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1.
Pediatr Blood Cancer ; 66(9): e27874, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31207059

RESUMO

Growth factor-independent 1B (GFI1B) variants are a rare cause of thrombocytopenia. We report on a male child who was initially diagnosed with immune thrombocytopenia. However, subtle clinical signs led to suspicion of a genetic cause of thrombocytopenia. Gene panel sequencing revealed a rare variant in GFI1B (C168F), which has recently been reported in several families with thrombocytopenia. We demonstrate that this variant significantly alters platelet parameters in population studies. This case highlights how diagnoses of exclusion, such as immune thrombocytopenia, can be confounded by genetic variation. Our understanding of blood disorders will undoubtedly evolve from an increased knowledge of human genetic variation.

2.
J Exp Med ; 216(5): 1050-1060, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30914438

RESUMO

Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five-amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.

4.
J Exp Clin Cancer Res ; 38(1): 28, 2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670049

RESUMO

BACKGROUND: Human microsatellite-stable (MSS) colorectal cancers (CRCs) are immunologically "cold" tumour subtypes characterized by reduced immune cytotoxicity. The molecular linkages between immune-resistance and human MSS CRC is not clear. METHODS: We used transcriptome profiling, in silico analysis, immunohistochemistry, western blot, RT-qPCR and immunofluorescence staining to characterize novel CRC immune biomarkers. The effects of selective antagonists were tested by in vitro assays of long term viability and analysis of kinase active forms using anti-phospho antibodies. RESULTS: We identified the lymphocyte antigen 6 complex, locus G6D (LY6G6D) as significantly overexpressed (around 15-fold) in CRC when compared with its relatively low expression in other human solid tumours. LY6G6D up-regulation was predominant in MSS CRCs characterized by an enrichment of immune suppressive regulatory T-cells and a limited repertoire of PD-1/PD-L1 immune checkpoint receptors. Coexpression of LY6G6D and CD15 increases the risk of metastatic relapse in response to therapy. Both JAK-STAT5 and RAS-MEK-ERK cascades act in concert as key regulators of LY6G6D and Fucosyltransferase 4 (FUT4), which direct CD15-mediated immune-resistance. Momelotinib, an inhibitor of JAK1/JAK2, consistently abrogated the STAT5/LY6G6D axis in vitro, sensitizing MSS cancer cells with an intact JAK-STAT signaling, to efficiently respond to trametinib, a MEK inhibitor used in clinical setting. Notably, colon cancer cells can evade JAK2/JAK1-targeted therapy by a reversible shift of the RAS-MEK-ERK pathway activity, which explains the treatment failure of JAK1/2 inhibitors in refractory CRC. CONCLUSIONS: Combined targeting of STAT5 and MAPK pathways has superior therapeutic effects on immune resistance. In addition, the new identified LY6G6D antigen is a promising molecular target for human MSS CRC.


Assuntos
Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Imunoglobulinas/genética , Fator de Transcrição STAT5/genética , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Fucosiltransferases/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Antígenos CD15/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Instabilidade de Microssatélites , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Pirimidinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
5.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

6.
Cell ; 173(1): 90-103.e19, 2018 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-29551269

RESUMO

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.

7.
Am J Hematol ; 92(9): E513-E519, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28568895

RESUMO

Immunodeficient mouse models have been valuable for studies of human hematopoiesis, but high-fidelity recapitulation of erythropoiesis in most xenograft recipients remains elusive. Recently developed immunodeficient and Kit mutant mice, however, have provided a suitable background to achieve higher-level human erythropoiesis after long-term hematopoietic engraftment. While there has been some characterization of human erythropoiesis in these models, a comprehensive analysis from various human developmental stages has not yet been reported. Here, we have utilized cell surface phenotypes, morphologic analyses, and molecular studies to fully characterize human erythropoiesis from multiple developmental stages in immunodeficient and Kit mutant mouse models following long-term hematopoietic stem and progenitor cell engraftment. We show that human erythropoiesis in such models demonstrates complete maturation and enucleation, as well as developmentally appropriate globin gene expression. These results provide a framework for future studies to utilize this model system for interrogating disorders affecting human erythropoiesis and for developing improved therapeutic approaches.


