RESUMO
Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.
Assuntos
Mama/citologia , Mama/enzimologia , Diferenciação Celular , Linhagem da Célula , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mama/patologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Receptor alfa de Estrogênio/agonistas , Feminino , Genes Supressores de Tumor , Humanos , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteólise , Transdução de Sinais , Fatores de Transcrição , Proteínas Supressoras de Tumor/deficiência , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Proteínas de Sinalização YAPRESUMO
The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, ß and γ, arise from the human gene. We now show that DMP1α, ß and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1ß and γ did not activate the ARF promoter, whereas only ß resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1ß reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1ß- and γ-isoforms share domains necessary for the inhibitory function of the ß-isoform. That DMP1ß may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/ß in the nucleus. Cells stably expressing DMP1ß, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and ß-ratios are tightly regulated in hematopoietic cells and DMP1ß antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.
Assuntos
Proliferação de Células/fisiologia , Regulação da Expressão Gênica/fisiologia , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p14ARF/genética , Animais , Linhagem Celular , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Isoformas de Proteínas/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Characterization of the molecular pathways that are required for the viability and maintenance of self-renewing tumor-initiating cells may ultimately lead to improved therapies for cancer. In this study, we show that a CD133(+)/CD44(+) population of cells enriched in prostate cancer progenitors (PCaPs) has tumor-initiating potential and that these progenitors can be expanded under nonadherent, serum-free, sphere-forming conditions. Cells grown under these conditions have increased in vitro clonogenic and in vivo tumorigenic potential. mRNA expression analysis of cells grown under sphere-forming conditions, compared with long-term monolayer cultures, revealed preferential activation of the PI3K/AKT signaling pathway. PI3K p110alpha and beta-protein levels were higher in cells grown under sphere-forming conditions, and phosphatase and tensin homolog (PTEN) knockdown by shRNA led to an increase in sphere formation as well as increased clonogenic and tumorigenic potential. Similarly, shRNA knockdown of FoxO3a led to an increase in tumorigenic potential. Consistent with these results, inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 led to growth inhibition of PCaPs. Taken together, our data strongly suggest that the PTEN/PI3K/Akt pathways are critical for prostate cancer stem-like cell maintenance and that targeting PI3K signaling may be beneficial in prostate cancer treatment by eliminating prostate cancer stem-like cells.
Assuntos
Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Antígeno AC133 , Antígenos CD , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Glicoproteínas , Humanos , Receptores de Hialuronatos , Masculino , Peptídeos , RNA Mensageiro/análise , RNA Neoplásico/análise , Transdução de Sinais/fisiologiaRESUMO
In the present study, we investigated the mechanism of CD44 ligation with the anti-CD44 monoclonal antibody A3D8 to inhibit the proliferation of human acute myeloid leukemia (AML) cells. The effects of A3D8 on myeloid cells were associated with specific disruption of cell cycle events and induction of G0/G1 arrest. Induction of G0/G1 arrest was accompanied by an increase in the expression of p21, attenuation of pRb phosphorylation and associated with decreased Cdk2 and Cdk4 kinase activities. Since c-Jun is an important regulator of proliferation and cell cycle progression, we analysed its role in A3D8-mediated growth arrest. We observed that A3D8 treatment of AML patient blasts and HL60/U937 cells led to the downregulation of c-Jun expression at mRNA and protein level. Transient transfection studies showed the inhibition of c-jun promoter activity by A3D8, involving both AP-1 sites. Furthermore, A3D8 treatment caused a decrease in JNK protein expression and a decrease in the level of phosphorylated c-Jun. Ectopic overexpression of c-Jun in HL60 cells was able to induce proliferation and prevent the antiproliferative effects of A3D8. In summary, these data identify an important functional role of c-Jun in the induction of cell cycle arrest and proliferation arrest of myeloid leukemia cells because of the ligation of the cell surface adhesion receptor CD44 by anti-CD44 antibody. Moreover, targeting of G1 regulatory proteins and the resulting induction of G1 arrest by A3D8 may provide new insights into antiproliferative and differentiation therapy of AML.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/fisiologia , Genes jun , Receptores de Hialuronatos/fisiologia , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Proteínas Proto-Oncogênicas , Doença Aguda , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/biossíntese , Quinases Ciclina-Dependentes/genética , Ciclinas/biossíntese , Ciclinas/genética , Fase G1/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60/citologia , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Receptores de Hialuronatos/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno , Leucemia Mieloide/patologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Células U937/citologia , Células U937/efeitos dos fármacos , Células U937/metabolismoRESUMO
