhancer activity and promoter activation. ChIP assays showed that C/EBPbeta bound to the HS12 Cdx site in vivo, and mutation of this site abrogated the binding of C/EBPbeta. Reduced expression of C/EBPbeta by transfection of small interfering RNA or interference with NF-kappaB activity decreased transcription from the bcl-2 promoters. These results demonstrate that the IgH 3' enhancers, particularly HS12, are important for the deregulation of bcl-2 promoter usage in t(14;18) lymphomas.
Assuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 18/genética , Elementos Facilitadores Genéticos/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Linfoma/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Translocação Genética , Acetilação , Animais , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Linfoma/patologia , Camundongos , Mutagênese Sítio-Dirigida , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Oct-1 and Oct-2 are members of the POU homeodomain family of transcriptional regulators and are critical for normal embryonic development. Gene-targeting studies showed that Oct-1 and Oct-2 are largely dispensable for B-cell development and immunoglobulin production, although both Oct-2 and Bob-1 are required for a proper immune response and germinal center formation. In these studies, we investigated the role of Oct factors in B-cell lymphomas. Recent investigations have shown increased expression of Oct-2 and Bob-1 in lymphomas, and we observed greatly increased levels of Oct-2 in lymphoma cells with the t(14;18) translocation. Decreased expression of Oct-1, Oct-2, or Bob-1 by RNA interference resulted in apoptosis and down-regulation of bcl-2 expression. Furthermore, Oct-2 induced bcl-2 promoter activity and mediated this effect through three regions in the bcl-2 P2 promoter. Although these regions did not contain canonical octamer motifs, we observed the direct interaction of Oct-2 with all three sites both in vitro by EMSA and in vivo by chromatin immunoprecipitation assay. Moreover, by mutation analysis we found that the ability of Oct-2 to activate bcl-2 required C/EBP, Cdx, and TATA-binding sites. Oct-2, therefore, acts as a cell survival factor in t(14;18) lymphoma cells by directly activating the antiapoptotic gene bcl-2.
Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma/genética , Fatores de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Translocação Genética , Apoptose/genética , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sobrevivência Celular/genética , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Linfoma/patologia , Fator 1 de Transcrição de Octâmero/genética , Fator 1 de Transcrição de Octâmero/metabolismo , Fator 2 de Transcrição de Octâmero/genética , Fator 2 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Células Tumorais CultivadasRESUMO
c-MYC is the prototype for oncogene activation by chromosomal translocation. In contrast to the tightly regulated expression of c-myc in normal cells, c-myc is frequently deregulated in human cancers. Herein, aspects of c-myc gene activation and the function of the c-Myc protein are reviewed. The c-myc gene produces an oncogenic transcription factor that affects diverse cellular processes involved in cell growth, cell proliferation, apoptosis and cellular metabolism. Complete removal of c-myc results in slowed cell growth and proliferation, suggesting that while c-myc is not required for cell proliferation, it acts as an integrator and accelerator of cellular metabolism and proliferation.
Assuntos
Genes myc/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fatores de Transcrição , Translocação Genética , Fatores de Transcrição de Zíper de Leucina Básica , Linfoma de Burkitt/genética , Adesão Celular , Diferenciação Celular , Divisão Celular , Senescência Celular , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Modelos Genéticos , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Transcrição Gênica , Ativação TranscricionalRESUMO
In neuronal cells, expression of the anti-apoptotic Bcl-2 gene is induced by hypoxia and produces a protective effect. We show here that this effect is dependent upon the cyclic AMP response element (CRE) in the Bcl-2 promoter since mutation of this element abolishes the response and the isolated CRE can confer the response on a heterologous promoter. Interestingly however, the CRE in the Bcl-2 promoter does not render the promoter responsive to cyclic AMP and is not essential for its response to nerve growth factor. Despite the lack of cyclic AMP responsiveness, activation of the Bcl-2 promoter via the CRE in response to hypoxia requires the CREB transcription factor and is associated with the enhanced phosphorylation of CREB on serine 133 and enhanced transcriptional activation by the CREB-binding protein, CBP, in response to hypoxia. This finding establishes the importance of the CRE in the induction of Bcl-2 gene expression by hypoxia, allowing the Bcl-2 protein to protect neuronal cells against this damaging stimulus.
