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1.
Cell Rep ; 35(2): 108994, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33852836

RESUMO

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumor for which new treatments are needed. CBL0137 is an anti-cancer compound developed from quinacrine that targets facilitates chromatin transcription (FACT), a chromatin remodeling complex involved in transcription, replication, and DNA repair. We show that CBL0137 displays profound cytotoxic activity against a panel of patient-derived DIPG cultures by restoring tumor suppressor TP53 and Rb activity. Moreover, in an orthotopic model of DIPG, treatment with CBL0137 significantly extends animal survival. The FACT subunit SPT16 is found to directly interact with H3.3K27M, and treatment with CBL0137 restores both histone H3 acetylation and trimethylation. Combined treatment of CBL0137 with the histone deacetylase inhibitor panobinostat leads to inhibition of the Rb/E2F1 pathway and induction of apoptosis. The combination of CBL0137 and panobinostat significantly prolongs the survival of mice bearing DIPG orthografts, suggesting a potential treatment strategy for DIPG.

2.
Cancer Res ; 80(19): 4129-4144, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32816860

RESUMO

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Cobre/metabolismo , Neuroblastoma/imunologia , Evasão Tumoral/fisiologia , Animais , Antígeno B7-H1/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Quelantes/farmacologia , Transportador de Cobre 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunoterapia/métodos , Células Matadoras Naturais , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos BALB C , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Trietilenofosforamida/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Stem Cells Dev ; 26(7): 461-470, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-27998208

RESUMO

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.


Assuntos
Diferenciação Celular/fisiologia , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/imunologia , Técnicas de Cultura de Células/métodos , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Camundongos , Linfócitos T/citologia , Transplante Homólogo/métodos
4.
Exp Hematol ; 43(12): 1001-1014.e5, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26384559

RESUMO

Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.


Assuntos
Antígenos CD19/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T , Animais , Criança , Pré-Escolar , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/imunologia , Linfócitos T/patologia , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Stem Cells Dev ; 24(6): 724-36, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25329250

RESUMO

Small-molecule inhibitors of glycogen synthase kinase 3ß (GSK3ß) have demonstrated strong anti-leukemia effects in preclinical studies. Here, we investigated the effect of GSK3ß inhibitor 6-Bromoindirubin-3-oxime (BIO) previously shown to inhibit leukemia cell growth in vitro and of animal models on hematopoietic regeneration in recipients of stem cell transplant. BIO administered to immunocompromised mice transplanted with human hematopoietic stem cells inhibited human stem cell engraftment in the bone marrow (BM) and peripheral blood. BIO reduced CD34(+) progenitor cells in the BM, and primitive lymphoid progenitors re-populated host thymus at later stages post-transplant. The development of all T-cell subsets in the thymus was suppressed in BIO-treated mice. Human cell engraftment was gradually restored after discontinuation of BIO treatment; however, T-cell depletion remained until the end of experiment, which correlated with the attenuated thymic function in the host. BIO delayed CD34(+) cell expansion in stroma-supported or cytokine-only cultures. BIO treatment delayed progenitor cell divisions and induced apoptosis in cultures with sub-optimal cytokine support. In addition, BIO inhibited B- and T-cell development in co-cultures with MS5 and OP9-DL1 BM stroma cells, respectively. These data suggest that administration of GKS3ß inhibitors may act to delay hematopoietic regeneration in patients who received stem cell transplant.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hematopoese , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Oximas/farmacologia , Animais , Apoptose , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
6.
PLoS One ; 9(11): e108994, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25369239

