RESUMO
The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBPα in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBPα mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBPα-ER chimeric proteins. We show here that S21D C/EBPα-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBPα-ER, while S21A C/EBPα-ER was less efficient. Furthermore, wild-type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBPα in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBPα in K562 cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Benzamidas/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Primers do DNA/genética , Fatores de Transcrição E2F/metabolismo , Granulócitos/metabolismo , Granulócitos/patologia , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Fosforilação , Regiões Promotoras Genéticas , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Ensaio Tumoral de Célula-TroncoRESUMO
MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.
Assuntos
Diferenciação Celular/genética , Inibidores do Crescimento/análise , MicroRNAs/análise , Regulação para Cima , Apoptose , Butiratos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inibidores do Crescimento/farmacologia , Humanos , Cloreto de Lítio/farmacologia , MicroRNAs/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis, and in neuroblastoma cells, expression of Slug is transcriptionally regulated by c-Myb via Myb binding sites in the 5'-flanking region and in the first intron of the slug gene. In embryonic kidney and neuroblastoma cells, c-Myb induced vimentin, fibronectin, and N-cadherin expression and membrane ruffling via actin polymerization consistent with the acquisition of a mesenchymal-like phenotype. Furthermore, down-regulation of endogenous c-Myb levels in colon carcinoma cells led to increased expression of E-cadherin and reduced levels of vimentin. Some of these changes are predominantly Slug-dependent as Slug silencing via RNA interference (RNAi) reverts the cells to a quasi-parental condition. Changes in gene expression and morphology induced by c-Myb-activated Slug correlated with increased ability to migrate (embryonic kidney) and to invade through a Matrigel membrane (embryonic kidney, colon carcinoma, neuroblastoma). c-Myb-dependent Slug expression was also essential for the homing of chronic myeloid leukemia K562 cells to the bone marrow. In summary, we show here that the proto-oncogene c-Myb controls Slug transcription in tumor cells of different origin. Such a regulatory pathway contributes to the acquisition of invasive properties that are important for the metastatic process.
Assuntos
Medula Óssea/patologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Íntrons/genética , Camundongos , Camundongos SCID , Metástase Neoplásica/genética , Metástase Neoplásica/fisiopatologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
The transcription factor C/EBPα is more potent than C/EBPß in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPß proteins. Wild-type and N-C/EBPα+ C/EBPß-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPß and N-C/EBPß+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPß-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPß inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPß-DBD, whereas C/EBPß induced a pattern similar to that of N-C/EBPß+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Apoptose/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Células K562 , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/farmacologia , Proteínas de Fusão bcr-abl/biossíntese , Fator de Transcrição GATA2/genética , Genes myb/efeitos dos fármacos , Sequência de Bases , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Primers do DNA/genética , Proteínas de Fusão bcr-abl/genética , Genes abl , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , RNA Interferente Pequeno/genética , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.
Assuntos
Neoplasias da Mama/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , Fator 1 de Elongação de Peptídeos/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNARESUMO
PURPOSE: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53. EXPERIMENTAL DESIGN: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fisher's exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI). RESULTS: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fisher's Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively). CONCLUSIONS: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.
Assuntos
Predisposição Genética para Doença , Neuroblastoma/genética , Neuroblastoma/patologia , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/genética , Criança , Pré-Escolar , Humanos , Lactente , Estimativa de Kaplan-Meier , Neuroblastoma/mortalidade , Projetos Piloto , Reação em Cadeia da PolimeraseRESUMO
We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.