Assuntos
Apoptose/fisiologia , Hipóxia Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Regulação da Expressão Gênica/genética , Genes bcl-2 , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas de Saccharomyces cerevisiae , Sistemas do Segundo Mensageiro/fisiologia , Animais , Apoptose/genética , Sítios de Ligação , Bucladesina/farmacologia , Proteína de Ligação a CREB , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Luciferases/biossíntese , Luciferases/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fator de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Estresse Oxidativo , Células PC12/efeitos dos fármacos , Células PC12/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
A synergistic interaction of Bcl-2 and c-Myc plays a role in lymphomagenesis in mice and in some patients as well. Progression of follicular lymphoma to a more aggressive lymphoma is seen in the majority of patients, and approximately 10% of the transformed lymphomas have a translocation of c-myc in addition to the translocation of bcl-2 found in the original follicular lymphoma. We investigated whether transcriptional deregulation of bcl-2 and c-myc could be examined in primary lymphoma cells by in vivo footprinting and in vitro protein-DNA binding studies. A matched pair of follicular and transformed lymphoma samples was examined. The transformed lymphoma had acquired a translocation of c-myc into the immunoglobulin heavy chain locus. High levels of bcl-2 expression were observed in both the follicular and transformed lymphomas, whereas the expression of c-myc was low in the follicular lymphoma and increased in the transformed lymphoma. In vivo footprint analysis revealed that a CRE site and a Cdx site in the bcl-2 promoter were occupied on the translocated alleles but not on the normal alleles in both the follicular and transformed lymphomas. Two nuclear factor kappaB sites were occupied on the translocated c-myc allele in the transformed lymphoma. Gel shift analysis revealed that these proteins bound to their respective sites in the bcl-2 or c-myc promoter. There was no evidence that the presence of one of the translocations in the immunoglobulin heavy chain locus influenced the expression of the other translocated gene.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes bcl-2/genética , Genes myc/genética , Linfoma Folicular/genética , Transcrição Gênica/genética , Alelos , Sítios de Ligação , Northern Blotting , Transformação Celular Neoplásica/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Pegada de DNA , Progressão da Doença , Eletroforese em Gel de Campo Pulsado , Humanos , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Translocação GenéticaRESUMO
OBJECTIVE: The retroviral oncogene v-Cbl causes pre-B cell lymphomas and myeloid leukemias in mice, and its Drosophila homologue is oncogenic, causing enhanced receptor tyrosine kinase signaling. The human Cbl gene resides at 11q23. The aim of this study is to determine the effect of oncogenic Cbl on growth-regulating responses. MATERIALS AND METHODS: The oncogenic mutant of Cbl (CblDelta1-357) was transfected into factor-dependent 32Dcl3 myeloid cells. Consequently, cell survival and differentiation were measured. Lyn, Syk, MAP kinase, and phosphatidylinositol 3'(PI3')-kinase activities, protein phosphorylation, Bcl-2 promoter activity, ubiquitination, and levels of Bcl-2, Bax, Bad, and Bcl-x(L) were determined. In addition, the effect of v-Cbl on TF-1 cell survival upon granulocyte-macrophage colony-stimulating factor withdrawal was studied. RESULTS: 32Dcl3 and TF-1 cells expressing v-Cbl showed resistance to apoptosis upon growth factor withdrawal, and 32Dcl3 cells completely failed to respond to granulocyte colony-stimulating factor's induction of differentiation. Basal activities of Lyn, Syk, and PI3'-kinase were elevated in the v-Cbl line. There was neither enhanced tyrosine phosphorylation of cellular protein content, Cbl, or Jak2, nor serine phosphorylation of MAP kinase or Akt. After factor withdrawal, the level of Bcl-2 was greater in v-Cbl cells than in control cells. CONCLUSIONS: Neither increased Bcl-2 promoter activity nor decreased ubiquitination of Bcl-2 could account for increased Bcl-2 levels. v-Cbl-expressing 32Dcl3 cells were resistant to differentiation. v-Cbl suppresses apoptosis and differentiation, possibly through enhancement of Lyn, Syk, and PI3'-kinase activities and Bcl-2.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Animais , Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromossomos Humanos Par 11 , Drosophila , Precursores Enzimáticos/metabolismo , Genes bcl-2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica v-cbl , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , Deleção de Sequência , Quinase Syk , Ubiquitinas/metabolismo , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-X , Quinases da Família src/metabolismoRESUMO
