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1.
Mol Cell ; 83(10): 1743-1760.e11, 2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37116497

RESUMO

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.


Assuntos
ADP-Ribosilação , Histonas , Histonas/genética , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Cromatina , Dano ao DNA , Anticorpos/genética , Transdução de Sinais
2.
Nucleic Acids Res ; 52(12): 6994-7011, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38828775

RESUMO

The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance.


Assuntos
Proteína BRCA1 , DNA Polimerase II , Replicação do DNA , Inibidores de Poli(ADP-Ribose) Polimerases , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase II/genética , Replicação do DNA/efeitos dos fármacos , Dano ao DNA , Linhagem Celular Tumoral , Recombinação Homóloga/genética , Recombinação Homóloga/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética
3.
Proc Natl Acad Sci U S A ; 116(33): 16479-16488, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346090

RESUMO

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-ß. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMP-induced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novel mechanism for therapeutic interventions in STING-associated inflammatory diseases.


Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Sequência Conservada , Regulação para Baixo , Evolução Molecular , Células HeLa/metabolismo , Humanos , Inflamação/patologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Lisossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Nucleotídeos Cíclicos/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Molécula 1 de Interação Estromal/metabolismo
4.
BMC Cancer ; 18(1): 872, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30185144

RESUMO

BACKGROUND: Adipose-tissue stem cells (ASCs) are subject of intensive research since their successful use in regenerative therapy. The drawback of ASCs is that they may serve as stroma for cancer cells and assist tumor progression. It is disquieting that ASCs frequently undergo genetic and epigenetic changes during their in vitro propagation. In this study, we describe the polyploidization of murine ASCs and the accompanying phenotypical, gene expressional and functional changes under long term culturing. METHODS: ASCs were isolated from visceral fat of C57BL/6 J mice, and cultured in vitro for prolonged time. The phenotypical changes were followed by microscopy and flow cytometry. Gene expressional changes were determined by differential transcriptome analysis and changes in protein expression were shown by Western blotting. The tumor growth promoting effect of ASCs was examined by co-culturing them with 4 T1 murine breast cancer cells. RESULTS: After five passages, the proliferation of ASCs decreases and cells enter a senescence-like state, from which a proportion of cells escape by polyploidization. The resulting ASC line is susceptible to adipogenic, osteogenic and chondrogenic differentiation, and expresses the stem cell markers CD29 and Sca-1 on an upregulated level. Differential transcriptome analysis of ASCs with normal and polyploid karyotype shows altered expression of genes that are involved in regulation of cancer, cellular growth and proliferation. We verified the increased expression of Klf4 and loss of Nestin on protein level. We found that elevated production of insulin-like growth factor 1 by polyploid ASCs rendered them more potent in tumor growth promotion in vitro. CONCLUSIONS: Our model indicates how ASCs with altered genetic background may support tumor progression.


Assuntos
Tecido Adiposo/citologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/biossíntese , Poliploidia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Antígenos de Superfície/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Cariótipo , Fator 4 Semelhante a Kruppel , Camundongos , Transcriptoma
5.
Int J Mol Sci ; 18(10)2017 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-28991167

RESUMO

Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.


Assuntos
Membranas Mitocondriais/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Curcumina/análogos & derivados , Curcumina/farmacologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
6.
J Biomol NMR ; 66(4): 227-232, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27885546

RESUMO

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.


Assuntos
Anticorpos Monoclonais/química , Ligantes , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio/química , Proteínas/química , Desenho de Fármacos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
7.
Cytotherapy ; 18(3): 360-70, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26857229

