RESUMO
The molecular processes governing hematopoiesis involve the interplay between lineage-specific transcription factors and a series of epigenetic tags, including DNA methylation and covalent histone tail modifications, such as acetylation, methylation, phosphorylation, SUMOylation and ubiquitylation. These post-translational modifications, which collectively constitute the 'histone code', are capable of affecting chromatin structure and gene transcription and are catalysed by opposing families of enzymes, allowing the developmental potential of hematopoietic stem cells to be dynamically regulated. The essential role of these enzymes in regulating normal blood development is highlighted by the finding that members from all families of chromatin regulators are targets for dysregulation in many hematological malignancies, and that patterns of histone modification are globally affected in cancer as well as the regulatory regions of specific oncogenes and tumor suppressors. The discovery that these epigenetic marks can be reversed by compounds targeting aberrant transcription factor/co-activator/co-repressor interactions and histone-modifying activities, provides the basis for an exciting field in which the epigenome of cancer cells may be manipulated with potential therapeutic benefits.
Assuntos
Transformação Celular Neoplásica/patologia , Epigênese Genética/fisiologia , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/patologia , Hematopoese/fisiologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Hematopoese/genética , HumanosRESUMO
Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Proteínas de Membrana/metabolismo , Neurônios/citologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Camundongos , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
The PLZF gene is one of five partners fused to the retinoic acid receptor alpha in acute promyelocytic leukemia. PLZF encodes a DNA-binding transcriptional repressor and the PLZF-RARalpha fusion protein like other RARalpha fusions can inhibit the genetic program mediated by the wild tpe retinoic acid receptor. However an increasing body of literature indicates an important role for the PLZF gene in growth control and development. This information suggests that loss of PLZF function might also contribute to leukemogenesis.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA/genética , Leucemia Promielocítica Aguda/etiologia , Leucemia Promielocítica Aguda/genética , Fatores de Transcrição/genética , Translocação Genética , Animais , Humanos , Fatores de Transcrição Kruppel-Like , Proteína com Dedos de Zinco da Leucemia Promielocítica , Dedos de ZincoRESUMO
To identify biologically functional regions in the product of the Drosophila melanogaster gene Kruppel, we cloned the Kruppel homolog from Drosophila virilis. Both the previously identified amino (N)-terminal repression region and the DNA-binding region of the D. virilis Kruppel protein are greater than 96% identical to those of the D. melanogaster Kruppel protein, demonstrating a selective pressure to maintain the integrity of each region during 60 million to 80 million years of evolution. An additional region in the carboxyl (C) terminus of Kruppel that was most highly conserved was examined further. A 42-amino-acid stretch within the conserved C-terminal region also encoded a transferable repression domain. The short, C-terminal repression region is a composite of three subregions of distinct amino acid composition, each containing a high proportion of either basic, proline, or acidic residues. Mutagenesis experiments demonstrated, unexpectedly, that the acidic residues contribute to repression function. Both the N-terminal and C-terminal repression regions were tested for the ability to affect transcription mediated by a variety of activator proteins. The N-terminal repression region was able to inhibit transcription in the presence of multiple activators. However, the C-terminal repression region inhibited transcription by only a subset of the activator proteins. The different activator specificities of the two regions suggest that they repress transcription by different mechanisms and may play distinct biological roles during Drosophila development.
Assuntos
Sequência Conservada/genética , Proteínas de Ligação a DNA/genética , Drosophila/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/genética , Transcrição Gênica , Sequência de Aminoácidos , Aminoácidos/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos/genética , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutação Puntual , Proteínas Recombinantes de Fusão , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The promyelocytic leukemia zinc finger (PLZF) gene, which is disrupted in therapy-resistant, t(11;17)(q23;q21)-associated acute promyelocytic leukemia (APL), is expressed in immature hematopoietic cells and is down-regulated during differentiation. To determine the role of PLZF in myeloid development, we engineered expression of PLZF in murine 32Dcl3 cells. Expression of PLZF had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle and an increased incidence of apoptosis. PLZF-expressing pools also secreted a growth-inhibitory factor, which could explain the severe growth suppression of PLZF-expressing pools that occurred despite the fact that only half of the cells expressed high levels of PLZF. PLZF overexpression inhibited myeloid differentiation of 32Dcl3 cells in response to granulocyte and granulocyte-macrophage colony-stimulating factors. Furthermore, cells that expressed PLZF appeared immature as demonstrated by morphology, increased expression of Sca-1, and decreased expression of Gr-1. These findings suggest that PLZF is an important regulator of cell growth, death, and differentiation. Disruption of PLZF function associated with t(11;17) may be a critical event leading to APL.