Assuntos
Eritropoese , Transplante de Células-Tronco Hematopoéticas , Mutação , Proteínas Proto-Oncogênicas c-kit/metabolismo , Animais , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-kit/genética
9.
Proc Natl Acad Sci U S A ; 113(16): 4434-9, 2016 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-27044088

RESUMO

Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.


Assuntos
Anemia de Diamond-Blackfan/metabolismo , Fator de Transcrição GATA1/metabolismo , Mutação , Elementos de Resposta , Transcrição Genética , Anemia de Diamond-Blackfan/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Motivos de Nucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
10.
Cell Stem Cell ; 18(1): 73-78, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26607381

RESUMO

Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.


Assuntos
Eritrócitos/citologia , Células-Tronco/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Citocinas/metabolismo , Células-Tronco Embrionárias/citologia , Sangue Fetal/citologia , Técnicas Genéticas , Variação Genética , Genoma Humano , Células-Tronco Hematopoéticas/citologia , Hemoglobinas/análise , Humanos , Mutação , Células-Tronco Pluripotentes/citologia , Medicina Regenerativa/métodos
11.
Biochim Biophys Acta ; 1853(1): 89-100, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25311384

RESUMO

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths worldwide; PDAC is characterized by poor prognosis, resistance to conventional chemotherapy and high mortality rate. TP53 tumor suppressor gene is frequently mutated in PDAC, resulting in the accumulation of mutated protein with potential gain-of-function (GOF) activities, such as genomic instability, hyperproliferation and chemoresistance. The purpose of this study was to assess the relevance of the p53 status on the PDAC cells response to the standard drug gemcitabine. We also examined the potential therapeutic effect of p53-reactivating molecules to restore the mutant p53 function in GEM treated PDAC cells. We showed that gemcitabine stabilized mutant p53 protein in the nuclei and induced chemoresistance, concurrent with the mutant p53-dependent expression of Cdk1 and CCNB1 genes, resulting in a hyperproliferation effect. Despite the adverse activation of mutant p53 by gemcitabine, simultaneous treatment of PDAC cells with gemcitabine and p53-reactivating molecules (CP-31398 and RITA) reduced growth rate and induced apoptosis. This synergistic effect was observed in both wild-type and mutant p53 cell lines and was absent in p53-null cells. The combination drug treatment induced p53 phosphorylation on Ser15, apoptosis and autophagosome formation. Furthermore, pharmacological inhibition of autophagy further increased apoptosis stimulated by gemcitabine/CP-31398 treatment. Together, our results show that gemcitabine aberrantly stimulates mutant p53 activity in PDAC cells identifying key processes with potential for therapeutic targeting. Our data also support an anti-tumoral strategy based on inhibition of autophagy combined with p53 activation and standard chemotherapy for both wild-type and mutant p53 expressing PDACs.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Apoptose/efeitos dos fármacos , Autofagia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias Pancreáticas/genética , Fosforilação , Pirimidinas/farmacologia
12.
Biochim Biophys Acta ; 1853(3): 549-60, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25533084

RESUMO

Onconase® (ONC) is a member of the RNase super-family that is secreted in oocytes and early embryos of Rana pipiens. Over the last years, research interest about this small and basic frog RNase, also called ranpirnase, constantly increased because of its high cytotoxicity and anticancer properties. Onconase is currently used in clinical trials for cancer therapy; however, the precise mechanisms determining cytotoxicity in cancer cells have not yet been fully investigated. In the present manuscript, we evaluate the antitumoral property of onconase in pancreatic adenocarcinoma cells and in non-tumorigenic cells as a control. We demonstrate that ONC stimulates a strong antiproliferative and proapoptotic effect in cancer cells by reporting for the first time that ONC triggers Beclin1-mediated autophagic cancer cell death. In addition, ONC inhibits the expression of mitochondrial uncoupling protein 2 (UCP2) and of manganese-dependent superoxide dismutase (MnSOD) triggering mitochondrial superoxide ion production. ONC-induced reactive oxygen species (ROS) are responsible for Akt/mTOR pathway stimulation determining the sensitivity of cancer cells to mTOR inhibitors and lessening autophagic stimulation. This indicates ROS/Akt/mTOR axis as a strategy adopted by cancer cells to reduce ONC-mediated cytotoxic autophagy stimulation. In addition, we demonstrate that ONC can sensitize pancreatic cancer cells to the standard chemotherapeutic agent gemcitabine allowing a reduction of drug concentration when used in combination settings, thus suggesting a lowering of chemotherapy-related side effects. Altogether, our results shed more light on the mechanisms lying at the basis of ONC antiproliferative effect in cancer cells and support its potential use to develop new anticancer strategies.


Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/patologia , Ribonucleases/farmacologia , Adenocarcinoma/metabolismo , Células Cultivadas , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteína Oncogênica v-akt/metabolismo , Neoplasias Pancreáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Am J Cancer Res ; 4(6): 907-15, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25520878

RESUMO

Since target therapy with mTOR inhibitors plays an important role in the current management of clear cell renal cell carcinoma (RCC), there is an increasing demand for predictive biomarkers, which may help to select patients that are most likely to benefit from personalized treatment. When dealing with formalin-fixed paraffin-embedded (FFPE) cancer tissue specimens, several techniques may be used to identify potential molecular markers, yielding different outcome in terms of accuracy. We sought to investigate and compare the capability of three main techniques to detect molecules performing an active function in mTOR pathway in RCC. Immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) analyses were performed on FFPE RCC tissue specimens from 16 patients by using the following mTOR pathway-related: mTOR (Ser235/236), phospho-mTOR (p-mTOR/Ser2448), phospho-p70S6k (p-p70S6k/Thr389), both monoclonal and polyclonal, phospho-S6Rb (p-S6Rb) and phospho-4EBP1 (p-4EBP1/Thr37/46). No single molecule was simultaneously revealed by all three techniques. Only p-p70S6k was detected by two methods (IHC and IF) using a monoclonal antibody. The other molecules were detected exclusively by one technique, as follows: p-mTOR and polyclonal p-p70S6K by IHC, p70S6K, p-S6Rb and p-4EBP1 by WB, and, finally, mTOR by IF. We found significant differences in detecting mTOR pathway-related active biomarkers by using three common techniques such as IHC, WB and IF on RCC samples. Such results have important implications in terms of predictive biomarker testing, and need to be related to clinical end-points such as responsiveness to targeted drugs by prospective studies.

14.
Front Immunol ; 5: 340, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25101082

RESUMO

The Wiskott-Aldrich syndrome (WAS) is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR and BCR). We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing (NGS), the T cell receptor ß and B cell immunoglobulin heavy chain (IGH) repertoires of eight patients with WAS and six controls were sequenced. Clonal expansions were identified within memory CD4(+) cells as well as in total, naïve and memory CD8(+) cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using NGS.

15.
Cancer Lett ; 349(2): 107-13, 2014 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-24755453

RESUMO

Among the wide family of microRNAs, microRNA 23b (miR-23b) intriguingly assumes opposite roles on regulation of reactive oxygen species (ROS) and on the development of human cancers. In this review we describe novel findings concerning the molecular events involved in miR-23b gene activation or repression and in both ROS regulation and tumour development. In particular, we define the molecular targets of miR-23b that determine its function as either a tumour suppressor or oncomir in different cell types. Finally, we analyze the involvement of miR-23b in cancer cell metabolism, including autophagy, and in biomarker signatures of microRNAs allowing a prognostic and therapeutic evaluation in various human cancers.


Assuntos
MicroRNAs/biossíntese , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias/genética
16.
Biochim Biophys Acta ; 1843(5): 976-84, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24487065

