Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42
Filtrar
1.
Biochem Biophys Res Commun ; 520(2): 297-303, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31601421

RESUMO

Clinical severity is heterogeneous among patients suffering from congenital erythropoietic porphyria (CEP) suggesting a modulation of the disease (UROS deficiency) by environmental factors and modifier genes. A KI model of CEP due to a missense mutation of UROS gene present in human has been developed on 3 congenic mouse strains (BALB/c, C57BL/6, and 129/Sv) in order to study the impact of genetic background on disease severity. To detect putative modifiers of disease expression in congenic mice, hematologic data, iron parameters, porphyrin content and tissue samples were collected. Regenerative hemolytic anemia, a consequence of porphyrin excess in RBCs, had various expressions: 129/Sv mice were more hemolytic, BALB/c had more regenerative response to anemia, C57BL/6 were less affected. Iron status and hemolysis level were directly related: C57BL/6 and BALB/c had moderate hemolysis and active erythropoiesis able to reduce iron overload in the liver, while, 129/Sv showed an imbalance between iron release due to hemolysis and erythroid use. The negative control of hepcidin on the ferroportin iron exporter appeared strain specific in the CEP mice models tested. Full repression of hepcidin was observed in BALB/c and 129/Sv mice, favoring parenchymal iron overload in the liver. Unchanged hepcidin levels in C57BL/6 resulted in retention of iron predominantly in reticuloendothelial tissues. These findings open the field for potential therapeutic applications in the human disease, of hepcidin agonists and iron depletion in chronic hemolytic anemia.


Assuntos
Hepcidinas/metabolismo , Ferro/metabolismo , Porfiria Eritropoética/genética , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Feminino , Hemólise , Hepcidinas/genética , Sobrecarga de Ferro/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Porfiria Eritropoética/etiologia , Porfiria Eritropoética/metabolismo , Porfirinas/metabolismo , Uroporfirinogênio III Sintetase/genética
2.
Sci Transl Med ; 10(459)2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232228

RESUMO

Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.


Assuntos
Ciclopirox/uso terapêutico , Reposicionamento de Medicamentos , Porfiria Eritropoética/tratamento farmacológico , Sítio Alostérico , Animais , Fenômenos Biofísicos , Linhagem Celular , Ciclopirox/farmacocinética , Modelos Animais de Doenças , Homeostase , Camundongos , Fenótipo , Porfiria Eritropoética/enzimologia , Porfiria Eritropoética/patologia , Uroporfirinogênio III Sintetase/antagonistas & inibidores , Uroporfirinogênio III Sintetase/química , Uroporfirinogênio III Sintetase/metabolismo
3.
Ann Biol Clin (Paris) ; 76(6): 705-715, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30257815

RESUMO

Hepcidin has progressively become essential in clinical practice for the diagnosis and follow-up of a large spectrum of diseases. Anyway, its own biochemical and structural characteristics have complicated and delayed the acquisition of a standardized quantifying tool of the peptide.


Assuntos
Hepcidinas/análise , Fatores Etários , Feminino , Regulação da Expressão Gênica , Hepcidinas/química , Hepcidinas/metabolismo , Hepcidinas/fisiologia , Humanos , Imunoensaio/métodos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino
4.
Hum Mol Genet ; 26(8): 1565-1576, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334762