The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/fisiologia , Linfócitos B/patologia , Linfócitos B/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Histona Desacetilase 1 , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leucemia Eritroblástica Aguda/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Complexo Correpressor Histona Desacetilase e Sin3 , Proteína de Ligação a TATA-Box , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Cytokine oncostatin M (OM) exerts growth-inhibitory and differentiative effects on breast cancer cells. Previously we showed that the transcription from the p53 gene in breast cancer cells was down regulated by OM. To elucidate the molecular mechanisms underlying the OM effect on p53 transcription, in this study, we dissected the p53 promoter region and analysed the p53 promoter activity in breast tumor cells. We showed that treatment of MCF-7 cells with OM induced a dose- and time-dependent suppression of p53 promoter activity. The p53 promoter activity was decreased to 35% of control at 24 h and further decreased to 20% at 48 h by OM at concentrations of 5 ng/ml and higher. Deletion of the 5'-flanking region of the p53 promoter from -426 to -97 did not affect the OM effect. However, further deletion to -40 completely abolished the repressive effect of OM. The p53 promoter region -96 to -41 contains NF-kappaB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kappaB binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at the PE21 motif totally abolished the OM effect. We further demonstrated that insertion of PE21 element upstream of the thymidine kinase minimal promoter generated an OM response analogous to that of the p53 promoter. Finally, we detected the specific binding of a nuclear protein with a molecular mass of 87 kDa to the PE21 motif. Taken together, we demonstrate that OM inhibits the transcription of the p53 gene through the PE21 element. Thus, the PE21 element is functionally involved in p53 transcription regulated by UV-induction and OM suppression.
Assuntos
Neoplasias da Mama/genética , Regulação para Baixo , Genes p53 , Peptídeos/farmacologia , Elementos de Resposta , Transcrição Gênica , Sítios de Ligação , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Genes Reporter , Humanos , Cinética , Proteínas Nucleares/metabolismo , Oncostatina M , Regiões Promotoras Genéticas , Deleção de Sequência , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The c-myc gene is translocated to one of the immunoglobulin genes in Burkitt's lymphoma resulting in deregulated expression of c-myc. Several enhancers have been shown to be important for expression of the immunoglobulin heavy chain gene. Four enhancer regions (murine-hypersensitive sites (MHS) 1, 2, 3, and 4) located 3' of the murine immunoglobulin heavy chain gene play a role in activating expression of the translocated c-myc gene. The enhancer regions also result in a shift in transcriptional initiation from the P2 promoter to P1 that is characteristic of the translocated c-myc allele. We found that the most 3' enhancer region (MHS4) activated the c-myc promoter by 46-fold in the Raji Burkitt's lymphoma cell line, and it was the most active enhancer in these cells. The addition of enhancer regions MHS1,2 and 3 to MHS4 increased c-myc transcription by an additional 3-fold and resulted in the full promoter shift from P2 to P1. By deletion analysis of enhancer region MHS4, we located a region that was critical for the transcriptional activity of MHS4. Electrophoretic mobility shift assay analysis revealed that NF-kappaB/Rel family members bound to this region. Mutation of the NF-kappaB binding site abolished both the enhancer activity and the promoter shift activity of MHS4. An active NF-kappaB site was also identified in the human HS4 enhancer. Inhibition of c-myc promoter activity driven by the immunoglobulin enhancers was observed with expression of a super-repressor IkappaBalpha construct. These results indicate that the NF-kappaB/Rel transcription factors play an important role in the deregulation of the translocated c-myc gene in Burkitt's lymphoma and suggest that interference with NF-kappaB function may represent a new approach to the treatment of Burkitt's lymphoma.