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultivo Condicionados , Proteínas de Ligação a DNA/biossíntese , Fase G1 , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inibidores do Crescimento/biossíntese , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-3/farmacologia , Fatores de Transcrição Kruppel-Like , Camundongos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Recombinantes/biossíntese , Fase de Repouso do Ciclo Celular , Fatores de Transcrição/biossíntese , Transfecção , Dedos de ZincoRESUMO
The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Repressoras/química , Fatores de Transcrição/química , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Aminoácidos , Arginina/química , Linhagem Celular , Dicroísmo Circular , DNA Complementar/metabolismo , Dimerização , Escherichia coli/metabolismo , Imunofluorescência , Genes Reporter , Glutamina/química , Humanos , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Mutação Puntual , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Temperatura , Tripsina/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Drosophila/metabolismo , Mutação Puntual , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Drosophila/genética , Proteínas de Drosophila , Fatores de Transcrição Kruppel-Like , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Dobramento de Proteína , Estrutura Secundária de Proteína , Fatores de Transcrição/biossíntese , Fatores de Transcrição/químicaRESUMO
The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide/genética , Proteínas Proto-Oncogênicas , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Animais , Células COS , Humanos , Fatores de Transcrição Kruppel-Like , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteína 1 Parceira de Translocação de RUNX1 , Transfecção , Dedos de ZincoRESUMO
The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Genes do Tumor de Wilms , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Transcrição Gênica/genética , Células Tumorais Cultivadas , Proteínas WT1RESUMO
A binding site selection from a CpG island library for the promyelocytic leukemia zinc finger protein (PLZF) identified two high affinity PLZF binding sites. These sequences also bound RARalpha/PLZF, a fusion protein formed in chromosomal translocation t(11;17)(q23;q21) associated with acute promyelocytic leukemia. PLZF bound DNA as a slowly migrating complex with an estimated mol. wt of 600 kDa whose formation was dependent on the POZ/dimerization domain of PLZF. The PLZF-DNA complex was unable to form in the presence of cdc2 antibodies. A PLZF-cdc2 interaction was further demonstrated by co-immunoprecipitation and a biotin-streptavidin pull-down assay. PLZF is a phosphoprotein and immunoprecipi-tates with a cdc2-like kinase activity. The PLZF-DNA complex was abolished with the addition of a phosphatase. These studies suggest that the activity of PLZF, a regulator of the cell cycle, may be modulated by cell cycle proteins. RARalpha/PLZF did not complex with cdc2, this potentially contributing to its aberrant transcriptional properties and potential role in leukemo-genesis.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Humanos , Fatores de Transcrição Kruppel-Like , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Proteína com Dedos de Zinco da Leucemia Promielocítica , Translocação Genética , Células Tumorais CultivadasRESUMO
Epigenetics, the modification of chromatin without changing the DNA sequence itself, determines whether a gene is expressed, and how much of a gene is expressed. Methylation of lysine 27 on histone 3 (H3K27me), a modification usually associated with gene repression, has established roles in regulating the expression of genes involved in lineage commitment and differentiation. Not surprisingly, alterations in the homeostasis of this critical mark have emerged as a recurrent theme in the pathogenesis of many cancers. Perturbations in the distribution or levels of H3K27me occur due to deregulation at all levels of the process, either by mutation in the histone itself, or changes in the activity of the writers, erasers, or readers of this mark. Additionally, as no single histone mark alone determines the overall transcriptional readiness of a chromatin region, deregulation of other chromatin marks can also have dramatic consequences. Finally, the significance of mutations altering H3K27me is highlighted by the poor clinical outcome of patients whose tumors harbor such lesions. Current therapeutic approaches targeting aberrant H3K27 methylation remain to be proven useful in the clinic. Understanding the biological consequences and gene expression pathways affected by aberrant H3K27 methylation may lead to identification of new therapeutic targets and strategies.
