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1.
BMC Med Inform Decis Mak ; 24(1): 134, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38789985

RESUMO

BACKGROUND: There are approximately 8,000 different rare diseases that affect roughly 400 million people worldwide. Many of them suffer from delayed diagnosis. Ciliopathies are rare monogenic disorders characterized by a significant phenotypic and genetic heterogeneity that raises an important challenge for clinical diagnosis. Diagnosis support systems (DSS) applied to electronic health record (EHR) data may help identify undiagnosed patients, which is of paramount importance to improve patients' care. Our objective was to evaluate three online-accessible rare disease DSSs using phenotypes derived from EHRs for the diagnosis of ciliopathies. METHODS: Two datasets of ciliopathy cases, either proven or suspected, and two datasets of controls were used to evaluate the DSSs. Patient phenotypes were automatically extracted from their EHRs and converted to Human Phenotype Ontology terms. We tested the ability of the DSSs to diagnose cases in contrast to controls based on Orphanet ontology. RESULTS: A total of 79 cases and 38 controls were selected. Performances of the DSSs on ciliopathy real world data (best DSS with area under the ROC curve = 0.72) were not as good as published performances on the test set used in the DSS development phase. None of these systems obtained results which could be described as "expert-level". Patients with multisystemic symptoms were generally easier to diagnose than patients with isolated symptoms. Diseases easily confused with ciliopathy generally affected multiple organs and had overlapping phenotypes. Four challenges need to be considered to improve the performances: to make the DSSs interoperable with EHR systems, to validate the performances in real-life settings, to deal with data quality, and to leverage methods and resources for rare and complex diseases. CONCLUSION: Our study provides insights into the complexities of diagnosing highly heterogenous rare diseases and offers lessons derived from evaluation existing DSSs in real-world settings. These insights are not only beneficial for ciliopathy diagnosis but also hold relevance for the enhancement of DSS for various complex rare disorders, by guiding the development of more clinically relevant rare disease DSSs, that could support early diagnosis and finally make more patients eligible for treatment.


Assuntos
Ciliopatias , Registros Eletrônicos de Saúde , Doenças Raras , Humanos , Ciliopatias/diagnóstico , Doenças Raras/diagnóstico , Sistemas de Apoio a Decisões Clínicas , Fenótipo
2.
Kidney Int ; 104(2): 378-387, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37230223

RESUMO

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.


Assuntos
Ciliopatias , Doenças Renais Císticas , Falência Renal Crônica , Doenças Renais Policísticas , Adulto , Humanos , Falência Renal Crônica/diagnóstico , Doenças Renais Policísticas/complicações , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Mutação , Ciliopatias/genética
3.
Proc Natl Acad Sci U S A ; 119(18): e2115960119, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35482924

RESUMO

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.


Assuntos
Ciliopatias , Doenças Renais Policísticas , Animais , Cílios/metabolismo , Ciliopatias/tratamento farmacológico , Ciliopatias/genética , Ciliopatias/metabolismo , Feminino , Humanos , Doenças Renais Císticas/congênito , Masculino , Camundongos , Doenças Renais Policísticas/metabolismo , Prostaglandinas/metabolismo , Receptores de Prostaglandina E/metabolismo , Peixe-Zebra
5.
Hum Mol Genet ; 31(13): 2121-2136, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35043953

RESUMO

Renal ciliopathies are the leading cause of inherited kidney failure. In autosomal dominant polycystic kidney disease (ADPKD), mutations in the ciliary gene PKD1 lead to the induction of CCL2, which promotes macrophage infiltration in the kidney. Whether or not mutations in genes involved in other renal ciliopathies also lead to immune cells recruitment is controversial. Through the parallel analysis of patients' derived material and murine models, we investigated the inflammatory components of nephronophthisis (NPH), a rare renal ciliopathy affecting children and adults. Our results show that NPH mutations lead to kidney infiltration by neutrophils, macrophages and T cells. Contrary to ADPKD, this immune cell recruitment does not rely on the induction of CCL2 in mutated cells, which is dispensable for disease progression. Through an unbiased approach, we identified a set of inflammatory cytokines that are upregulated precociously and independently of CCL2 in murine models of NPH. The majority of these transcripts is also upregulated in NPH patient renal cells at a level exceeding those found in common non-immune chronic kidney diseases. This study reveals that inflammation is a central aspect in NPH and delineates a specific set of inflammatory mediators that likely regulates immune cell recruitment in response to NPH genes mutations.