Assuntos
Linfoma de Burkitt/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Proteínas I-kappa B , Cadeias Pesadas de Imunoglobulinas/genética , NF-kappa B/metabolismo , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Mutação/genética , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Fator de Transcrição RelA , Transfecção , Translocação Genética/genética , Células Tumorais CultivadasRESUMO
In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Translocação Genética/genética , Animais , Linfócitos B , Sítios de Ligação , Fator de Transcrição CDX2 , Proteínas de Ligação a DNA/genética , Humanos , Linfoma Folicular , Mutagênese , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Transfecção , Células Tumorais Cultivadas , Raios Ultravioleta , Regulação para Cima/genéticaRESUMO
In our previous study we showed that insulin-like growth factor-I induces a cAMP-response element (CRE) site-containing Bcl-2 promoter through a novel signaling pathway involving mitogen-activated protein kinase kinase 6/p38beta mitogen-activated protein kinase/MAP kinase-activated protein kinase-3/cAMP-response element-binding protein (CREB) (Pugazhenthi, S., Miller, E., Sable, C., Young, P., Heidenreich, K. A., Boxer, L. M., and Reusch, J. E.-B. (1999) J. Biol. Chem. 274, 27529-27535). In the present investigation, we define a second pathway contributing to CREB-dependent up-regulation of Bcl-2 expression as a novel anti-apoptotic function of Akt signaling. To examine the role of Akt on Bcl-2 expression, a series of transient transfections using a luciferase reporter gene driven by the promoter region of Bcl-2 containing a CRE were carried out. Pharmacological inhibition of phosphatidylinositol (PI) 3-kinase, the upstream kinase of Akt, with LY294002 led to a 45% decrease in Bcl-2 promoter activity. The reporter activity was enhanced 2.3-fold by overexpression of active p110 subunit of PI 3-kinase and inhibited 44% by the dominant negative p85 subunit of PI 3-kinase. Cotransfection with 3-phosphoinositide-dependent kinase (PDK1), which is required for the full activation of Akt, resulted in enhanced luciferase activity. Insulin-like growth factor-I-mediated induction of Bcl-2 promoter activity was decreased significantly (p < 0.01) by the dominant negative forms of p85 subunit of PI 3-kinase, PDK1, and Akt. These data indicate that regulation of Bcl-2 expression by IGF-I involves a signaling cascade mediated by PI 3-kinase/PDK1/Akt/CREB. Furthermore, we measured the Bcl-2 mRNA in PC12 cells overexpressing Akt by real-time quantitative reverse transcription-polymerase chain reaction using the TaqMan(TM) fluorogenic probe system. We observed a 2.1-fold increase in Bcl-2 mRNA levels in the Akt cell line compared with control PC12 cells, supporting the observation that enhanced CREB activity by Akt signaling leads to increased Bcl-2 promoter activity and cell survival.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas/fisiologia , Animais , Fator de Crescimento Insulin-Like I/farmacologia , Células PC12 , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Regulação para CimaRESUMO
PC12 and INS-1 cells both express the nerve growth factor (NGF) receptors trkA and p75NTR and the epidermal growth factor receptor (EGF). In PC12 cells, NGF treatment initiates a signaling cascade that ultimately leads to a change of the genetic program of the cell. We have investigated the role of NGF in regulating gene transcription in PC12 and INS-1 cells, in order to define if there are NGF-regulated genes per se. Furthermore, to distinguish between growth factor stimulation via receptor tyrosine kinases in general and NGF-specific changes in gene transcription, we analyzed the effects of EGF on gene transcription. First, we tested the biological activities of fusion proteins consisting of the DNA-binding domain of the yeast transcription factor GAL4 and the phosphorylation-dependent activation domains of the transcription factors Elk1, CREB, ATF2 and c-jun in NGF- or EGF-treated PC12 cells. We found a striking increase in the transcriptional activity of the GAL4-Elk1 fusion protein that is a major substrate for the extracellular signal-regulated protein kinase (ERK). This effect was observed in NGF- as well as in EGF-treated PC12 cells. In INS-1 cells, however, the activity of the GAL4-Elk1 fusion protein was induced by NGF, but not by EGF. The effects of NGF and EGF on gene transcription were subsequently studied with plasmids containing reporter genes under control of the Egr-1, c-jun, HES-1 or Bc12 regulatory sequences. NGF stimulated Egr-1 promoter activities in PC12 and INS-1 cells, although the effect was much more pronounced in PC12 cells than in INS-1 cells. EGF also stimulated Egr-1 promoter activity in both PC12 and INS-1 cells. Stimulation of c-jun promoter activity by NGF was observed only in PC12 cells. Deletion mutagenesis demonstrated the importance of the 12-O-tetradecanoylphorbol-13-acetate response elements within the c-jun promoter for basal and NGF-mediated transcriptional induction. Likewise, NGF activated HES1 and Bcl2 P1 promoter activities in PC12 cells but not in INS-1 cells and EGF did not show any effects on these promoters. We conclude that in PC12 and INS-1 cells, NGF signaling leads to an activation of the ERK subtype of mitogen-activated protein kinases in the nucleus and a subsequent activation of Egr-1 gene transcription. The NGF-induced transcription of the c-jun, HES1 and Bc12 genes is, in contrast, cell type-specific, indicating that NGF can trigger different gene expression programs dependent on the signaling pathways present in a particular cell type. EGF is clearly able to activate gene transcription, suggesting that the differences in the biological activities of EGF and NGF cannot be explained by the inability of EGF to stimulate gene transcription.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces , Fator de Crescimento Neural/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Ativador da Transcrição , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Insulinoma , Fator de Crescimento Neural/farmacologia , Células PC12 , Feocromocitoma , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Receptor de Fator de Crescimento Neural , Receptor trkA/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