RESUMO

BACKGROUND AND OBJECTIVE: Sodium glucose cotransporter 2 (SGLT2) is the main luminal glucose transporter in the kidney. SGLT2 inhibition results in glycosuria and improved glycaemic control. Drugs inhibiting this transporter have recently been approved for clinical use and have been suggested to have potential renoprotective benefits by limiting glycotoxicity in the proximal tubule. We aimed to determine the renoprotective benefits of empagliflozin, an SGLT2 inhibitor, independent of its glucose lowering effect. RESEARCH DESIGN AND METHODS: We induced diabetes using a low dose streptozotocin protocol in 7-8 week old endothelial nitric oxide (eNOS) synthase knockout mice. We measured fasting blood glucose on a monthly basis, terminal urinary albumin/creatinine ratio. Renal histology was assessed for inflammatory and fibrotic changes. Renal cortical mRNA transcription of inflammatory and profibrotic cytokines, glucose transporters and protein expression of SGLT2 and GLUT1 were determined. Outcomes were compared to diabetic animals receiving the angiotensin receptor blocker telmisartan (current best practice). RESULTS: Diabetic mice had high matched blood glucose levels. Empagliflozin did not attenuate diabetes-induced albuminuria, unlike telmisartan. Empagliflozin did not improve glomerulosclerosis, tubular atrophy, tubulointerstitial inflammation or fibrosis, while telmisartan attenuated these. Empagliflozin did not modify tubular toll-like receptor-2 expression in diabetic mice. Empagliflozin did not reduce the upregulation of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor ß1 and fibronectin mRNA observed in the diabetic animals, while telmisartan decreased transcription of MCP-1 and fibronectin. Empagliflozin increased GLUT1 mRNA expression and telmisartan increased SGLT2 mRNA expression in comparison to untreated diabetic mice. However no significant difference was found in protein expression of GLUT1 or SGLT2 among the different groups. CONCLUSION: Hence SGLT2 inhibition does not have renoprotective benefits independent of glucose lowering.


Assuntos
Glicemia/metabolismo , Nefropatias Diabéticas/prevenção & controle , Túbulos Renais Proximais/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Albuminúria/etiologia , Animais , Compostos Benzidrílicos/farmacologia , Compostos Benzidrílicos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Glicemia/análise , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , RNA Mensageiro/metabolismo , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose , Estreptozocina/toxicidade , Telmisartan , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
7.
Nephrol Dial Transplant ; 29(2): 313-24, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24166472

RESUMO

BACKGROUND: Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes. METHODS: As TGF-ß1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-ß1+/- the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1-/- mice, and secondly eNOS-/- mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined. RESULTS: Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-ß1 and in kidneys from diabetic mice. TRAM34 reduced TGF-ß1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts. CONCLUSIONS: These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.


Assuntos
Nefropatias Diabéticas/genética , Regulação da Expressão Gênica , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Córtex Renal/patologia , RNA/genética , Animais , Biópsia , Western Blotting , Proliferação de Células , Células Cultivadas , Diabetes Mellitus Experimental , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Imuno-Histoquímica , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Córtex Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
8.
Pathol Res Pract ; 209(11): 731-4, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24064283

RESUMO

Structural alteration to the microanatomical organization of the glomerular filtration barrier results in proteinuria. Conventional transmission electron microscopy is an important diagnostic tool to assess the degree of ultrastructural damage of the corpusclar filtration unit. However, this approach lacks the ability to collect accurate stereological insights in a relative large tissue volume. Transmission electron tomography offers the ability to gather three-dimensional information with relative ease. Therefore, this contribution aims to highlight what electron tomography can bring to the pathologist in this challenging area of diagnostic practice. Kidney tissue was prepared for routine ultrastructural transmission electron microscopy investigation. Three-dimensional data stacks were automatically acquired by tilting semi-thin sections of 270 nm in an angular range of typically -60° to +60° with 1° increment. Subsequently, models of the filtration unit were produced by computer-assisted tracking of structures of interest. This short report illustrates the capability that transmission electron tomography can offer in the fine structure-function assessment of the porous fenestrated glomerular capillary endothelium, the underlying basement membrane and the podocyte filtration slits. Furthermore, this approach allows the generation of morphometric data about size, shape and volume alterations of the kidney's filtration barrier at the nanoscale.


Assuntos
Tomografia com Microscopia Eletrônica , Barreira de Filtração Glomerular/ultraestrutura , Modelos Anatômicos , Animais , Gráficos por Computador , Simulação por Computador , Células Endoteliais/ultraestrutura , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/ultraestrutura
9.
Exp Hematol ; 41(12): 1016-27.e1, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24018603