Assuntos
Ciliopatias , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Adulto , Animais , Criança , Ciliopatias/genética , Fibrose , Humanos , Rim , Camundongos , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética
6.
J Biomed Inform ; 100: 103308, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622800

RESUMO

Rare diseases are often hard and long to be diagnosed precisely, and most of them lack approved treatment. For some complex rare diseases, precision medicine approach is further required to stratify patients into homogeneous subgroups based on the clinical, biological or molecular features. In such situation, deep phenotyping of these patients and comparing their profiles based on subjacent similarities are thus essential to help fast and precise diagnoses and better understanding of pathophysiological processes in order to develop therapeutic solutions. In this article, we developed a new pipeline of using deep phenotyping to define patient similarity and applied it to ciliopathies, a group of rare and severe diseases caused by ciliary dysfunction. As a French national reference center for rare and undiagnosed diseases, the Necker-Enfants Malades Hospital (Necker Children's Hospital) hosts the Imagine Institute, a research institute focusing on genetic diseases. The clinical data warehouse contains on one hand EHR data, and on the other hand, clinical research data. The similarity metrics were computed on both data sources, and were evaluated with two tasks: diagnoses with EHRs and subtyping with ciliopathy specific research data. We obtained a precision of 0.767 in the top 30 most similar patients with diagnosed ciliopathies. Subtyping ciliopathy patients with phenotypic similarity showed concordances with expert knowledge. Similarity metrics applied to rare disease offer new perspectives in a translational context that may help to recruit patients for research, reduce the length of the diagnostic journey, and better understand the mechanisms of the disease.


Assuntos
Ciliopatias/diagnóstico , Fenótipo , Doenças Raras/diagnóstico , Ciliopatias/classificação , Data Warehousing , Registros Eletrônicos de Saúde , Humanos , Doenças Raras/classificação
7.
Am J Physiol Renal Physiol ; 315(1): F57-F73, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29537311

RESUMO

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.


Assuntos
Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase Ialfa/antagonistas & inibidores , Rim/efeitos dos fármacos , Doenças Renais Policísticas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Piridinas/farmacologia , Roscovitina/farmacologia , Animais , Caseína Quinase 1 épsilon/genética , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Ialfa/genética , Caseína Quinase Ialfa/metabolismo , Catálise , Cromatografia de Afinidade/métodos , Modelos Animais de Doenças , Humanos , Rim/enzimologia , Rim/patologia , Camundongos Transgênicos , Doenças Renais Policísticas/enzimologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Purinas/metabolismo , Piridinas/metabolismo , Roscovitina/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Hum Mol Genet ; 25(11): 2245-2255, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27053712

RESUMO

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.


Assuntos
Cílios/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/genética , Falência Renal Crônica/tratamento farmacológico , Doenças Renais Policísticas/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Cílios/patologia , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Purinas/administração & dosagem , Roscovitina , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
9.
Cancer Res ; 74(23): 6820-32, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25297632

RESUMO

Clear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading to increased level of hypoxia-inducible factors (HIF). In this study, we investigated the potential role of HIF2α in regulating RCC susceptibility to natural killer (NK) cell-mediated killing. We demonstrated that the RCC cell line 786-0 with mutated VHL was resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7). This resistance was found to require HIF2α stabilization. On the basis of global gene expression profiling and chromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a direct novel target of HIF2α and that targeting ITPR1 significantly increased susceptibility of 786-0 cells to NK-mediated lysis. Mechanistically, HIF2α in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, both ITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increased granzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediated tumor regression. Our data provide insight into the link between HIF2α, the ITPR1-related pathway, and natural immunity and strongly suggest a role for the HIF2α/ITPR1 axis in regulating RCC cell survival.