Assuntos
Metilação de DNA , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Neoplasias/genética , Fatores de Transcrição/metabolismo , Animais , Histonas/genética , Humanos , LisinaRESUMO
MMSET/WHSC1 is a histone methyltransferase (HMT) overexpressed in t(4;14)+ multiple myeloma (MM) patients, believed to be the driving factor in the pathogenesis of this MM subtype. MMSET overexpression in MM leads to an increase in histone 3 lysine 36 dimethylation (H3K36me2), and a decrease in histone 3 lysine 27 trimethylation (H3K27me3), as well as changes in proliferation, gene expression and chromatin accessibility. Prior work linked methylation of histones to the ability of cells to undergo DNA damage repair. In addition, t(4;14)+ patients frequently relapse after regimens that include DNA damage-inducing agents, suggesting that MMSET may play a role in DNA damage repair and response. In U2OS cells, we found that MMSET is required for efficient non-homologous end joining as well as homologous recombination. Loss of MMSET led to loss of expression of several DNA repair proteins, as well as decreased recruitment of DNA repair proteins to sites of DNA double-strand breaks (DSBs). By using genetically matched MM cell lines that had either high (pathological) or low (physiological) expression of MMSET, we found that MMSET-high cells had increased damage at baseline. Upon addition of a DNA-damaging agent, MMSET-high cells repaired DNA damage at an enhanced rate and continued to proliferate, whereas MMSET-low cells accumulated DNA damage and entered cell cycle arrest. In a murine xenograft model using t(4;14)+ KMS11 MM cells harboring an inducible MMSET shRNA, depletion of MMSET enhanced the efficacy of chemotherapy, inhibiting tumor growth and extending survival. These findings help explain the poorer prognosis of t(4;14) MM and further validate MMSET as a potential therapeutic target in MM and other cancers.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute promyelocytic leukemia (APL) has been recognized as a distinct clinical entity for over 40 years. Although relatively rare among hematopoietic malignancies (approximately 10% of AML cases), this disease has attracted a particularly good share of attention by becoming the first human cancer in which all-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, was able to induce complete remission (CR). ATRA induced remission is not associated with rapid cell death, as in the case of conventional chemotherapy, but with a restoration of the 'normal' granulocytic differentiation pathway. With this remarkable medical success story APL has overnight become a paradigm for the differentiation therapy of cancer. A few years later, excitement with APL was further enhanced by the discovery that a cytogenetic marker for this disease, the t(15:17) reciprocal chromosomal translocation, involves a fusion between the retinoic acid receptor alpha (RARalpha) gene and a previously unknown locus named promyelocytic leukemia (PML). Consequence of this gene rearrangement is expression of the PML-RARalpha chimeric oncoprotein, which is responsible for the cellular transformation as well as ATRA response that is observed in APL. Since this initial discovery, a number of different translocation partner genes of RARalpha have been reported in rarer cases of APL, strongly suggesting that disruption of RARalpha underlies its pathogenesis. This article reviews various rearrangements of the RARalpha gene that have so far been described in literature, functions of the proteins encoded by the different RARalpha partner loci, and implications that these may have for the molecular pathogenesis of APL.
Assuntos
Leucemia Promielocítica Aguda/genética , Proteínas do Leite , Receptores do Ácido Retinoico/genética , Translocação Genética/genética , Antígenos Nucleares , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Proteínas de Neoplasias/genética , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/genética , Nucleofosmina , Proteínas de Fusão Oncogênica/genética , Receptor alfa de Ácido Retinoico , Fator de Transcrição STAT5 , Transativadores/genética , TretinoínaRESUMO
Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARalpha. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRalpha), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARalpha to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT-PCR, cloning, and sequence studies identified NPM as the RARalpha partner on chromosome 5, and both NPM-RARalpha and RARalpha-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RARalpha chimeric protein on the subcellular localization of PML, RXRalpha, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10-20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRalpha, and NPM-RARalpha in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARalpha partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Promielocítica Aguda/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Translocação Genética , Nucléolo Celular , Criança , Células HL-60 , Humanos , Fatores de Transcrição Kruppel-Like , Masculino , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Fatores de Transcrição/análise , Células U937 , Regulação para CimaRESUMO
Wilms' tumor belongs to a small group of pediatric neoplasms that have served as paradigms of human cancers in which recessive mutations play a primary role in tumorigenesis. WT1 is a candidate tumor suppressor gene that is mutationally inactivated in a proportion of both familial and sporadic Wilms' tumors. Recent studies demonstrated that WT1 can partially suppress growth of a Wilms' tumor cell line in vitro and in vivo. We investigated the ability of WT1 to inhibit the expression of the transformed phenotype in non-Wilms' tumor cells. The expression of WT1 cDNA in ras-transformed NIH3T3 cells yielded large, flat cells that exhibited complete contact-inhibition. These morphologic changes were associated with decreased proliferation, suppression of clonogenicity in soft agar and inhibition of tumor growth in nude mice. Moreover, expression of WT1 in non-transformed NIH3T3 cells resulted in similar morphologic changes and profound resistance to transformation by an activated ras oncogene. These studies suggest that tumor inhibition by WT1 in these cells may be achieved by interference with the ras-mediated signalling pathway.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Genes ras , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Células 3T3 , Animais , Divisão Celular , DNA Complementar , Expressão Gênica , Camundongos , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Mapeamento por Restrição , Transdução de Sinais , Proteínas WT1RESUMO
Acute promyelocytic leukemia (APL) associated with chromosomal rearrangement t(11;17) is a distinct syndrome which, unlike typical t(15;17) APL, fails to respond to all-trans retinoic acid (ATRA) therapy. In t(11;17) the PLZF gene, encoding a Krüppel-like zinc finger protein, is fused to the retinoic acid receptor-alpha (RAR alpha) gene, yielding two classes of chimeric proteins. PLZF protein was found in the nucleus in a punctate speckled pattern that differed from the nuclear body expression pattern of the PML protein affected in t(15;17) APL. The reciprocal PLZF-RAR alpha and RAR alpha-PLZF fusion proteins were localized to the nucleus both in the presence and absence of ATRA. PLZF-RAR alpha, in combination with the retinoid X receptor (RXR) bound to a retinoic acid-responsive element (RARE) less efficiently than RAR alpha and formed multimeric DNA-protein complexes. PLZF-RAR alpha stimulated ATRA-dependent transcription of RARE-containing reporter genes with diminished activity compared to wild-type RAR alpha. In addition, PLZF-RAR alpha antagonized the function of coexpressed wild-type RAR alpha, an effect relieved by over-expression of RXR. Leukemogenesis in t(11;17) APL may be related to interference with ATRA-mediated differentiation due to sequestration of RXR by the PLZF-RAR alpha chimera. However, disruption of the function of the myeloid-specific PLZF protein may also play an important role.
