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1.
Mol Cell ; 42(1): 62-74, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21474068

RESUMO

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Proteínas de Transporte/genética , Caseína Quinase II/metabolismo , Linhagem Celular , Proteínas Correpressoras , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Modelos Moleculares , Chaperonas Moleculares , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína SUMO-1/metabolismo , Estresse Fisiológico
2.
EMBO J ; 31(6): 1427-39, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22274616

RESUMO

Studies have suggested that the clock regulator PER2 is a tumour suppressor. A cancer network involving PER2 raises the possibility that some tumour suppressors are directly involved in the mammalian clock. Here, we show that the tumour suppressor promyelocytic leukaemia (PML) protein is a circadian clock regulator and can physically interact with PER2. In the suprachiasmatic nucleus (SCN), PML expression and PML-PER2 interaction are under clock control. Loss of PML disrupts and dampens the expression of clock regulators Per2, Per1, Cry1, Bmal1 and Npas2. In the presence of PML and PER2, BMAL1/CLOCK-mediated transcription is enhanced. In Pml(-/-) SCN and mouse embryo fibroblast cells, the cellular distribution of PER2 is primarily perinuclear/cytoplasmic. PML is acetylated at K487 and its deacetylation by SIRT1 promotes PML control of PER2 nuclear localization. The circadian period of Pml(-/-) mice displays reduced precision and stability consistent with PML having a role in the mammalian clock mechanism.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Acetilação , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Relógios Circadianos/fisiologia , Citoplasma/metabolismo , Fibroblastos/metabolismo , Masculino , Camundongos , Proteína da Leucemia Promielocítica , Sirtuína 1/metabolismo , Transcrição Gênica/genética
3.
Br J Haematol ; 169(3): 344-51, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25612675

RESUMO

Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re-expression. Furthermore, relapse-free survival of non-inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias.


Assuntos
Inversão Cromossômica , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Metilação de DNA , Leucemia Mieloide Aguda/genética , Regiões Promotoras Genéticas , Adulto , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Decitabina , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Pessoa de Meia-Idade , Ativação Transcricional/efeitos dos fármacos
4.
Mol Cell Biol ; 23(12): 4247-56, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12773567

RESUMO

The PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. Promyelocytic leukemia protein (PML) is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation, and genome stability. Our study here shows that PML colocalizes and associates in vivo with the DNA damage response protein TopBP1 in response to ionizing radiation (IR). Both PML and TopBP1 colocalized with the IR-induced bromodeoxyuridine single-stranded DNA foci. PML and TopBP1 also colocalized with Rad50, Brca1, ATM, Rad9, and BLM. IR and interferon (IFN) coinduce the expression levels of both TopBP1 and PML. In PML-deficient NB4 cells, TopBP1 was unable to form IR-induced foci. All-trans-retinoic acid induced reorganization of the PML nuclear body (NB) and reappearance of the IR-induced TopBP1 foci. Inhibition of PML expression by siRNA is associated with a significant decreased in TopBP1 expression. Furthermore, PML-deficient cells express a low level of TopBP1, and its expression cannot be induced by IR or IFN. Adenovirus-mediated overexpression of PML in PML(-/-) mouse embryo fibroblasts substantially increased TopBP1 expression, which colocalized with the PML NBs. These studies demonstrated a mechanism of PML-dependent expression of TopBP1. PML overexpression induced TopBP1 protein but not the mRNA expression. Pulse-chase labeling analysis demonstrated that PML overexpression stabilized the TopBP1 protein, suggesting that PML plays a role in regulating the stability of TopBP1 in response to IR. Together, our findings demonstrate that PML regulates TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage.


Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Motivos de Aminoácidos , Northern Blotting , Western Blotting , Bromodesoxiuridina/farmacologia , Reparo do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA , Humanos , Interferons/farmacologia , Microscopia de Fluorescência , Plasmídeos/metabolismo , Testes de Precipitina , Proteína da Leucemia Promielocítica , Ligação Proteica , Radiação Ionizante , Fatores de Tempo , Tretinoína/farmacologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor
5.
Cancer Res ; 65(4): 1277-84, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15735013

RESUMO

The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/fisiologia , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Doença Aguda , Linhagem Celular , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica , Humanos , Interleucina-3/genética , Leucemia Mieloide/enzimologia , Leucemia Mieloide/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Transfecção
6.
Oncogene ; 21(24): 3925-33, 2002 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-12032831

RESUMO

The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled-coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267-332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Transcrição Gênica , Apoptose , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Microscopia de Fluorescência , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Plasmídeos/metabolismo , Testes de Precipitina , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor
7.
PLoS One ; 9(4): e94450, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24728382