RESUMO

Congenital erythropoietic porphyria (CEP) is an inborn error of heme biosynthesis characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in deleterious porphyrin accumulation in blood cells responsible for hemolytic anemia and cutaneous photosensitivity. We analyzed here the molecular basis of UROS impairment associated with twenty nine UROS missense mutations actually described in CEP patients. Using a computational and biophysical joint approach we predicted that most disease-causing mutations would affect UROS folding and stability. Through the analysis of enhanced green fluorescent protein-tagged versions of UROS enzyme we experimentally confirmed these data and showed that thermodynamic instability and premature protein degradation is a major mechanism accounting for the enzymatic deficiency associated with twenty UROS mutants in human cells. Since the intracellular loss in protein homeostasis is in excellent agreement with the in vitro destabilization, we used molecular dynamic simulation to rely structural 3D modification with UROS disability. We found that destabilizing mutations could be clustered within three types of mechanism according to side chain rearrangements or contact alterations within the pathogenic UROS enzyme so that the severity degree correlated with cellular protein instability. Furthermore, proteasome inhibition using bortezomib, a clinically available drug, significantly enhanced proteostasis of each unstable UROS mutant. Finally, we show evidence that abnormal protein homeostasis is a prevalent mechanism responsible for UROS deficiency and that modulators of UROS proteolysis such as proteasome inhibitors or chemical chaperones may represent an attractive therapeutic option to reduce porphyrin accumulation and prevent skin photosensitivity in CEP patients when the genotype includes a missense variant.


Assuntos
Mutação de Sentido Incorreto/genética , Porfiria Eritropoética/genética , Relação Estrutura-Atividade , Uroporfirinogênio III Sintetase/genética , Biologia Computacional , Homeostase , Humanos , Porfiria Eritropoética/metabolismo , Porfiria Eritropoética/patologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/química , Inibidores de Proteassoma/uso terapêutico , Dobramento de Proteína , Uroporfirinogênio III Sintetase/química
5.
Haematologica ; 102(2): 260-270, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28143953

RESUMO

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.


Assuntos
Anemia Hemolítica/metabolismo , Hepatócitos/metabolismo , Hepcidinas/metabolismo , Ferro/metabolismo , Anemia Hemolítica/sangue , Anemia Hemolítica/complicações , Anemia Hemolítica/genética , Animais , Apoptose , Transporte Biológico , Biomarcadores , Modelos Animais de Doenças , Eritrócitos/metabolismo , Eritropoese , Expressão Gênica , Heme/metabolismo , Hepcidinas/sangue , Hepcidinas/genética , Humanos , Ferro/urina , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/metabolismo , Macrófagos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Baço/fisiologia , Estresse Fisiológico
6.
Stem Cells Transl Med ; 6(2): 382-393, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28191782

RESUMO

Iatrogenic tumorigenesis is a major limitation for the use of human induced pluripotent stem cells (hiPSCs) in hematology. The teratoma risk comes from the persistence of hiPSCs in differentiated cell populations. Our goal was to evaluate the best system to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSCs expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in immune-deficient mice was tracked by in vivo bioimaging. We observed that systemic injection of hiPSCs produced multisite teratoma as soon as 5 weeks after injection. To eliminate hiPSCs before grafting, we tested the embryonic-specific expression of suicide genes under the control of the pmiR-302/367 promoter. This promoter was highly active in hiPSCs but not in differentiated cells. The gene/prodrug inducible Caspase-9 (iCaspase-9)/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that iCaspase-9-expressing hiPSCs died in a dose-dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly, nonspecific toxicity of AP20187 on iCaspase-9-negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+ -derived human hematopoiesis in adoptive transfers. Survivin inhibition is an alternative to the suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155 was more efficient than AP20187/iCaspase-9 for killing hiPSCs, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSC purge by survivin inhibitor fully eradicated teratoma formation in immune-deficient mice. This will be useful to improve the safety management for hiPSC-based medicine. Stem Cells Translational Medicine 2017;6:382-393.