Previously (J. Liu, et al., Cell Growth Differ., 8: 667-676, 1997), we showed that oncostatin M (OM), a cytokine produced by activated T cells and macrophages, inhibited the proliferation of breast cancer cells derived from solid tumors and malignant effusions. OM-treated cells showed reduced growth rates and differentiated phenotypes. Because the p53 tumor suppressor protein plays an important role in cellular proliferation, we examined p53 protein expression in three OM-responsive breast cancer cell lines, MCF-7, MDA-MB231, and H3922. Western blot analysis showed that p53 protein levels in all three of the cell lines were decreased by OM treatment. Reduction of p53 protein was detected after 1 day of OM treatment and reached maximal suppression of 10-20% of control after 3 days in H3922 and 40% of control after 4 days in MCF-7 cells. A comparison of p53 mRNA in OM-treated cells versus untreated control cells showed that exposure to OM reduced the steady-state levels of p53 mRNA transcripts to an extent similar to that of the p53 protein levels. This observation suggests that the effect of OM on p53 protein expression does not occur at the posttranslational level. Nuclear run-on assays verified that OM decreased the number of actively transcribed p53 mRNAs, which suggests a transcriptional regulatory mechanism. The effect of OM on p53 expression seems to be mediated through the extracellular signal-regulated kinase (ERK) pathway, inasmuch as the inhibition of ERK activation with a specific inhibitor (PD98059) to the ERK upstream kinase mitogen/extracellular-regulated protein kinase kinase abrogated the OM inhibitory activity on p53 expression in a dose-dependent manner. In addition to OM, we showed that the p53 protein expression in MCF-7 cells was also decreased by phorbol 12-myristate 13-acetate treatment (PMA). Because both OM and PMA induce MCF-7 cells to differentiate, our data suggest that p53 expression in breast cancer cells is down-regulated during the differentiation process.
Assuntos
Neoplasias da Mama/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Peptídeos/fisiologia , Western Blotting , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oncostatina M , Fenótipo , Transfecção , Células Tumorais CultivadasRESUMO
Transforming growth factor-beta (TGF-beta) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc-finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF-beta in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF-beta effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl-2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co-expression of Bcl-2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro-apoptotic properties of TGF-beta.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Pulmão/citologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Dedos de Zinco/genética , Animais , Divisão Celular/genética , Linhagem Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Fragmentação do DNA , Proteínas de Ligação a DNA/genética , Dactinomicina/farmacologia , Epitélio/metabolismo , Epitélio/patologia , Genes Precoces , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity of bcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from -1640 to -1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhanced the bcl-2 promoter activity by 2.3-fold, which was inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of the bcl-2 promoter with MAPK kinase 6 and the beta isozyme of p38 MAPK resulted in 2-3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38beta MAPK significantly (p < 0.01). IGF-I increased the activity of p38beta MAPK introduced into the cells by adenoviral infection. Thus, we have characterized a novel signaling pathway (MAPK kinase 6/p38beta MAPK/MAPKAP-K3) that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes bcl-2/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Regiões Promotoras Genéticas/genética , Animais , Linhagem Celular , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Genes Reporter , Genes bcl-2/efeitos dos fármacos , Humanos , Cinética , Luciferases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células PC12 , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Genes bcl-2/genética , Genes bcl-2/fisiologia , Genes p53/fisiologia , Fatores de Transcrição/fisiologia , Células Cultivadas , Regiões Promotoras Genéticas , Fator de Transcrição Brn-3RESUMO
The Bcl-2 protein has an anti-apoptotic effect in neuronal and other cell types. We show for the first time that the Bcl-2 promoter is activated by the neuronal survival factor nerve growth factor (NGF) and that this effect is dependent on a region of the promoter from -1472 to -1414. This activation requires the Rap-1 G protein and the MEK-1 and p42/p44 MAPK enzymes but is independent of other NGF-activated signalling pathways involving protein kinase A or protein kinase C.