RESUMO

Promyelocytic leukemia nuclear bodies (PML NBs) are comprised of PML and a striking variety of its associated proteins. Various cellular functions have been attributed to PML NBs, including the regulation of gene expression. We report here that induced expression of PML recruits Sp1 into PML NBs, leading to the reduction of Sp1 transactivation function. Specifically, Chromatin immunoprecipitation (ChIP) assay demonstrated that induced expression of PML significantly diminishes the amount of Sp1 binding to its target gene promoter, immunofluorescence staining showed dramatic increase in the co-localization between PML and Sp1 upon induction of PML expression, moreover, PML and Sp1 co-fractionated in the core nuclear matrix. Our study further showed that PML promotes SUMOylation of Sp1 in a RING-motif-dependent manner, SUMOylation of Sp1 facilitates physical interaction between Sp1 and PML and recruitment of Sp1 into the PML NBs, the SUMO binding motif of PML was also important for its interaction with Sp1. The results of this study demonstrate a novel mechanism by which PML regulates gene expression through sequestration of the transcription factor into PML NBs.


Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Fator de Transcrição Sp1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Dados de Sequência Molecular , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Transcrição Sp1/química , Sumoilação , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo
8.
Cancer ; 116(2): 369-76, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19950129

RESUMO

BACKGROUND: The authors evaluated the utility of immunofluorescence staining with an antipromyelocytic leukemia (anti-PML) antibody for patients with a suspected diagnosis of new or relapsed acute promyelocytic leukemia (APL) and correlated the findings with the results of other established diagnostic modalities. METHODS: Bone marrow (BM) and/or peripheral blood (PB) smears from 349 patients in whom the diagnosis of APL was considered were assessed with the anti-PML antibody using immunofluorescence. The study group included 199 patients with confirmed APL and 150 with other conditions. The results of conventional cytogenetics, reverse transcription polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) performed on these patients were correlated with the PML results. RESULTS: Among patients with confirmed APL, anti-PML antibody was positive in 182 of 184 BM and 32 of 33 PB smears. Conventional cytogenetics demonstrated t(15;17)(q22;q12) in 166 of 182 (91%) patients; 10 had a normal karyotype, 4 had insufficient mitoses to grow in culture, 1 was inconclusive, and 1 was 48, XX, +8, +8. Anti-PML staining was positive in 9 of 10 with a normal karyotype and in all 4 cases with insufficient mitoses. RT-PCR and FISH were positive for PML-retinoic acid receptor-alpha in 169 of 172 (98%) and 90 of 94 (96%) cases, respectively. Among the patients without APL, 148 of 150 (98.6%) were negative with anti-PML antibody. The sensitivity and specificity of the test were 98.9% and 98.7%, respectively. CONCLUSIONS: PML immunofluorescence staining is a rapid (<4 hours turnaround time) and reliable frontline diagnostic approach that can facilitate initiation of targeted therapy, particularly in clinical settings where cytogenetic and molecular testing are not readily available.


Assuntos
Anticorpos Antineoplásicos , Imunofluorescência/métodos , Leucemia Promielocítica Aguda/diagnóstico , Proteínas Nucleares , Fatores de Transcrição , Proteínas Supressoras de Tumor , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Cariotipagem , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Proteína da Leucemia Promielocítica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
9.
Mol Biol Cell ; 19(7): 3020-7, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18463162

RESUMO

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-alpha treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-alpha promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-alpha-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.


Assuntos
Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Animais , Apoptose , Células HeLa , Humanos , Fatores de Transcrição MEF2 , Metaloproteinase 10 da Matriz/metabolismo , Camundongos , Microscopia Confocal , Fatores de Regulação Miogênica/metabolismo , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Mol Cell Biol ; 28(3): 997-1006, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18039859

RESUMO

Promyelocytic leukemia protein (PML) is an important regulator due to its role in numerous cellular processes including apoptosis, viral infection, senescence, DNA damage repair, and cell cycle regulation. Despite the role of PML in many cellular functions, little is known about the regulation of PML itself. We show that PML stability is regulated through interaction with the peptidyl-prolyl cis-trans isomerase Pin1. This interaction is mediated through four serine-proline motifs in the C terminus of PML. Binding to Pin1 results in degradation of PML in a phosphorylation-dependent manner. Furthermore, our data indicate that sumoylation of PML blocks the interaction, thus preventing degradation of PML by this pathway. Functionally, we show that in the MDA-MB-231 breast cancer cell line modulating levels of Pin1 affects steady-state levels of PML. Furthermore, degradation of PML due to Pin1 acts both to protect these cells from hydrogen peroxide-induced death and to increase the rate of proliferation. Taken together, our work defines a novel mechanism by which sumoylation of PML prevents Pin1-dependent degradation. This interaction likely occurs in numerous cell lines and may be a pathway for oncogenic transformation.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Peptidilprolil Isomerase/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Neoplasias da Mama/etiologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Fenótipo , Proteína da Leucemia Promielocítica , Ligação Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
11.
Mol Cell ; 24(3): 341-54, 2006 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-17081986

RESUMO

Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Trióxido de Arsênio , Arsenicais/farmacologia , Células COS , Proteínas de Transporte/química , Chlorocebus aethiops , Proteínas Correpressoras , Dexametasona/farmacologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Chaperonas Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Óxidos/farmacologia , Proteína da Leucemia Promielocítica , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo
12.
Mol Cell ; 17(5): 721-32, 2005 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-15749021