Overexpression of proto-oncogene c-jun and constitutive activation of the Jun N-terminal kinase (JNK) signaling pathway have been implicated in the leukemic transformation process. However, c-jun expression and the role of the JNK signaling pathway have not been investigated in primary acute myeloid leukemia (AML) cells with frequently observed balanced rearrangements such as t(8;21). In the present study, we report elevated c-jun mRNA expression in AML patient bone marrow cells with t(8;21), t(15;17) or inv(16), and a high correlation in mRNA expression levels of AML1-ETO and c-jun within t(8;21)-positive AML patient cells. In myeloid U937 cells, c-jun mRNA and protein expression increase upon inducible expression of AML1-ETO. AML1-ETO transactivates the human c-jun promoter through the proximal activator protein (AP-1) site by activating the JNK pathway. Overexpression of JNK-inhibitor JIP-1 and chemical JNK inhibitors reduce the transactivation capacity of AML1-ETO on the c-jun promoter and the proapoptotic function of AML1-ETO in U937 cells. An autocrine mechanism involving granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSF-R) might participate in AML1-ETO mediated JNK-signaling, because AML1-ETO induces G-CSF and G-CSF-R expression, and G-CSF-R-neutralizing antibodies reduce AML1-ETO-induced JNK phosphorylation. These data suggest a model in which AML1-ETO induces proto-oncogene c-jun expression via the proximal AP-1 site of the c-jun promoter in a JNK-dependent manner.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Genes jun , Leucemia Mieloide Aguda/genética , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/fisiologia , Fatores de Transcrição/genética , Translocação Genética , Subunidade alfa 2 de Fator de Ligação ao Core , Fator Estimulador de Colônias de Granulócitos/fisiologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Fosforilação , Proto-Oncogene Mas , Proteína 1 Parceira de Translocação de RUNX1 , Transdução de Sinais , Ativação Transcricional , Células U937RESUMO
Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Células-Tronco Neoplásicas/imunologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Antígeno CD24/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Integrina beta1/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Microscopia de Fluorescência , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Transdução de SinaisRESUMO
Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Benzilaminas , Adesão Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Ciclamos , Docetaxel , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Taxoides/farmacologiaRESUMO
C-type lectin domain family 5, member A (CLEC5A), also known as myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), is a cell surface receptor strongly associated with the activation and differentiation of myeloid cells. CLEC5A associates with its adaptor protein DAP12 to activate a signaling cascade resulting in activation of downstream kinases in inflammatory responses. Currently, little is known about the transcriptional regulation of CLEC5A. We identified CLEC5A as one of the most highly induced genes in a microarray gene profiling experiment of PU.1 restored myeloid PU.1-null cells. We further report that CLEC5A expression is significantly reduced in several myeloid differentiation models upon PU.1 inhibition during monocyte/macrophage or granulocyte differentiation. In addition, CLEC5A mRNA expression was significantly lower in primary acute myeloid leukemia (AML) patient samples than in macrophages and granulocytes from healthy donors. Moreover, we found activation of a CLEC5A promoter reporter by PU.1 as well as in vivo binding of PU.1 to the CLEC5A promoter. Our findings indicate that CLEC5A expression in monocyte/macrophage and granulocytes is regulated by PU.1.
Assuntos
Diferenciação Celular/genética , Lectinas Tipo C/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/genética , Transativadores/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Lectinas Tipo C/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
PURPOSE: The cancer stem cell hypothesis predicts that standard prostate cancer monotherapy eliminates bulk tumor cells but not a tumor-initiating cell population, eventually leading to relapse. Many studies have sought to determine the underlying differences between bulk tumor and cancer stem cells. EXPERIMENTAL DESIGN: Our previous data suggest that the PTEN/PI3K/AKT pathway is critical for the in vitro maintenance of CD133(+)/CD44(+) prostate cancer progenitors and, consequently, that targeting PI3K signaling may be beneficial in treatment of prostate cancer. RESULTS: Here, we show that inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 leads to a decrease in the population of CD133(+)/CD44(+) prostate cancer progenitor cells in vivo. Moreover, the combination of the PI3K/mTOR modulator NVP-BEZ235, which eliminates prostate cancer progenitor populations, and the chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors in a prostate cancer xenograft model than monotherapy. CONCLUSION: This combination treatment ultimately leads to the expansion of cancer progenitors with a PTEN E91D mutation, suggesting that the analysis of PTEN mutations could predict therapeutic response to the dual therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sistemas de Liberação de Medicamentos/métodos , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Imidazóis/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , PTEN Fosfo-Hidrolase/genética , Peptídeos/metabolismo , Neoplasias da Próstata/patologia , Quinolinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.
