RESUMO
Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.
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The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, â¼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Células Mieloides/citologia , Regiões Promotoras Genéticas , Transativadores/metabolismo , Ativação Transcricional , Sequência de Bases , Linhagem Celular , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Células Mieloides/metabolismo , Ligação ProteicaRESUMO
The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.
Assuntos
Antígenos CD34/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Animais , Transplante de Medula Óssea , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Sangue Fetal/citologia , Genótipo , Células HL-60 , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/biossíntese , Talidomida/análogos & derivados , Adulto , Animais , Antimetabólitos Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Citarabina/farmacologia , Mutação da Fase de Leitura , Humanos , Fatores Imunológicos/uso terapêutico , Células K562 , Lenalidomida , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/fisiologia , Talidomida/farmacologia , Talidomida/uso terapêutico , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A genome-wide association study of papillary thyroid carcinoma (PTC) pinpointed two independent SNPs (rs944289 and rs965513) located in regions containing no annotated genes (14q13.3 and 9q22.33, respectively). Here, we describe a unique, long, intergenic, noncoding RNA gene (lincRNA) named Papillary Thyroid Carcinoma Susceptibility Candidate 3 (PTCSC3) located 3.2 kb downstream of rs944289 at 14q.13.3 and the expression of which is strictly thyroid specific. By quantitative PCR, PTCSC3 expression was strongly down-regulated (P = 2.84 × 10(-14)) in thyroid tumor tissue of 46 PTC patients and the risk allele (T) was associated with the strongest suppression (genotype [TT] (n = 21) vs. [CT] (n = 19), P = 0.004). In adjacent unaffected thyroid tissue, the genotype [TT] was associated with up-regulation of PTCSC3 ([TT] (n = 21) vs. [CT] (n = 19), P = 0.034). The SNP rs944289 was located in a binding site for the CCAAT/enhancer binding proteins (C/EBP) α and ß. The risk allele destroyed the binding site in silico. Both C/EBPα and C/EBPß activated the PTCSC3 promoter in reporter assays (P = 0.0009 and P = 0.0014, respectively) and the risk allele reduced the activation compared with the nonrisk allele (C) (P = 0.026 and P = 0.048, respectively). Restoration of PTCSC3 expression in PTC cell line cells (TPC-1 and BCPAP) inhibited cell growth (P = 0.002 and P = 0.019, respectively) and affected the expression of genes involved in DNA replication, recombination and repair, cellular movement, tumor morphology, and cell death. Our data suggest that SNP rs944289 predisposes to PTC through a previously uncharacterized, long intergenic noncoding RNA gene (PTCSC3) that has the characteristics of a tumor suppressor.
Assuntos
Carcinoma Papilar/genética , Polimorfismo de Nucleotídeo Único , RNA não Traduzido/genética , Neoplasias da Glândula Tireoide/genética , Animais , Sítios de Ligação/genética , Northern Blotting , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células COS , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Proliferação de Células , Chlorocebus aethiops , Cromossomos Humanos Par 14/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Predisposição Genética para Doença/genética , Genótipo , Células HEK293 , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/patologiaRESUMO
The transcription factor C/EBPα is a critical mediator of myeloid differentiation and is often functionally impaired in acute myeloid leukemia. Recent studies have suggested that oncogenic FLT3 activity disrupts wild-type C/EBPα function via phosphorylation on serine 21 (S21). Despite the apparent role of pS21 as a negative regulator of C/EBPα transcription activity, the mechanism by which phosphorylation tips the balance between transcriptionally competent and inhibited forms remains unresolved. In the present study, we used immuno-affinity purification combined with quantitative mass spectrometry to delineate the proteins associated with C/EBPα on chromatin. We identified DEK, a protein with genetic links to leukemia, as a member of the C/EBPα complexes, and demonstrate that this association is disrupted by S21 phosphorylation. We confirmed that DEK is recruited specifically to chromatin with C/EBPα to enhance GCSFR3 promoter activation. In addition, we demonstrated that genetic depletion of DEK reduces the ability of C/EBPα to drive the expression of granulocytic target genes in vitro and disrupts G-CSF-mediated granulocytic differentiation of fresh human BM-derived CD34(+) cells. Our data suggest that C/EBPα and DEK coordinately activate myeloid gene expression and that S21 phosphorylation on wild-type C/EBPα mediates protein interactions that regulate the differentiation capacity of hematopoietic progenitors.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Diferenciação Celular/genética , Proteínas Cromossômicas não Histona/fisiologia , Células Mieloides/fisiologia , Proteínas Oncogênicas/fisiologia , Anticorpos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Humanos , Células K562 , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