RESUMO

Among the large number of variants belonging to the pancreatic-type secretory ribonuclease (RNase) superfamily, bovine pancreatic ribonuclease (RNase A) is the proto-type and bovine seminal RNase (BS-RNase) represents the unique natively dimeric member. In the present manuscript, we evaluate the anti-tumoral property of these RNases in pancreatic adenocarcinoma cell lines and in nontumorigenic cells as normal control. We demonstrate that BS-RNase stimulates a strong anti-proliferative and pro-apoptotic effect in cancer cells, while RNase A is largely ineffective. Notably, we reveal for the first time that BS-RNase triggers Beclin1-mediated autophagic cancer cell death, providing evidences that high proliferation rate of cancer cells may render them more susceptible to autophagy by BS-RNase treatment. Notably, to improve the autophagic response of cancer cells to BS-RNase we used two different strategies: the more basic (as compared to WT enzyme) G38K mutant of BS-RNase, known to interact more strongly than wt with the acidic membrane of cancer cells, or BS-RNase oligomerization (tetramerization or formation of larger oligomers). Both mutant BS-RNase and BS-RNase oligomers potentiated autophagic cell death as compared to WT native dimer of BS-RNase, while the various RNase A oligomers remained completely ineffective. Altogether, our results shed more light on the mechanisms lying at the basis of BS-RNase antiproliferative effect in cancer cells, and support its potential use to develop new anti-cancer strategies.


Assuntos
Adenocarcinoma/patologia , Proteínas Reguladoras de Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Neoplasias Pancreáticas/patologia , Ribonuclease Pancreático/farmacologia , Sêmen/enzimologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Proteína Beclina-1 , Bovinos , Linhagem Celular Tumoral , Masculino
17.
Cell Mol Life Sci ; 71(7): 1171-90, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23807210

RESUMO

An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.


Assuntos
Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Canais Iônicos/química , Canais Iônicos/genética , Metformina/farmacologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Proteína Desacopladora 2
18.
J Allergy Clin Immunol ; 131(4): 1136-45, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23384681

RESUMO

BACKGROUND: Mutations in Janus kinase 3 (JAK3) are a cause of severe combined immunodeficiency, but hypomorphic JAK3 defects can result in a milder clinical phenotype, with residual development and function of autologous T cells. Maternal T-cell engraftment is a common finding in infants with severe combined immunodeficiency but is not typically observed in patients with residual T-cell development. OBJECTIVE: We sought to study in detail the molecular, cellular, and humoral immune phenotype and function of 3 patients with hypomorphic JAK3 mutations. METHODS: We analyzed the distribution and function of T and B lymphocytes in 3 patients and studied the in vitro and in vivo responses of maternal T lymphocytes in 1 patient with maternal T-cell engraftment and residual production of autologous T lymphocytes. RESULTS: B cells were present in normal numbers but with abnormal distribution of marginal zone-like and memory B cells. B-cell differentiation to plasmablasts in vitro in response to CD40 ligand and IL-21 was abolished. In 2 patients the T-cell repertoire was moderately restricted. Surprisingly, 1 patient showed coexistence of maternal and autologous T lymphocytes. By using an mAb recognizing the maternal noninherited HLA-A2 antigen, we found that autologous cells progressively accumulated in vivo but did not compete with maternal cells in vitro. CONCLUSION: The study of 3 patients with hypomorphic JAK3 mutations suggests that terminal B-cell maturation/differentiation requires intact JAK3 function, even if partially functioning T lymphocytes are present. Maternal T-cell engraftment can occur in patients with JAK3 mutations despite the presence of autologous T cells.


Assuntos
Linfócitos B/patologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Variação Genética/imunologia , Janus Quinase 3/genética , Imunodeficiência Combinada Severa/genética , Linfócitos T/patologia , Linfócitos B/imunologia , Sequência de Bases , Diferenciação Celular/imunologia , Proliferação de Células , Pré-Escolar , Feminino , Humanos , Imunidade Materno-Adquirida , Lactente , Janus Quinase 3/imunologia , Masculino , Dados de Sequência Molecular , Linhagem , Cultura Primária de Células , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Transdução de Sinais , Linfócitos T/imunologia
19.
Biochim Biophys Acta ; 1833(3): 672-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23124112

RESUMO

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.


Assuntos
Adenocarcinoma/patologia , Autofagia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Canais Iônicos/antagonistas & inibidores , Iridoides/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colagogos e Coleréticos/farmacologia , Imunofluorescência , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 2
20.
Apoptosis ; 18(3): 337-46, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23238993

RESUMO

TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática , Furanos/farmacologia , Humanos , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/genética
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