Assuntos
Genoma Humano , Mitocôndrias/enzimologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Microscopia ImunoeletrônicaRESUMO
In the present study, we employed 2-DE to characterize the effect of the acute promyelocytic leukemia (APL)-specific PML-RARalpha fusion protein on the proteome. Differentially expressed proteins, a number of which are related to the cell cycle function, including oncoprotein18 (OP18), heat shock protein70, glucose-regulated protein75, and peptidyl-prolyl isomerase, were identified by MS. Subsequent bioinformatic pathway discovery revealed an integrated network constituting SMARCB1, MYC, and TP53-regulated pathways. The data from the DNA microarray and proteomic experiments demonstrated the correlation between the translocation and higher expression of OP18 at mRNA and protein levels. Transient cotransfection assay revealed that PML-RARalpha is a potent activator of OP18 promoter and this transcriptional activation is retinoic acid sensitive. PML-RARalpha induction also leads to decreased phosphorylation on Ser63 residue of OP18, which is okadaic acid sensitive suggesting the involvement of a phosphatase pathway. Overexpression of a constitutively phosphorylated Ser63 mutant of OP18 in PML-RARalpha expressing APL patient, PR9, and NB4 cells led to a G2/M-phase arrest in contrast to a phosphorylation-deficient Ser63 mutant and untransfected control. Taken together, our results demonstrate the significance of decreased Ser63 phosphorylation of OP18 in PML-RARalpha-mediated effects on cell cycle.
Assuntos
Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/análise , Proteoma/análise , Serina/metabolismo , Estatmina/metabolismo , Linhagem Celular , Células Clonais , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Genes Reporter , Humanos , Leucemia Promielocítica Aguda/patologia , Luciferases/metabolismo , Espectrometria de Massas , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Mapeamento de Peptídeos , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Proteômica/métodos , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Estatmina/química , Estatmina/genética , Transfecção , Células U937 , Sulfato de ZincoRESUMO
Recent results indicate that interactions of transcription factors with other nuclear proteins play an important role in stem cell development, lineage commitment and differentiation in the haematopoietic system, and the pathogenesis of myeloid leukaemias. High-throughput proteomics by mass spectrometric analysis of gel-separated proteins can identify multi-protein complexes and changes in the expression of multiple proteins simultaneously. This review describes an application of proteomic methods (2D gel electrophoresis (GE) and mass spectrometry (MS)), which can be used to identify regulated protein targets of transcription factors important in myeloid differentiation and leukaemia. This global high-throughput functional proteomics approach could lead to new insights into the network of protein-protein interactions and target proteins involved in myeloid stem cell development and leukaemia as well as provide new targets for rational pathogenesis-based therapies of leukaemia and cancer.
Assuntos
Leucemia/genética , Células Mieloides/fisiologia , Proteínas de Neoplasias/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/análise , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/análise , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/fisiologia , Peptídeos/análise , Proteômica , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/fisiologia , Proteína 1 Parceira de Translocação de RUNX1 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transativadores/análise , Transativadores/fisiologia , Fatores de Transcrição/análiseRESUMO
Several transcription factors have been implicated as playing a role in myelopoiesis. PU.1, an ets-family transcription factor, is required for the development of myeloid and lymphoid lineages, whereas the transcription factor CCAAT-enhancer binding protein family member C/EBPalpha is essential for granulocyte development. We present here the first evidence that C/EBPalpha blocks the function of PU.1. PU.1 and C/EBPalpha interact physically and colocalize in myeloid cells. As a consequence of this interaction, C/EBPalpha can inhibit the function of PU.1 to activate a minimal promoter containing only PU.1 DNA-binding sites. We further demonstrate that the leucine zipper in the DNA-binding domain of C/EBPalpha interacts with the beta3/beta4 region in the DNA-binding domain of PU.1 and as a result displaces the PU.1 coactivator c-Jun. Finally, C/EBPalpha blocks PU.1-induced dendritic cell development from CD34+ human cord blood cells. The functional blocking of PU.1 by C/EBPalpha could be the mechanism by which C/EBPalpha inhibits cell fates specified by PU.1 and directs cell development to the granulocyte lineage.