Assuntos
Caspase 9/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Genes Transgênicos Suicidas , Doenças Hematológicas/cirurgia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imidazóis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/transplante , Naftoquinonas/farmacologia , Medicina Regenerativa/métodos , Survivina/antagonistas & inibidores , Tacrolimo/análogos & derivados , Teratoma/prevenção & controle , Animais , Caspase 9/metabolismo , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Medição de Risco , Survivina/metabolismo , Tacrolimo/farmacologia , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Fatores de Tempo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Cancer Lett ; 390: 91-102, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28089829

RESUMO

Pancreatic adenocarcinoma, highly resistant to all current anti-cancer treatments, necessitates new approaches promoting cell death. We hypothesized that combined actions of several Bioactive Food Components (BFCs) might provide specific lethal effect towards tumor cells, sparing healthy cells. Human tumor pancreatic cell lines were tested in vitro for sensitivity to resveratrol, capsaicin, piceatannol, and sulforaphane cytotoxic effects. Combination of two or three components showed striking synergetic effect with gemcitabine in vitro. Each BFC used alone did not affect pancreatic tumor growth in a preclinical in vivo model, whereas couples of BFCs had anti-tumor activity. In addition, tumor toxicity was similar using gemcitabine alone or a combination of BFCs and two thirds of gemcitabine dose. Moreover, BFCs enhanced fibrotic response as compared to gemcitabine treatment alone. Reactive oxygen species (ROS) and apoptosis increases were observed, while cell cycle was very mildly affected. This study raises the possibility to use BFCs as beneficial food complements in the therapy of pancreatic adenocarcinoma, especially for patients unable to receive full doses of chemotherapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Capsaicina/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Estilbenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Nus , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Gencitabina , Neoplasias Pancreáticas
8.
Biomed Res Int ; 2016: 2180946, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27413738

RESUMO

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder. Considering that XP patients have a defect of the nucleotide excision repair (NER) pathway which enables them to repair DNA damage caused by UV light, they have an increased risk of developing skin and eyes cancers. In the present study, we investigated the involvement of the prevalent XPA and XPC genes mutations-nonsense mutation (c.682C>T, p.Arg228X) and a two-base-pair (2 bp) deletion (c.1643_1644delTG or p.Val548Ala fsX25), respectively-in 19 index cases from 19 unrelated families in the West of Algeria. For the genetic diagnosis of XPA gene, we proceeded to PCR-RFLP. For the XPC gene, we validated a routine analysis which includes a specific amplification of a short region surrounding the 2 bp deletion using a fluorescent primer and fragment sizing (GeneScan size) on a sequencing gel. Among the 19 index cases, there were 17 homozygous patients for the 2 bp deletion in the XPC gene and 2 homozygous patients carrying the nonsense XPA mutation. Finally, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. The use of fragment sizing is the simplest method to analyze this 2 bp deletion for the DNA samples coming from countries where the mutation c.1643_1644delTG of XPC gene is prevalent.


Assuntos
Análise Mutacional de DNA/métodos , Proteínas de Ligação a DNA/genética , Genótipo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Xeroderma Pigmentoso/genética , Adolescente , Argélia , Criança , Pré-Escolar , Dano ao DNA , Éxons , Feminino , Corantes Fluorescentes/química , Deleção de Genes , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Pele/metabolismo , Xeroderma Pigmentoso/diagnóstico , Xeroderma Pigmentoso/etnologia , Adulto Jovem
9.
Hum Mol Genet ; 23(17): 4479-90, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24714983

RESUMO

Hemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type 4A versus type 4B). However, the vast majority of SLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance of combining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management.


Assuntos
Proteínas de Transporte de Cátions/deficiência , Hemocromatose/genética , Anotação de Sequência Molecular , Mutação de Sentido Incorreto/genética , Adulto , Idoso , Substituição de Aminoácidos/genética , Transporte Biológico , Proteínas de Transporte de Cátions/sangue , Proteínas de Transporte de Cátions/genética , Simulação por Computador , Feminino , Ferritinas/sangue , Frequência do Gene/genética , Estudos de Associação Genética , Células HEK293 , Hemocromatose/sangue , Hepcidinas/farmacologia , Humanos , Espaço Intracelular/metabolismo , Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Splicing de RNA/genética , Relação Estrutura-Atividade , População Branca/genética , Adulto Jovem
10.
Am J Hum Genet ; 94(4): 611-7, 2014 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-24680888

RESUMO

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.