RESUMO

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have powerful immunosuppressive activity. This function of MSCs is attributed to plethora of the expressed immunosuppressive factors, such as galectin-1 (Gal-1), a pleiotropic lectin with robust anti-inflammatory effect. Nevertheless, whether Gal-1 renders or contributes to the immunosuppressive effect of MSCs has not been clearly established. Therefore, this question was the focus of a complex study. METHODS: MSCs were isolated from bone marrows of wild-type and Gal-1 knockout mice and their in vitro anti-proliferative and apoptosis-inducing effects on activated T cells were examined. The in vivo immunosuppressive activity was tested in murine models of type I diabetes and delayed-type hypersensitivity. RESULTS: Both Gal-1-expressing and -deficient MSCs inhibited T-cell proliferation. Inhibition of T-cell proliferation by MSCs was mediated by nitric oxide but not PD-L1 or Gal-1. In contrast, MSC-derived Gal-1 triggered apoptosis in activated T cells that were directly coupled to MSCs, representing a low proportion of the T-cell population. Furthermore, absence of Gal-1 in MSCs did not affect their in vivo immunosuppressive effect. CONCLUSIONS: These results serve as evidence that Gal-1 does not play a role in the systemic immunosuppressive effect of MSCs. However, a local contribution of Gal-1 to modulation of T-cell response by direct cell-to-cell interaction cannot be excluded. Notably, this study serves a good model to understand how the specificity of a pleiotropic protein depends on the type and localization of the producing effector cell and its target.


Assuntos
Comunicação Celular/genética , Galectina 1/fisiologia , Fatores Imunológicos/fisiologia , Células-Tronco Mesenquimais/metabolismo , Animais , Apoptose/genética , Medula Óssea/metabolismo , Proliferação de Células/genética , Células Cultivadas , Galectina 1/genética , Fatores Imunológicos/genética , Imunossupressores/metabolismo , Ativação Linfocitária/genética , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia
8.
Acta Biol Hung ; 62(1): 106-11, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21388924

RESUMO

Galectin-1 (Gal-1), a mammalian lectin induces apoptosis of T lymphocytes. Contradictory data have resulted in confusing knowledge regarding mechanism of Gal-1 induced T-cell apoptosis. In this paper we aimed to resolve this controversy by comparing cell death induced by low (1.8 µM, lowGal-1) and high (18 µM, highGal-1) concentration of soluble Gal-1. We show that lowGal-1 and highGal-1 trigger phosphatidylserine exposure, generation of rafts and mitochondrial membrane depolarization. In contrast, lowGal-1 but not highGal-1 is dependent on the presence of p56lck and ZAP70 and activates caspase cascade. The results allow the conclusion that the cell-death mechanism strictly depends on the concentration of Gal-1.


Assuntos
Apoptose/efeitos dos fármacos , Galectina 1/farmacologia , Linfócitos T/efeitos dos fármacos , Apoptose/fisiologia , Caspases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/citologia
9.
Chembiochem ; 11(2): 228-34, 2010 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-19938027

RESUMO

Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.


Assuntos
Assialoglicoproteínas/metabolismo , Galectina 1/metabolismo , Glicoproteínas/metabolismo , Peptídeos/farmacologia , Tirosina/química , alfa-Fetoproteínas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Assialoglicoproteínas/antagonistas & inibidores , Fetuínas , Galectina 1/antagonistas & inibidores , Galectina 1/genética , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-Fetoproteínas/antagonistas & inibidores
10.
Front Oncol ; 10: 157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32133294

RESUMO

Background: The close proximity of adipose tissue and mammary epithelium predispose involvement of adipose cells in breast cancer development. Adipose-tissue stem cells (ASCs) contribute to tumor stroma and promote growth of cancer cells. In our previous study, we have shown that murine ASCs, which undergo polyploidization during their prolonged in vitro culturing, enhanced the proliferation of 4T1 murine breast cancer cells in IGF1 dependent manner. Aims: In the present study, our aim was to clarify the regulation of ASC-derived IGF1. Methods: 4T1 murine breast carcinoma cells were co-transplanted with visceral fat-derived ASCs (vASC) or with the polyploid ASC.B6 cell line into female BALB/c mice and tumor growth and lung metastasis were monitored. The conditioned media of vASCs and ASC.B6 cells were subjected to LC-MS/MS analysis and the production of IGFBP2 was verified by Western blotting. The regulatory effect was examined by adding recombinant IGFBP2 to the co-culture of ASC.B6 and 4T1. Akt/protein kinase B (PKB) activation was detected by Western blotting. Results: Polyploid ASCs promoted the tumor growth and metastasis more potently than vASCs with normal karyotype. vASCs produced the IGF1 regulator IGFBP2, which inhibited proliferation of 4T1 cells. Downregulation of IGFBP2 by polyploidization of ASCs and enhanced secretion of IGF1 allowed survival signaling in 4T1 cells, leading to Akt phosphorylation. Conclusions: Our results implicate that ASCs in the tumor microenvironment actively regulate the growth of breast cancer cells through the IGF/IGFBP system.