C/EPBα proteins, encoded by the CCAAT-enhancer-binding protein α gene, play a crucial role in granulocytic development, and defects in this transcription factor have been reported in acute myeloid leukemia. Here, we defined the C/EBPα signature characterized by a set of genes up-regulated upon C/EBPα activation. We analyzed expression of the C/EBPα signature in a cohort of 525 patients with acute myeloid leukemia and identified a subset characterized by low expression of this signature. We referred to this group of patients as the C/EBPα dysfunctional subset. Remarkably, a large percentage of samples harboring C/EBPα biallelic mutations clustered within this subset. We hypothesize that re-activation of the C/EBPα signature in the C/EBPα dysfunctional subset could have therapeutic potential. In search for small molecules able to reverse the low expression of the C/EBPα signature we applied the connectivity map. This analysis predicted positive connectivity between the C/EBPα activation signature and histone deacetylase inhibitors. We showed that these inhibitors reactivate expression of the C/EBPα signature and promote granulocytic differentiation of primary samples from the C/EBPα dysfunctional subset harboring biallelic C/EBPα mutations. Altogether, our study identifies histone deacetylase inhibitors as potential candidates for the treatment of certain leukemias characterized by down-regulation of the C/EBPα signature.
Assuntos
Antineoplásicos/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Transcriptoma , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Mutação/efeitos dos fármacos , Mutação/genética , Ativação TranscricionalRESUMO
While pentacyclic triterpenoids have a rich history in chemistry and biology, the challenges associated with their asymmetric synthesis contribute to the current reality that medicinal exploration in the area is largely constrained to natural product derivatization. To address this deficiency, a function-oriented synthesis of pentacyclic triterpenoids was pursued. Overall, we report a divergent synthesis of 26-norgermanicol and 26-norlupeol and we have identified a new class of androgen receptor antagonist that is â¼6× more potent than lupeol.
Assuntos
Produtos Biológicos , Triterpenos , Triterpenos Pentacíclicos , Triterpenos/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Produtos Biológicos/farmacologiaRESUMO
Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação Puntual , Tirosina Quinase 3 Semelhante a fms/genética , Idoso , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Ativação Enzimática/genética , Feminino , Granulócitos/patologia , Humanos , Células K562 , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Masculino , Células Mieloides/patologia , Fosforilação , Piperazinas/farmacologia , Quinazolinas/farmacologia , Serina/metabolismo , Células Tumorais Cultivadas , Células U937 , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Background: Among women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERß plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERß agonists without ERα activity has limited the exploration of ERß activation as a strategy for ERα+ breast cancer. Methods: We measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERß agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERß agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERß agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes. Results: In this study, we demonstrate the efficacy of highly selective ERß agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERß agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERß agonists and ERα antagonists suggested that the efficacy of ERß agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERß gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples. Conclusion: Our results demonstrate that highly selective ERß agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.
RESUMO
Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERß, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17ß-estradiol geometry in the design of ERß selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERß selective structure-activity relationship. We report ERß agonists with low nanomolar potency, greater than 200-fold selectivity for ERß over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERß selective agonists measure favorably against clinically developed ERß agonists and support further evaluation of carborane-based selective estrogen receptor modulators.