Assuntos
Apoptose/efeitos dos fármacos , DNA Topoisomerase IV/administração & dosagem , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Amantadina , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Expressão Gênica , Células HL-60 , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Células K562 , Leucemia Mieloide Aguda/patologia , Macrófagos/fisiologia , Piperidinas/farmacologia , Mapas de Interação de Proteínas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Tamoxifeno/farmacologia , Tretinoína/farmacologiaRESUMO
Most triple-negative breast cancers (TNBCs) exhibit gene expression patterns associated with epithelial-to-mesenchymal transition (EMT), a feature that correlates with a propensity for metastatic spread. Overexpression of the EMT regulator Slug is detected in basal and mesenchymal-type TNBCs and is associated with reduced E-cadherin expression and aggressive disease. The effects of Slug depend, in part, on the interaction of its N-terminal SNAG repressor domain with the chromatin-modifying protein lysine demethylase 1 (LSD1); thus, we investigated whether tranylcypromine [also known as trans-2-phenylcyclopropylamine hydrochloride (PCPA) or Parnate], an inhibitor of LSD1 that blocks its interaction with Slug, suppresses the migration, invasion, and metastatic spread of TNBC cell lines. We show here that PCPA treatment induces the expression of E-cadherin and other epithelial markers and markedly suppresses migration and invasion of TNBC cell lines MDA-MB-231 and BT-549. These effects were phenocopied by Slug or LSD1 silencing. In two models of orthotopic breast cancer, PCPA treatment reduced local tumor growth and the number of lung metastases. In mice injected directly in the blood circulation with MDA-MB-231 cells, PCPA treatment or Slug silencing markedly inhibited bone metastases but had no effect on lung infiltration. Thus, blocking Slug activity may suppress the metastatic spread of TNBC and, perhaps, specifically inhibit homing/colonization to the bone.
Assuntos
Inativação Gênica/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Tranilcipromina/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Histona Desmetilases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Fluorescência , Inibidores da Monoaminoxidase/farmacologia , Invasividade Neoplásica , Metástase Neoplásica/prevenção & controle , Plasmídeos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família Snail , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
The process of epithelial-mesenchymal transition (EMT) which is required for cancer cell invasion is regulated by a family of E-box-binding transcription repressors, which include Snail (SNAIL1) and Slug (SNAI2). Snail appears to repress the expression of the EMT marker E-cadherin by epigenetic mechanisms dependent on the interaction of its N-terminal SNAG domain with chromatin-modifying proteins including lysine-specific demethylase 1 (LSD1/KDM1A). We assessed whether blocking Snail/Slug-LSD1 interaction by treatment with Parnate, an enzymatic inhibitor of LSD1, or TAT-SNAG, a cell-permeable peptide corresponding to the SNAG domain of Slug, suppresses the motility and invasiveness of cancer cells of different origin and genetic background. We show here that either treatment blocked Slug-dependent repression of the E-cadherin promoter and inhibited the motility and invasion of tumor cell lines without any effect on their proliferation. These effects correlated with induction of epithelial and repression of mesenchymal markers and were phenocopied by LSD1 or Slug downregulation. Parnate treatment also inhibited bone marrow homing/engraftment of Slug-expressing K562 cells. Together, these studies support the concept that targeting Snail/Slug-dependent transcription repression complexes may lead to the development of novel drugs selectively inhibiting the invasive potential of cancer cells.
Assuntos
Histona Desmetilases/metabolismo , Invasividade Neoplásica , Fatores de Transcrição/metabolismo , Animais , Caderinas/metabolismo , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Humanos , Imunoprecipitação , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Tranilcipromina/farmacologiaRESUMO
The p53 gene is rarely mutated in neuroblastoma, but codon 72 polymorphism that modulates its proapoptotic activity might influence cancer risk and clinical outcome. We investigated whether this polymorphism affects neuroblastoma risk and disease outcome and assessed the biologic effects of the p53-72R and p53-72P isoforms in p53-null cells. Comparison of 288 healthy subjects and 286 neuroblastoma patients revealed that the p53-72 polymorphism had no significant impact on the risk of developing neuroblastoma; however, patients with the Pro/Pro genotype had a shorter survival than those with the Arg/Arg or the Arg/Pro genotypes even in the stage 3 and 4 subgroup without MYCN amplification. By Cox regression analysis, the p53 Pro/Pro genotype seems to be an independent marker of poor prognosis (hazard ratio = 2.74; 95% confidence interval = 1.14-6.55, P = .014) together with clinical stage, MYCN status, and age at diagnosis. In vitro, p53-72P was less effective than p53-72R in inducing apoptosis and inhibiting survival of p53-null LAN-1 cells treated with etoposide, topotecan, or ionizing radiation but not taxol. By contrast, p53-72P was more effective in promoting p21-dependent accelerated senescence, alone or in the presence of etoposide. Thus, the p53-72 Pro/Pro genotype might be a marker of poor outcome independent of MYCN amplification, possibly improving risk stratification. Moreover, the lower apoptosis and the enhanced accelerated senescence by the p53-72P isoform in response to DNA damage suggest that patients with neuroblastoma with the p53-72 Pro/Pro genotype may benefit from therapeutic protocols that do not rely only on cytotoxic drugs that function, in part, through p53 activation.