Assuntos
Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Leucemia Promielocítica Aguda/genética , Receptores do Ácido Retinoico/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Fatores de Transcrição/fisiologia , Translocação Genética , Sequência de Bases , Proteínas de Ligação a DNA/análise , Rearranjo Gênico , Humanos , Fatores de Transcrição Kruppel-Like , Leucemia Promielocítica Aguda/etiologia , Dados de Sequência Molecular , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores do Ácido Retinoico/análise , Proteínas Recombinantes de Fusão/análise , Fatores de Transcrição/análise , Transcrição GênicaRESUMO
The PLZF gene was identified by its fusion with the RARalpha locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARalpha-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARalpha-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.
Assuntos
Ciclo Celular/genética , Ciclina A/metabolismo , Proteínas de Ligação a DNA/fisiologia , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/genética , Vetores Genéticos , Humanos , Interfase/genética , Fatores de Transcrição Kruppel-Like , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Camundongos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Fase S/genética , Fatores de Transcrição/genética , Translocação Genética , Dedos de Zinco/fisiologiaRESUMO
Primary blasts of a t(11;17)(q23;q21) acute promyelocytic leukaemia (APL) patient were analysed with respect to retinoic acid (RA) and arsenic trioxide (As2O3) sensitivity as well as PLZF/RARalpha status. Although RA induced partial monocytic differentiation ex vivo, but not in vivo, As203 failed to induce apoptosis in culture, contrasting with t(15;17) APL and arguing against the clinical use of As203 in t(11;17)(q23;q21) APL. Prior to cell culture, PLZF/RARalpha was found to exactly co-localize with PML onto PML nuclear bodies. However upon cell culture, it quickly shifted towards microspeckles, its localization found in transfection experiments. Arsenic trioxide, known to induce aggregation of PML nuclear bodies, left the microspeckled PLZF/RARalpha localization completely unaffected. RA treatment led to PLZF/RARalpha degradation. However, this complete PLZF/RARalpha degradation was not accompanied by differentiation or apoptosis, which could suggest a contribution of the reciprocal RARalpha/PLZF fusion product in leukaemogenesis or the existence of irreversible changes induced by the chimera.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/efeitos dos fármacos , Óxidos/farmacologia , Receptores do Ácido Retinoico/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Tretinoína/farmacologia , Apoptose , Trióxido de Arsênio , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Imunofluorescência , Humanos , Fatores de Transcrição Kruppel-Like , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Proteínas de Fusão Oncogênica/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/metabolismo , Translocação Genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The WT1 tumor suppressor gene, implicated in hereditofamilial and sporadic Wilms' tumor, is required for normal renal development and is up-regulated during the mesenchymal-epithelial transition. NIH3T3 fibroblasts overexpressing WT1 were less proliferative, larger in size and more firmly attached to tissue culture plastic, suggesting an alteration of their state of differentiation. These cells were studied in vivo by subcutaneous injection into nude mice. The resulting tumors exhibited epithelioid histopathology and formed desmosome-like structures. Molecular analyses of these WT1 expressing fibroblasts grown in culture and in nude mice revealed significant alterations in the expression of many kidney epithelial markers. These studies indicate that WT1 expression can initiate features of a program of epithelial differentiation consistent with a prominent role for WT1 in the mesenchymal epithelial transition that occurs during renal development. Through this work we identified a number of novel target genes for the WT1 transcription factor, including uvomorulin, integrin alpha8 and perlecan, and suggest that WTI may activate the IGF-II gene, also implicated in the development of Wilms' tumor.