Assuntos
Ferroquelatase/genética , Oligonucleotídeos Antissenso/uso terapêutico , Protoporfiria Eritropoética/terapia , Linhagem Celular , Feminino , Humanos , Masculino , Linhagem , Polimorfismo Genético , Protoporfirinas/metabolismo , Splicing de RNA , RNA Mensageiro/genética
11.
Cell Stem Cell ; 13(5): 549-63, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24095676

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) are exposed to low levels of oxygen in the bone marrow niche, and hypoxia-inducible factors (HIFs) are the main regulators of cellular responses to oxygen variation. Recent studies using conditional knockout mouse models have unveiled a major role for HIF-1α in the maintenance of murine HSCs; however, the role of HIF-2α is still unclear. Here, we show that knockdown of HIF-2α, and to a much lesser extent HIF-1α, impedes the long-term repopulating ability of human CD34(+) umbilical cord blood cells. HIF-2α-deficient HSPCs display increased production of reactive oxygen species (ROS), which subsequently stimulates endoplasmic reticulum (ER) stress and triggers apoptosis by activation of the unfolded-protein-response (UPR) pathway. HIF-2α deregulation also significantly decreased engraftment ability of human acute myeloid leukemia (AML) cells. Overall, our data demonstrate a key role for HIF-2α in the maintenance of human HSPCs and in the survival of primary AML cells.


Assuntos
Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Proc Natl Acad Sci U S A ; 110(45): 18238-43, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24145442

RESUMO

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones.


Assuntos
Modelos Moleculares , Porfiria Eritropoética/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Uroporfirinogênio III Sintetase/genética , Uroporfirinogênio III Sintetase/metabolismo , Animais , Western Blotting , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Dicroísmo Circular , Primers do DNA/genética , Células Eritroides/metabolismo , Humanos , Camundongos , Mutação de Sentido Incorreto/genética , Porfiria Eritropoética/genética , Porfirinas/sangue , Porfirinas/urina , Dobramento de Proteína , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Fluorescência , Uroporfirinogênio III Sintetase/química
13.
PLoS One ; 8(8): e71596, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24058405

RESUMO

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Hematopoese/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células Tumorais Cultivadas
14.
Mol Cancer ; 12: 83, 2013 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-23902722

RESUMO

BACKGROUND: Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical. RESULTS: FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors. CONCLUSION: In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.


Assuntos
Células Epiteliais/metabolismo , Genes Supressores de Tumor , Fenótipo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Ligantes , Camundongos , Modelos Biológicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Transplante Heterólogo
15.
Mol Cancer ; 11: 81, 2012 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-23088623

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. RESULTS: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. CONCLUSION: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.


Assuntos
Antígenos de Superfície/metabolismo , Carcinoma Ductal Pancreático/terapia , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Terapia Genética/métodos , Neoplasias Pancreáticas/terapia , Animais , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Claudinas/genética , Claudinas/metabolismo , Sistemas de Liberação de Medicamentos , Ganciclovir/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/genética , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos SCID , Mucina-4/genética , Mucina-4/metabolismo , Neoplasias Pancreáticas/genética , Timidina Quinase/genética , Timidina Quinase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Am J Hum Genet ; 91(1): 109-21, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22795135

RESUMO

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.


Assuntos
Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/transplante , Porfiria Eritropoética/terapia , Uroporfirinogênio III Sintetase/genética , Diferenciação Celular , Estudos de Viabilidade , Vetores Genéticos , Células-Tronco Hematopoéticas/citologia , Humanos , Queratinócitos/citologia , Lentivirus/genética , Porfiria Eritropoética/genética , Transdução Genética
17.
J Cell Sci ; 124(Pt 24): 4172-83, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22193962