Assuntos
Compostos de Boro/farmacologia , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Compostos de Boro/síntese química , Compostos de Boro/química , Relação Dose-Resposta a Droga , Estrogênios/síntese química , Estrogênios/química , Células HEK293 , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper 'Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs' (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3'UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3'UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.
Assuntos
Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/genética , Células HCT116 , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , RNA Mensageiro/metabolismoRESUMO
Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb -14 site confers lineage-specific reporter gene expression in vivo. This kb -14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Elementos de Resposta/genética , Transativadores/genética , Transativadores/metabolismo , Animais , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem da Célula , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação/genética , Células Mieloides/metabolismo , Especificidade de Órgãos , Alinhamento de SequênciaRESUMO
OBJECTIVE: We investigated the role of CCAAT enhancer-binding protein-alpha (C/EBPalpha) during zebrafish embryonic blood development. METHODS: Whole-mount mRNA in situ hybridization was performed to determine the spatio-temporal expression pattern of zebrafish cebpa in developing hematopoietic progenitors. A deletion mutation of cebpa (zD420), which mimics the human dominant-negative mutations of C/EBPalpha, was transfected into CV1 cell line to evaluate its transcriptional activity in vitro and injected into zebrafish embryos at the one- to two-cell stage to examine its effects on primitive hematopoiesis during early zebrafish development. RESULTS: Zebrafish cebpa is expressed in the anterior and posterior lateral plate mesoderm at 12 hours postfertilization, along with scl, pu.1, and gata1 in developing hematopoietic progenitors. In vitro, the deletion mutation of cebpa (zD420) prevents expression of the full-length protein, allowing the expression of truncated isoforms from internal translational initiation sites. As in the human, the truncated zebrafish C/EBPalpha proteins did not activate the expression of known target granulocytic genes, and in fact suppressed transactivation that was induced in vitro by the full-length protein. Forced expression of the zD420 mRNA in zebrafish embryos led to an expansion of primitive erythropoiesis, without a discernible effect on granulopoiesis. CONCLUSION: Expression of the truncated isoforms of cebpa alters the developmental pattern of hematopoietic progenitor cells during embryogenesis.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Peixe-Zebra/genética , Animais , Sequência de Bases , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , DNA Complementar/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Deleção de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , Translocação Genética/genética , Translocação Genética/fisiologia , Transplante Heterólogo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Standard chemotherapy is not curative for many patients with acute myeloid leukemia (AML). New treatment strategies combining demethylating agents, such as decitabine, and drugs that induce myelomonocytic differentiation (i.e. Vitamin D3) may re-establish functional hematopoiesis in these patients. We studied the effects of decitabine alone or in combination with Vitamin D3 (VD3) on U937 cells and AML blasts. Preincubation with decitabine (0.1-1 microM) and subsequent exposure to VD3 (3 nM) synergistically induced monocytic differentiation. To elucidate the mechanisms of decitabine- and VD3-induced monocytic differentiation, we investigated the effects of the two drugs on transcription factors implicated in monocytic differentiation. Northern and Western blotting showed that decitabine induced transcription of c-jun but not PU.1, while VD3 increased PU.1, IRF8, and C/EBPbeta but not c-jun. Using electromobility shift assays, we demonstrated increased DNA binding of nuclear proteins from decitabine- and VD3-induced U937 cells to the CD11b promoter. In addition, we investigated whether the myeloid transcription factor Sp1 played a role in decitabine- and VD3-induced CD14 expression. Indeed, we found that mithramycin A, a specific inhibitor of Sp1, inhibited both VD3- and decitabine-induced upregulation of CD14, which is in line with previous data showing that Sp1 is critical for CD14 promoter activity. Induction of CD11b and/or CD14 by decitabine and/or VD3 was confirmed in primary AML patient samples at the time of diagnosis. In conclusion, decitabine synergizes with Vitamin D3 to induce CD11b and CD14 expression, likely by enhancing PU.1/c-jun and Sp1 transcriptional activity.