RESUMO

Hematopoietic stem cell transplantation (HSCT) is used in the treatment of hematologic and nonhematologic disorders. PostHSCT immunologic reconstitution is a critical component for successful outcome. Pretransplant conditioning impairs thymic function, leading to delayed T cell regeneration. Thymus-independent T cell expansion is associated with defective generation of naive T cells and memory T cell skewing, resulting in decreased diversity in the T cell repertoire, thus attenuating the immune responses and increasing the risk of opportunistic infections and leukemia relapse. Wingless (Wnt) signaling has been identified as an important regulator of T cell development and function. Activated Wnt signaling inhibited differentiation of mature T cells in transgenic mouse models. The effect of Wnt activation on T cell regeneration following HSCT was not investigated. In this study, we demonstrate that the GSK-3ß inhibitor 6-bromoindirubin 3'-oxime (BIO) activates Wnt/ß-catenin signaling, elevates the proportion of naive T cells, and delays T cell differentiation during homeostatic T cell expansion in lymphodepleted mice transplanted with human hematopoietic stem cells. In vitro BIO-treatment promoted naive T cell expansion following mitogenic stimulation and improved proliferative responses of T cells to allogeneic stimuli. Treatment with BIO acts to expand the IL7Rα(+) subset of naive T cells, suggesting the potential mechanism driving T cell expansion during IL-7-dependent T cell proliferation. BIO downregulated expression of genes activated during effector cell differentiation and preserved naive T cell gene expression. We propose that administration of GSK-3ß inhibitor increases the potency of T cells in recipients of HSCT by expansion of naive T cell subsets with a diverse T cell receptor repertoire.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Transplante de Células-Tronco Hematopoéticas , Indóis/farmacologia , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Humanos , Ativação Linfocitária , Camundongos , Fenótipo , Linfócitos T/citologia , Transplante Heterólogo
10.
PLoS One ; 8(8): e73428, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23991195

RESUMO

NADPH oxidase 4 (Nox4) is reported to be the major source of reactive oxygen species (ROS) in the kidneys during the early stages of diabetic nephropathy. It has been shown to mediate TGFß1-induced differentiation of cardiac fibroblasts into myofibroblasts. Despite TGFß1 being recognised as a mediator of renal fibrosis and functional decline role in diabetic nephropathy, the renal interaction between Nox 4 and TGFß1 is not well characterised. The aim of this study was to investigate the role of Nox4 inhibition on TGFß1-induced fibrotic responses in proximal tubular cells and in a mouse model of diabetic nephropathy. Immortalised human proximal tubular cells (HK2) were incubated with TGFß1 ± plumbagin (an inhibitor of Nox4) or specific Nox4 siRNA. Collagen IV and fibronectin mRNA and protein expression were measured. Streptozotocin (STZ) induced diabetic C57BL/6J mice were administered plumbagin (2 mg/kg/day) or vehicle (DMSO; 50 µl/mouse) for 24 weeks. Metabolic, physiological and histological markers of nephropathy were determined. TGFß1 increased Nox4 mRNA expression and plumbagin and Nox4 siRNA significantly inhibited TGF-ß1 induced fibronectin and collagen IV expression in human HK2 cells. STZ-induced diabetic C57BL/6J mice developed physiological features of diabetic nephropathy at 24 weeks, which were reversed with concomitant plumbagin treatment. Histologically, plumbagin ameliorated diabetes induced upregulation of extracellular matrix protein expression compared to control. This study demonstrates that plumbagin ameliorates the development of diabetic nephropathy through pathways that include Nox4 signalling.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , NADPH Oxidases/metabolismo , Naftoquinonas/uso terapêutico , Animais , Sequência de Bases , Linhagem Celular Transformada , Colágeno Tipo IV/genética , Primers do DNA , Nefropatias Diabéticas/enzimologia , Fibronectinas/genética , Glucose/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Estreptozocina , Superóxido Dismutase/genética , Fator de Crescimento Transformador beta1/metabolismo
11.
Diabetes ; 62(8): 2923-34, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23656889