Assuntos
Autofagia/imunologia , Carcinoma de Células Renais/imunologia , Receptores de Inositol 1,4,5-Trifosfato/imunologia , Neoplasias Renais/imunologia , Células Matadoras Naturais/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Beclina-1 , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transcriptoma
10.
Proc Natl Acad Sci U S A ; 111(41): 14794-9, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25267645

RESUMO

TNFα is a potent cytokine that plays a critical role in numerous cellular processes, particularly immune and inflammatory responses, programmed cell death, angiogenesis, and cell migration. Thus, understanding the molecular mechanisms that mediate TNFα-induced cellular responses is a crucial issue. It is generally accepted that global DNA binding activity of the NF-κB avian reticuloendotheliosis viral (v-rel) oncogene related B (RelB) subunit is not induced upon TNFα treatment in fibroblasts, despite its TNFα-induced nuclear accumulation. Here, we demonstrate that RelB plays a critical role in promoting fibroblast migration upon prolonged TNFα treatment. We identified the two kinases IκB kinase α (IKKα) and IκB kinase ß (IKKß) as RelB interacting partners whose activation by TNFα promotes RelB phosphorylation at serine 472. Once phosphorylated on serine 472, nuclear RelB dissociates from its interaction with the inhibitory protein IκBα and binds to the promoter of critical migration-associated genes, such as the matrix metallopeptidase 3 (MMP3). Further, we show that RelB serine 472 phosphorylation status controls MMP3 expression and promigration activity downstream of TNF receptors. Our findings provide new insights into the regulation of RelB activity and reveal a novel link between selective NF-κB target gene expression and cellular response in response to TNFα.


Assuntos
Movimento Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Quinase I-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Transcrição RelB/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Fibroblastos/efeitos dos fármacos , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
11.
Blood ; 122(23): 3713-22, 2013 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-24089328

RESUMO

Monogenic interleukin-10 (IL-10) and IL-10 receptor (IL-10R) deficiencies cause very early onset severe inflammatory bowel disease. Here, we report that 5 patients with an IL-10R1 (n = 1) or IL-10R2 (n = 4) deficiency developed B-cell non-Hodgkin lymphoma between the ages of 5 and 6 years (which was recurrent in 1 patient). These lymphomas had some of the characteristics of diffuse large B-cell lymphomas and contained monoclonal, Epstein-Barr virus-negative germinal center B cells. The tumors displayed a remarkably homogeneous signature, with original activation of the nuclear factor κB pathway and a decrease in intratumor T-cell infiltration. Hence, IL-10R deficiency is associated with a high risk of developing B-cell lymphoma. Our results revealed an unexpected role of the IL-10R pathway in lymphomagenesis.


Assuntos
Subunidade alfa de Receptor de Interleucina-10/deficiência , Subunidade alfa de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/deficiência , Subunidade beta de Receptor de Interleucina-10/genética , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Adolescente , Idade de Início , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Genes rel , Predisposição Genética para Doença , Centro Germinativo/imunologia , Centro Germinativo/patologia , Humanos , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Interleucina-10/metabolismo , Linfoma de Células B/etiologia , Linfoma Difuso de Grandes Células B/etiologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Masculino , Mutação , NF-kappa B/metabolismo , Linhagem , Transdução de Sinais
12.
Mol Cancer Res ; 11(7): 724-35, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23604035

RESUMO

The death domain containing TNF receptor 6 (CD95/Fas) is a direct target for the NF-κB transcription factor and is repressed in solid tumors such as colon carcinomas. Previously, we reported that the Fas death receptor, while overexpressed in low-risk myelodysplastic syndromes (MDS), becomes undetectable on CD34(+) progenitors when the disease progresses to secondary acute myeloid leukemia (AML). This study determined the interplay between NF-κB and Fas during MDS progression. We first observed that Fas was induced by TNF-α in the HL60 cell line. In these cells, p65 (RELA) was associated with the FAS promoter, and inhibition of the NF-κB pathway by an IKKα inhibitor (BAY11-7082) or lentiviral expression of a nondegradable mutant of IκBα (IκSR) blocked Fas expression. In contrast, TNF-α failed to induce Fas expression in the colon carcinoma cell line SW480, due to hypermethylation of the FAS promoter. Azacitidine rescued p65 binding on FAS promoter in vitro, and subsequently Fas expression in SW480 cells. Furthermore, inhibition of the NF-κB pathway decreased the expression of Fas in MDS CD45(lo)CD34(+) bone marrow cells. However, despite the nuclear expression of p65, Fas was often low on CD45(lo)CD34(+) AML cells. TNF-α failed to stimulate its expression, while azacitidine efficiently rescued p65 binding and Fas reexpression. Overall, these data suggest that DNA methylation at NF-κB sites is responsible for FAS gene silencing.