Assuntos
Códon , Genótipo , Neuroblastoma/genética , Proteína Supressora de Tumor p53/genética , Adulto , Envelhecimento/genética , Apoptose/genética , Linhagem Celular Tumoral , Pré-Escolar , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Polimorfismo de Nucleotídeo Único , Prognóstico , Isoformas de Proteínas/genética , Interferência de RNA , Proteína Supressora de Tumor p53/metabolismoRESUMO
Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Fusão bcr-abl/genética , Fatores de Transcrição/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Divisão Celular , Ensaio de Unidades Formadoras de Colônias , Primers do DNA , Regulação para Baixo , Amplificação de Genes , Regulação da Expressão Gênica , Genes Reporter , Humanos , Sequências Repetidas Invertidas/genética , Células K562 , Luciferases/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.
Assuntos
Diferenciação Celular , Quinase 3 da Glicogênio Sintase/farmacologia , Células Mieloides/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/antagonistas & inibidores , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Linhagem Celular , Regulação para Baixo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Inibidores de Proteases/farmacologia , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Frações SubcelularesRESUMO
C/EBPalpha and C/EBPbeta, two transcription factors of the C/EBP family play important roles in the proliferation and differentiation of various cell types including myeloid progenitors. Expression of C/EBPalpha and C/EBPbeta is repressed in myeloid blast crisis of Chronic Myelogenous Leukemia by mechanisms that involve translation repression which depends on the interaction of RNA-binding proteins with conserved binding sites in the 5'UTR of c/ebpalpha and c/ebpbeta mRNA. Ectopic expression of C/EBPalpha and C/EBPbeta in myeloid progenitors expressing the BCR/ABL oncogene inhibits proliferation, induces differentiation and suppresses leukemogenesis in mice, but C/EBPalpha is markedly more effective than C/EBPbeta. The more potent effects of C/EBPalpha probably depends on protein-protein interaction with cell-cycle regulatory proteins, but the pattern of genes modulated by C/EBPalpha and C/EBPbeta is not completely overlapping. This suggests that transcription-dependent and -independent effects are both involved and support the therapeutic potential of reactivating C/EBPalpha and C/EBPbeta expression in leukemic cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana/metabolismoRESUMO
Chronic phase-to-blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPalpha, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region-Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPalpha activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPalpha eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPalpha, which were more potent than those of K298E C/EBPalpha, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPalpha in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPalpha has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPalpha activation.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/biossíntese , Diferenciação Celular/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Crise Blástica/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Granulócitos/metabolismo , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Mutação , Piperazinas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores de Proteínas Quinases/metabolismo , Pirimidinas/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Translational regulation by oncogenic proteins may be a rapid and efficient mechanism to modulate gene expression. We report here the identification of the CEBPB gene as a target of translational regulation in myeloid precursor cells transformed by the BCR/ABL oncogene. Expression of CEBPB was repressed in 32D-BCR/ABL cells and reinduced by imatinib (STI571) via a mechanism that appears to depend on expression of the CUG-repeat RNA-binding protein CUGBP1 and the integrity of the CUG-rich intercistronic region of c/ebpbeta mRNA. Constitutive expression or conditional activation of wild-type CEBPB induced differentiation and inhibited proliferation of 32D-BCR/ABL cells in vitro and in mice, but a DNA binding-deficient CEBPB mutant had no effect. The proliferation-inhibitory effect of CEBPB was, in part, mediated by the CEBPB-induced GADD45A gene. Because expression of CEBPB (and CEBPA) is low in the blast crisis (BC) stage of chronic myelogenous leukemia (CML) and is inversely correlated with BCR/ABL tyrosine kinase levels, these findings point to the therapeutic potential of restoring C/EBP activity in CML-BC and, perhaps, other types of acute leukemia.