RESUMO

The Ca(2+)-activated K(+) channel KCa3.1 mediates cellular signaling processes associated with dysfunction of vasculature. However, the role of KCa3.1 in diabetic nephropathy is unknown. We sought to assess whether KCa3.1 mediates the development of renal fibrosis in two animal models of diabetic nephropathy. Wild-type and KCa3.1(-/-) mice, and secondly eNOS(-/-) mice, had diabetes induced with streptozotocin and then were treated with/without a selective inhibitor of KCa3.1 (TRAM34). Our results show that the albumin-to-creatinine ratio significantly decreased in diabetic KCa3.1(-/-) mice compared with diabetic wild-type mice and in diabetic eNOS(-/-) mice treated with TRAM34 compared with diabetic mice. The expression of monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule 1 (ICAM1), F4/80, plasminogen activator inhibitor type 1 (PAI-1), and type III and IV collagen significantly decreased (P < 0.01) in kidneys of diabetic KCa3.1(-/-) mice compared with diabetic wild-type mice. Similarly, TRAM34 reduced the expression of the inflammatory and fibrotic markers described above in diabetic eNOS(-/-) mice. Furthermore, blocking the KCa3.1 channel in both animal models led to a reduction of transforming growth factor-ß1 (TGF-ß1) and TGF-ß1 type II receptor (TßRII) and phosphorylation of Smad2/3. Our results provide evidence that KCa3.1 mediates renal fibrosis in diabetic nephropathy through the TGF-ß1/Smad signaling pathway. Blockade of KCa3.1 may be a novel target for therapeutic intervention in patients with diabetic nephropathy.


Assuntos
Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Pirazóis/uso terapêutico , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Quimiocina CCL2/metabolismo , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Fibrose , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Knockout , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Pirazóis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
12.
Am J Physiol Renal Physiol ; 304(10): F1266-73, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23408168

RESUMO

MicroRNAs (miRNAs) comprise of a novel class of endogenous small noncoding RNAs that frequently downregulate the expression of target genes. Recent reports suggest that miRNA-200b prevents epithelial-to-mesenchymal transition (EMT) in cancer cells by targeting the E-box binding transcription factors Zinc finger E-box-binding homeobox 1 (ZEB1) and Zinc finger E-box-binding homeobox 2 (ZEB2). About 35% of active fibroblasts are derived from EMT which is central to the development of progressive renal fibrosis. Hence, this study was designed to assess the effect of miRNA-200b on transforming growth factor-ß (TGF-ß1)-induced fibrotic responses in renal tubular cells. Morphologically, human kidney-2 cells transfected with miRNA-200b retained their epithelial cell characteristics when exposed to TGF-ß1. miRNA-200b significantly increased E-cadherin (P < 0.001) and reduced fibronectin mRNA and protein expression (both P < 0.01) independent of phospho-Smad2/3 and phospho-p38 and p42/44 signaling. Increased E-cadherin expression was associated with decreased expression of ZEB1 and ZEB2 and repression of fibronectin was mediated through direct targeting of the fibronectin mRNA, demonstrated using pMIR luciferase reporter assay and site-directed mutagenesis. These results suggest that miRNA-200b suppresses TGF-ß1-induced EMT via inhibition of ZEB1 and ZEB2 and the extracellular matrix protein fibronectin by directing targeting of its 3'UTR mRNA, independent of pathways directly involved in TGF-ß1 signaling.


Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fibronectinas/metabolismo , Túbulos Renais Proximais/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibrose , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , MicroRNAs/genética , Fosforilação/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Transfecção
13.
Exp Hematol ; 41(1): 39-55.e10, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22999867

RESUMO

Graft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3'-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Doença Enxerto-Hospedeiro/prevenção & controle , Indóis/farmacologia , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Transplante de Células-Tronco Hematopoéticas , Humanos , Indóis/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Oximas/uso terapêutico , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos T/imunologia
14.
Virol J ; 6: 184, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19878571

RESUMO

BACKGROUND: Multiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes. RESULTS: The aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs. CONCLUSION: Deletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6x shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.


Assuntos
Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/genética , Mutagênese Insercional/métodos , RNA Interferente Pequeno/genética , Deleção de Sequência , Simulação por Computador , Humanos
15.
PLoS One ; 4(7): e6461, 2009 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-19649289

RESUMO

BACKGROUND: Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34(+) HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. METHODOLOGY/PRINCIPAL FINDINGS: Using commercially available G-CSF mobilized peripheral blood (PB) CD34(+) cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. CONCLUSIONS/SIGNIFICANCE: This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34(+) cells.