Assuntos
Progressão da Doença , Epigênese Genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , NF-kappa B/genética , Transcrição Gênica , Receptor fas/genética , Azacitidina/farmacologia , Sequência de Bases , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Nitrilas/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Sulfonas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Receptor fas/metabolismo
13.
PLoS One ; 8(3): e59127, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555623

RESUMO

The NF-κB family of transcription factors has emerged as a key player in the pathogenesis of multiple myeloma (MM). NF-κB is activated by at least two major signaling pathways. The classical pathway results in the activation of mainly RelA containing dimers, whereas the alternative pathway leads to the activation of RelB/p52 and RelB/p50 heterodimers. Activating mutations in regulators of the alternative pathway have been identified in 17% of MM patients. However, the status of RelB activation per se and its role in the regulation of cell survival in MM has not been investigated. Here, we reveal that 40% of newly diagnosed MM patients have a constitutive RelB DNA-binding activity in CD138(+) tumor cells, and we show an association with increased expression of a subset of anti-apoptotic NF-κB target genes, such as cIAP2. Furthermore, we demonstrate that RelB exerts a crucial anti-apoptotic activity in MM cells. Our findings indicate that RelB activation is key for promoting MM cell survival through the upregulation of anti-apoptotic proteins. Altogether, our study provides the framework for the development of new molecules targeting RelB in the treatment of MM.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Inibidoras de Apoptose/genética , Mieloma Múltiplo/genética , Fator de Transcrição RelB/genética , Transcrição Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Proteína 3 com Repetições IAP de Baculovírus , Sobrevivência Celular , DNA/metabolismo , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Cultura Primária de Células , Transdução de Sinais , Sindecana-1/genética , Sindecana-1/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelB/metabolismo , Ubiquitina-Proteína Ligases
14.
PLoS One ; 7(6): e38808, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719951

RESUMO

BACKGROUND: Isolated limb perfusion with TNF-α and melphalan is used with remarkable efficiency to treat unresectable limb sarcomas. Here we tested the ability of TNF-α to directly induce apoptosis of sarcoma cells. In addition, we investigated the impact of p53 in the regulation of such effect. METHODOLOGY/PRINCIPAL FINDINGS: We first analysed the ability of TNF-α to induce apoptosis in freshly isolated tumour cells. For this purpose, sarcoma tumours (n = 8) treated ex vivo with TNF-α were processed for TUNEL staining. It revealed substantial endothelial cell apoptosis and levels of tumour cell apoptosis that varied from low to high. In order to investigate the role of p53 in TNF-α-induced cell death, human sarcoma cell lines (n = 9) with different TP53 and MDM2 status were studied for their sensitivity to TNF-α. TP53(Wt) cell lines were sensitive to TNF-α unless MDM2 was over-expressed. However, TP53(Mut) and TP53(Null) cell lines were resistant. TP53 suppression in TP53(Wt) cell lines abrogated TNF-α sensitivity and TP53 overexpression in TP53(Null) cell lines restored it. The use of small molecules that restore p53 activity, such as CP-31398 or Nutlin-3a, in association with TNF-α, potentiated the cell death of respectively TP53(Mut) and TP53(Wt)/MDM2(Ampl). In particular, CP-31398 was able to induce p53 as well as some of its apoptotic target genes in TP53(Mut) cells. In TP53(Wt)/MDM2(Ampl) cells, Nutlin-3a effects were associated with a decrease of TNF-α-induced NF-κB-DNA binding and correlated with a differential regulation of pro- and anti-apoptotic genes such as TP53BP2, GADD45, TGF-ß1 and FAIM. CONCLUSION/SIGNIFICANCE: More effective therapeutic approaches are critically needed for the treatment of unresectable limb sarcomas. Our results show that restoring p53 activity in sarcoma cells correlated with increased sensitivity to TNF-α, suggesting that this strategy may be an important determinant of TNF-α-based sarcomas treatment.