RESUMO

The promyelocytic leukemia gene (PML), which is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL), encodes a multifunctional protein involved in several important cellular functions. Herein, we demonstrate that PML is localized to centrosomes and that PML deficiency leads to centrosome amplification. By using PML isoform-specific antibodies, we found PML3-specific association with the centrosome and the pole of the mitotic spindle. PML3 deficiency leads to dysregulation of the centrosome duplication checkpoint. Furthermore, PML3 physically interacts with Aurora A and regulates its kinase activity. Specific knockdown of PML3 activates Cdk2/cyclin kinase activity. The results of this study implicate a direct role for PML3 in the control of centrosome duplication through suppression of Aurora A activation to prevent centrosome reduplication.


Assuntos
Genoma , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Aurora Quinases , Medula Óssea/metabolismo , Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Quinase 2 Dependente de Ciclina , Citoplasma/metabolismo , Humanos , Imunoprecipitação , Mitose , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plasmídeos/metabolismo , Proteína da Leucemia Promielocítica , Isoformas de Proteínas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/metabolismo , Fuso Acromático , Frações Subcelulares/metabolismo , Fatores de Tempo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Células U937 , Proteínas de Xenopus
13.
J Biol Chem ; 277(35): 31734-9, 2002 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-12080044

RESUMO

The promyelocytic leukemia (PML) protein is a tumor suppressor that is disrupted by the chromosomal translocation t(15;17), a consistent cytogenetic feature of acute promyelocytic leukemia. A role of PML in multiple pathways of apoptosis was conclusively demonstrated using PML(-/-) animal and cell culture models. In a previous study, we found that PML sensitizes tumor necrosis factor-induced apoptosis in tumor necrosis factor (TNF)-resistant U2OS cells. This finding helped to explain the mechanism of PML-induced apoptosis. The zinc finger protein A20 is a target gene of NF kappa B inducible by TNF alpha, and it is a potent inhibitor of TNF-induced apoptosis. In the this study, we demonstrated that PML is a transcriptional repressor of the A20 promoter and that PML represses A20 expression induced by TNF alpha. We showed that PML inhibits A20 transactivation through the NF kappa B site by interfering with its binding to the promoter. We also showed that stable overexpression of A20 inhibits apoptosis and caspase activation induced by PML/TNF alpha. The results of this study suggest that A20 is a downstream target of PML-induced apoptosis and supports a role of A20 in modulating cell death induced by PML/TNF alpha in TNF-resistant cells.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas/genética , Fatores de Transcrição/genética , Apoptose , Sequência de Bases , Cádmio/farmacologia , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Promielocítica Aguda , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteína da Leucemia Promielocítica , Transcrição Gênica , Ativação Transcricional , Translocação Genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Supressoras de Tumor , Dedos de Zinco
14.
J Biol Chem ; 278(14): 12294-304, 2003 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-12540841

RESUMO

The promyelocytic leukemia protein (PML) is a growth/tumor suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death by apoptosis in the tumor necrosis factor alpha (TNFalpha)-resistant cell line U2OS and other cell lines. Treatment with TNFalpha significantly sensitized these cells to apoptosis in a p53-independent manner. PML/TNFalpha-induced cell death is associated with DNA fragmentation, activation of caspase-3, -7, and -8, and degradation of DNA fragmentation factor/inhibitor of CAD. PML/TNFalpha-induced cell death could be blocked by the caspase-8 inhibitors CrmA and c-FLIP but not by Bcl-2. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. PML is a transcriptional repressor of NF-kappaB by interacting with RelA/p65 and prevents its binding to the cognate enhancer through the C terminus. Coimmunoprecipitation and double-color immunofluorescence staining demonstrated that PML physically interacts with RelA/p65 in vivo and the two proteins colocalized at the endogenous levels. Overexpression of NF-kappaB rescued cell death induced by PML/TNFalpha. Furthermore, PML(-/-) mouse embryo fibroblasts are more resistant to TNFalpha-induced apoptosis. Together this study defines a novel mechanism by which PML induces apoptosis through repression of the NF-kappaB survival pathway.


Assuntos
Apoptose/fisiologia , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Caspases/metabolismo , Desoxirribonucleases/metabolismo , Expressão Gênica , Humanos , Camundongos , NF-kappa B/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Osteossarcoma , Proteína da Leucemia Promielocítica , Estrutura Terciária de Proteína , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Transcrição RelA , Fatores de Transcrição/química , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor
15.
J Biol Chem ; 279(3): 1838-44, 2004 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-14597622

RESUMO

The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFkappaB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML-/- mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.


Assuntos
Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Ciclo Celular , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/fisiologia , Regiões Promotoras Genéticas , Proteína da Leucemia Promielocítica , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Survivina , Ativação Transcricional , Proteínas Supressoras de Tumor
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