RESUMO

In mouse and human skin, HIF-1α is constitutively expressed in the epidermis, mainly in the basal layer. HIF-1α has been shown to have crucial systemic functions: regulation of kidney erythropoietin production in mice with constitutive HIF-1α epidermal deletion, and hypervascularity following epidermal HIF-1α overexpression. However, its local role in keratinocyte physiology has not been clearly defined. To address the function of HIF-1α in the epidermis, we used the mouse model of HIF-1α knockout targeted to keratinocytes (K14-Cre/Hif1a(flox/flox)). These mice had a delayed skin phenotype characterized by skin atrophy and pruritic inflammation, partly mediated by basement membrane disturbances involving laminin-332 (Ln-332) and integrins. We also investigated the relevance of results of studies in mice to human skin using reconstructed epidermis and showed that HIF-1α knockdown in human keratinocytes impairs the formation of a viable reconstructed epidermis. A diminution of keratinocyte growth potential, following HIF-1α silencing, was associated with a decreased expression of Ln-322 and α6 integrin and ß1 integrin. Overall, these results indicate a role of HIF-1α in skin homeostasis especially during epidermal aging.


Assuntos
Envelhecimento/fisiologia , Epiderme/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Queratinócitos/metabolismo , Animais , Apoptose , Moléculas de Adesão Celular/metabolismo , Pontos de Checagem do Ciclo Celular , Regulação para Baixo , Técnicas de Inativação de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Integrinas/metabolismo , Queratinócitos/citologia , Camundongos , Fenótipo , Pele/anatomia & histologia , Cicatrização , Calinina
18.
J Invest Dermatol ; 131(9): 1793-805, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21633368

RESUMO

Besides lung, postnatal human epidermis is the only epithelium in direct contact with atmospheric oxygen. Skin epidermal oxygenation occurs mostly through atmospheric oxygen rather than tissue vasculature, resulting in a mildly hypoxic microenvironment that favors increased expression of hypoxia-inducible factor-1α (HIF-1α). Considering the wide spectrum of biological processes, such as angiogenesis, inflammation, bioenergetics, proliferation, motility, and apoptosis, that are regulated by this transcription factor, its high expression level in the epidermis might be important to HIF-1α in skin physiology and pathophysiology. Here, we review the role of HIF-1α in cutaneous angiogenesis, skin tumorigenesis, and several skin disorders.


Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Epiderme/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neovascularização Fisiológica/fisiologia , Neoplasias Cutâneas/fisiopatologia , Carcinoma de Células Escamosas/metabolismo , Humanos , Oxigênio/metabolismo , Dermatopatias/metabolismo , Dermatopatias/fisiopatologia , Neoplasias Cutâneas/metabolismo
19.
Blood ; 118(6): 1443-51, 2011 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-21653323

RESUMO

Mutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.


Assuntos
5-Aminolevulinato Sintetase/genética , Mutação de Sentido Incorreto , Porfiria Eritropoética/genética , Uroporfirinogênio III Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Sequência de Aminoácidos , Anemia Sideroblástica/genética , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Sequência de Bases , Pré-Escolar , Eletroforese em Gel de Poliacrilamida , Saúde da Família , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Genótipo , Humanos , Lactente , Cinética , Masculino , Dados de Sequência Molecular , Linhagem , Porfiria Eritropoética/metabolismo , Porfiria Eritropoética/patologia , Protoporfiria Eritropoética/genética , Protoporfiria Eritropoética/metabolismo , Homologia de Sequência de Aminoácidos , Índice de Gravidade de Doença , Espectrofotometria , Uroporfirinogênio III Sintetase/metabolismo , Uroporfirinogênios/metabolismo
20.
Biochim Biophys Acta ; 1807(6): 609-19, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21167810

RESUMO

Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Queratinócitos/metabolismo , NADPH Oxidases/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/efeitos adversos , Antioxidantes/metabolismo , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Modelos Biológicos , Dados de Sequência Molecular , NADPH Oxidase 1 , NADPH Oxidases/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Interferência de RNA/efeitos dos fármacos , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência do Ácido Nucleico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...