Assuntos
Antígenos CD34/imunologia , Vetores Genéticos , Células-Tronco Hematopoéticas/imunologia , Lentivirus/genética , Transdução Genética , Sangue , Células Cultivadas , Meios de Cultura Livres de Soro , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Humanos
16.
Retrovirology ; 6: 55, 2009 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-19497094

RESUMO

BACKGROUND: The RNA interference (RNAi) pathway is a mechanism of gene-suppression with potential gene therapy applications for treating viral disease such as HIV-1. The most suitable inducer of RNAi for this application is short hairpin RNA (shRNA) although it is limited to suppressing a single target. A successful anti-HIV-1 therapy will require combinations of multiple highly active, highly conserved shRNAs to adequately counter the emergence of resistant strains. RESULTS: We calculated the percentage conservations of 8, 846 unique 19 nucleotide HIV-1 targets amongst 37, 949 HIV-1 gene sequence fragments containing 24.8 million 19 mers. We developed a novel method of determining conservation in 'profile' sets of 5 overlapping 19 mer sequences (covering 23 nucleotides in total) to ensure that the intended conservation of each shRNA would be unaffected by possible variations in shRNA processing. Ninety six of the top ranking targets from 22 regions were selected based on conservation profiles, predicted activities, targets and specific nucleotide inclusion/exclusion criteria. We constructed 53 shRNAs with 20 bp stems and 43 shRNAs with 21 bp stems which we tested and ranked using fluorescent reporter and HIV-1 expression assays. Average suppressive activities ranged from 71 - 75%, with 65 hairpins classed as highly active (> 75% activity). Overall we found little difference in activities from minor changes in stem length (20 cf. 21), or between neighboring targets differing by a single nucleotide in start position. However, there were several exceptions which suggest that all sequences, irrespective of similarities in target site or design, may be useful candidates. We encountered technical limitations with GFP reporter assays when the target domain was long and or when the distance between the target site and fusion junction was large. Assay performance was improved by dividing large targets into several shorter domains. CONCLUSION: In summary, our novel selection process resulted in a large panel of highly active shRNAs spanning the HIV-1 genome, representing excellent candidates for use in multiple shRNA gene therapies. Our core selection method ensuring maximal conservation in the processed product(s) is also widely applicable to other shRNA applications.


Assuntos
Infecções por HIV/genética , HIV-1/genética , Sequências Repetidas Invertidas , Interferência de RNA , RNA Interferente Pequeno/genética , Linhagem Celular , Sequência Conservada , Genes Reporter , Terapia Genética/métodos , Variação Genética , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , RNA Interferente Pequeno/farmacologia , RNA Viral/antagonistas & inibidores , RNA Viral/genética , Análise de Sequência de DNA/métodos , Inativação de Vírus/efeitos dos fármacos
17.
Int J Oncol ; 33(1): 175-83, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18575764

RESUMO

Interferon regulatory factor (IRF) 1 and its functional antagonist IRF2 were originally discovered as transcription factors that regulate the interferon-beta gene. Control of cell growth has led to the definition of IRF1 as a tumour suppressor gene and IRF2 as an oncogene. Clinically, approximately 70% of cases of acute myeloid leukaemia demonstrate dysregulated expression of IRF1 and/or IRF2. Our previous studies have shown that human leukaemic TF-1 cells exhibit abnormally high expression of both IRF1 and IRF2, the latter acting to abrogate IRF1 tumour suppression, making these cells ideal for analysis of down-regulation of IRF2 expression. A novel G418 screening protocol was developed and used for identifying effective siRNA that targets IRF2 (siIRF2). Using optimized siIRF2 in leukaemic TF-1 cells, IRF2 was down-regulated by approximately 70% at both mRNA and protein levels. Phenotypically, this resulted in growth inhibition associated with G2/M arrest as well as induction of polyploidy, differentiation and apoptosis. In contrast to these results, siIRF2 targeting did not affect normal haematopoietic stem/progenitor cell growth. These results indicate the potential utility of IRF2 inhibition as a therapeutic approach to cancer.