Assuntos
Azacitidina/análogos & derivados , Colecalciferol/biossíntese , Monócitos/citologia , Transcrição Gênica , Azacitidina/farmacocinética , Antígeno CD11b/biossíntese , Diferenciação Celular , Decitabina , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Modelos Biológicos , Monócitos/metabolismo , Plicamicina/farmacologia , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Células U937RESUMO
CCAAT/enhancer-binding protein alpha (C/EBPalpha) is one of the key transcription factors that mediate lineage specification and differentiation of multipotent myeloid progenitors into mature granulocytes. Although C/EBPalpha is known to induce granulopoiesis while suppressing monocyte differentiation, it is unclear how C/EBPalpha regulates this cell fate choice at the mechanistic level. Here we report that inducers of monocyte differentiation inhibit the alternate cell fate choice, that of granulopoiesis, through inhibition of C/EBPalpha. This inhibition is mediated by extracellular signal-regulated kinases 1 and/or 2 (ERK1/2), which interact with C/EBPalpha through an FXFP docking site and phosphorylate serine 21. As a consequence of C/EBPalpha phosphorylation, induction of granulocyte differentiation by C/EBPalpha or retinoic acid is inhibited. Our analysis of C/EBPalpha by fluorescent resonance energy transfer revealed that phosphorylation induces conformational changes in C/EBPalpha, increasing the distance between the amino termini of C/EBPalpha dimers. Thus, myeloid development is partly regulated by an ERK1/2-mediated change in the conformation of C/EBPalpha that favors monocyte differentiation by blocking granulopoiesis.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Leucopoese/fisiologia , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Granulócitos/efeitos dos fármacos , Humanos , Células K562 , Leucopoese/efeitos dos fármacos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Serina/química , Tretinoína/farmacologia , Células U937RESUMO
BACKGROUND: C/EBPalpha is a transcription factor essential for terminal differentiation of several cell types. It has not known if C/EBPalpha protein is expressed and functions in the prostate gland. METHODS: The presence of C/EBPalpha in normal and cancerous prostate epithelium was examined by immunochemistry. Over expression of C/EBPalpha in LNCaP cells was conducted with retrovirus-mediated transduction. PSA expression was examined by RT-PCR and western blot and PSA promoter activity by luciferase reporter assay. RESULTS: In normal prostate C/EBPalpha was expressed in the basal layer of the epithelium. In prostate cancer C/EBPalpha was detected at low levels throughout the cancers and in advanced prostate cancer C/EBPalpha expression was associated with decreased expression of AR and PSA. Overexpression of C/EBPalpha inhibited epigenetically PSA expression and was accompanied by the loss of expression of AR. Transient increase of C/EBPalpha inhibited the PSA promoter/enhancer activity independently of expression of AR. CONCLUSION: In LNCaP cells C/EBPalpha over expression inhibits expression of PSA by AR -dependent and independent mechanisms and by extinguishing AR expression provides a model for hormonal independent cell growth.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Antígeno Prostático Específico/genética , Receptores Androgênicos/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Masculino , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Transcrição Gênica/genéticaRESUMO
Multiple myeloma (MM) is a hematologic malignancy of plasma cells (PCs). In the United States, MM accounts for approximately 1% of all diagnoses and 2% of all cancer-related deaths. Although MM is a treatable disease, most patients eventually relapse, and despite the development of numerous treatment options it is still considered incurable. Mechanisms of communication between MM-PCs and bone marrow microenvironment, including cell-cell contacts and release of pro-survival factors, promote cancer cell survival and drug resistance. Recently, the importance of extracellular vesicles (EVs) as mechanisms of communication between MM cells and other cells in the microenvironment has been reported. In this review, the authors provide the update on the biology and clinical aspects of EVs in MM.
Assuntos
Biomarcadores Tumorais/metabolismo , Vesículas Extracelulares/patologia , Mieloma Múltiplo/patologia , Animais , Vesículas Extracelulares/metabolismo , Humanos , Mieloma Múltiplo/metabolismo , Microambiente TumoralRESUMO
Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorable prognosis in the near future.