Assuntos
Genes p53 , Sarcoma/fisiopatologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Sarcoma/patologia
15.
Blood ; 117(6): 1917-27, 2011 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-21139082

RESUMO

Chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Aiolos, a member of the Ikaros family of zinc-finger transcription factors, plays an important role in the control of mature B lymphocyte differentiation and maturation. In this study, we showed that Aiolos expression is up-regulated in B-CLL cells. This overexpression does not implicate isoform imbalance or disturb Aiolos subcellular localization. The chromatin status at the Aiolos promoter in CLL is defined by the demethylation of DNA and an enrichment of euchromatin associated histone markers, such as the dimethylation of the lysine 4 on histone H3. These epigenetic modifications should allow its upstream effectors, such as nuclear factor-κB, constitutively activated in CLL, to gain access to promoter, resulting up-regulation of Aiolos. To determine the consequences of Aiolos deregulation in CLL, we analyzed the effects of Aiolos overexpression or down-regulation on apoptosis. Aiolos is involved in cell survival by regulating the expression of some Bcl-2 family members. Our results strongly suggest that Aiolos deregulation by epigenetic modifications may be a hallmark of CLL.


Assuntos
Epigênese Genética , Fator de Transcrição Ikaros/genética , Leucemia Linfocítica Crônica de Células B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Apoptose/fisiologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Cromatina/genética , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Primers do DNA/genética , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator de Transcrição Ikaros/antagonistas & inibidores , Fator de Transcrição Ikaros/metabolismo , Técnicas In Vitro , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Frações Subcelulares/metabolismo
16.
Leuk Res ; 34(3): 289-93, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19540588

RESUMO

The Aiolos transcription factor plays a crucial role in the control of lymphocyte differentiation and proliferation. The expression of Aiolos isoform has been studied in lymphoid pathologies but nothing is known about its expression in unaffected human hematopoietic subpopulations. In this manuscript we show for the first time the differential Aiolos expression at the RNA and protein level in hematopoietic cell subpopulations. B cells express higher levels of Aiolos than NK and T cells while monocytes express almost undetectable levels of Aiolos. Moreover, human CD34 (+) progenitors do not express Aiolos. We did not observe significant difference when comparing naive to memory T and B cells, but we observed an important difference between Bright and Dim NK cells. Furthermore, we show that, in addition to hematopoietic cells, non hematopoietic cell lines such as MCF-7, SW480, HEK, PC3 and HeLa also express Aiolos.


Assuntos
Linfócitos B/metabolismo , Fator de Transcrição Ikaros/biossíntese , Células Matadoras Naturais/metabolismo , Monócitos/metabolismo , Linfócitos T/metabolismo , Western Blotting , Linhagem Celular , Separação Celular , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Subpopulações de Linfócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
FEBS Lett ; 582(3): 457-67, 2008 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-18206652

RESUMO

In order to investigate the epigenetic component of Aiolos regulation, we analyzed the methylation status of its 5' CpG island in relation to histone modifications. Inhibition of CpG methylation restores Aiolos expression, as well as euchromatin-associated markers, in U937 and 1106 mel cell lines. DNA methylation and low levels of euchromatin-associated signatures are observed in U937 and 1106 mel cell lines, while the opposite characterizes Daudi, Jurkat, T and B cells. CpG methylation is not necessary to repress transcription in monocytes and melanocytes where silencing mechanism involves heterochromatin-associated signature. We show that DNA methylation directs Aiolos silencing and chromatin status in tumor cell lines, while in primary cells is mainly regulated by histone modifications.


Assuntos
Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Fatores de Transcrição/genética , Acetilação , Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatina/metabolismo , Ilhas de CpG/fisiologia , Metilação de DNA , Decitabina , Combinação de Medicamentos , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator de Transcrição Ikaros , Células Jurkat , Neoplasias/patologia , Regiões Promotoras Genéticas , Ativação Transcricional , Células U937
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