Assuntos
Fator Regulador 2 de Interferon/antagonistas & inibidores , Leucemia/terapia , RNA Interferente Pequeno/genética , Antígenos CD34/análise , Ciclo Celular , Linhagem Celular Tumoral , Hematopoese , Humanos , Fator Regulador 2 de Interferon/genética , Leucemia/patologia , Receptores de Lipopolissacarídeos/análise
18.
Stem Cells ; 26(5): 1288-97, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18323411

RESUMO

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Células-Tronco Hematopoéticas/enzimologia , Leucemia/enzimologia , Leucemia/patologia , Animais , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos , Camundongos SCID , Oximas/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fatores de Tempo , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Cordão Umbilical/enzimologia , beta Catenina/metabolismo
19.
Exp Hematol ; 35(6): 908-19, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17533045

RESUMO

OBJECTIVES: Mutations in ras oncogenes occur at high frequency in acute myeloid leukemia and myelodysplastic syndromes; however, the role of ras genes in leukemogenesis has not been clearly defined. Our previous studies have shown that expression of mutant N-ras (N-rasG13R, G to C transversion) in human hematopoietic progenitor cells (HPC) promotes myeloid differentiation and proliferation both in vitro and in a NOD/SCID mouse model. In the present study, we performed expression profiling to identify the transcriptome induced by N-rasG13R in human HPC, and analyzed the effect of mutant N-ras in sorted specific subpopulations of HPC. METHODS: cDNA microarray analysis was performed on cord blood CD34(+) cells transduced with a retrovirus containing GFP alone or in combination with mutant N-ras. Transduced cells were also sorted into factorial subpopulations according to CD34 and transgene expression, and analyzed in suspension or semi-solid methylcellulose culture. RESULTS: Among a variety of changes, including upregulation of cytokine genes, we found that N-rasG13R induced expression of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1/WAF1). Analysis by RT-PCR revealed that increased p16(INK4a) and p21(CIP1/WAF1) occurred in the most primitive, CD34(+)/Ras(+) population but not in the more mature CD34(-)/Ras(+) cells or in the CD34(+)/Ras(-) cells. Moreover, N-rasG13R inhibited the proliferation of the primitive CD34(+)/Ras(+) cells, both in liquid culture and in colony assays. This growth suppression correlated with an increased proportion of myelomonocytic colonies and a decrease of erythroid colonies. In contrast, the growth of CD34(-)/Ras(+) cells and CD34(+)/Ras(-) HPC was not inhibited. CONCLUSIONS: These findings demonstrated the mutant N-ras induced transcriptome, and that this is associated with HPC growth suppression/myelomonocytic differentiation, and identify upregulation of cyclin inhibitors as key events in this process. The results indicate that ras mutation alone is not sufficient to induce leukemogenesis; collaborative secondary event(s) are involved in the process.


Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Genes ras/genética , Células Progenitoras Mieloides/metabolismo , Regulação para Cima/genética , Animais , Antígenos CD34/biossíntese , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Citocinas/biossíntese , Citocinas/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Células Progenitoras Mieloides/patologia
20.
Curr Gene Ther ; 6(5): 543-50, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17073600

RESUMO

Acute myeloid leukaemia (AML) is the most common form of leukaemia in adults. Although of the order of 75-85% of patients will achieve complete remission after induction chemotherapy, long-term survival is still relatively low. Despite the progress in the rational design of drugs in disorders such as chronic myeloid leukaemia, AML lacks a single specific pathogenomic event to act as a drug target. Interferon regulatory factor 1 (IRF1) is a member of a family of related proteins that act as transcriptional activators or repressors. IRF1 and its functional antagonist IRF2 originally discovered as transcription factors regulating the interferon-beta (IFN-beta) gene, are involved in the regulation of normal haematopoiesis and leukaemogenesis. IRF1 appears to act as a tumour suppressor gene and IRF2 as an oncogene. IRF1 acts to repress IRF2 function through the repression of cyclin-dependent kinase (CDK) inhibitor p21WAF1 critical for cell growth control. It appears that the tumour suppression function of IRF1 is abolished by IRF2. This review focuses on the interaction between IRF1 and IRF2 in myeloid development and leukaemogenesis, particularly in relation to the Ras signalling pathway. IRF2 may be a viable and specific therapeutic target in human leukaemia.


Assuntos
Fator Regulador 1 de Interferon/fisiologia , Fator Regulador 2 de Interferon/fisiologia , Leucemia Mieloide/terapia , Doença Aguda , Animais , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 2 de Interferon/genética , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos , Modelos Animais , Fenótipo , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismo
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