11.
Exp Hematol ; 35(2): 305-13, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17258079

RESUMO

OBJECTIVE: The immunosuppressive and anti-inflammatory activity of mammalian galectin-1 (Gal-1) has been well established in experimental in vivo animal models and in vitro studies. Since the proliferation and migration of leukocytes represent a necessary and important step in response to the inflammatory insult, we have investigated whether Gal-1 affects the mobilization of hematopoietic progenitor cells (HPC) induced by cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). METHODS: Bone marrow HPCs were mobilized with CY/G-CSF or CY/G-CSF plus human recombinant Gal-1 in BDF1 mice. Bone marrow (BM) and blood cells were taken at different time points and analyzed for their in vivo repopulating ability in lethally irradiated syngeneic animals. The number of myeloid progenitor cells in BM and blood samples was determined by colony-forming cell assay. Expression of surface markers (Sca-1, CD3epsilon, CD45R/B220, Ter-119, GR-1, and CD11b) on nucleated marrow cells was measured by flow cytometry. The lymphocytes, granulocytes, and monocytes in blood samples were counted after Giemsa staining. RESULTS: Gal-1 dramatically inhibited CY/G-CSF-induced HPC migration to the periphery as well as decreased peripheral neutrophilia and monocytosis in a dose- and time-dependent manner. In contrast, Gal-1 itself stimulated HPC expansion and accumulation within the BM. The presence of the lectin for inhibition of HPC mobilization was essential during the second half of the treatment. Moreover, Gal-1 inhbited transendothelial migration of BM-derived HPCs in response to SDF-1 in vitro. CONCLUSION: Gal-1 blocked BM progenitor cell migration induced by CY/G-CSF treatment, indicating a novel anti-inflammatory function of the lectin. We suggest that the inhibition of HPC mobilization occurs mainly via obstructing the transendothelial migration of BM-derived cells including primitive hematopoietic and committed myeloid progenitor cells and mature granulocytes and monocytes.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Galectina 1/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Biomarcadores , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/imunologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/antagonistas & inibidores , Ensaio de Unidades Formadoras de Colônias/métodos , Ciclofosfamida/farmacologia , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Células-Tronco Hematopoéticas/imunologia , Injeções Intraperitoneais , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Recombinantes/farmacologia , Fatores de Tempo
12.
Cell Signal ; 18(11): 1887-96, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16549336

RESUMO

The mechanism of apoptosis induced by human galectin-1, a mammalian beta-galactoside-binding protein with a remarkable cytotoxic effect on activated peripheral T cells and tumor T cell lines has been extensively investigated in this study. Here we first show that galectin-1 initiate the acid sphingomyelinase mediated release of ceramide and this event is critical in the further steps. Elevation of ceramide level coincides with exposure of phosphatidylserine on the outer cell membrane. The downstream events, decrease of Bcl-2 protein amount, depolarization of the mitochondria and activation of the caspase 9 and caspase 3 depend on production of ceramide. All downstream steps, including production of ceramide, require the generation of membrane rafts and the presence of two tyrosine kinases, p56(lck) and ZAP70. Based on our findings we suggest a model of the mechanism of galectin-1 triggered cell death.


Assuntos
Apoptose , Ceramidas/biossíntese , Galectina 1/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/fisiologia , Linhagem Celular , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo
13.
Immunobiology ; 220(4): 483-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25468561

RESUMO

Secreted, extracellular galectin-1 (exGal-1) but not intracellular Gal-1 (inGal-1) has been described as a strong immunosuppressive protein due to its major activity of inducing apoptosis of activated T-cells. It has previously been reported that T-cells express Gal-1 upon activation, however its participation in T-cell functions has remained largely elusive. To determine function of Gal-1 expressed by activated T-cells we have carried out a series of experiments. We have shown that Gal-1, expressed in Gal-1-transgenic Jurkat cells or in activated T-cells, remained intracellularly indicating that Gal-1-induced T-cell death was not a result of an autocrine effect of the de novo expressed Gal-1. Rather, a particular consequence of the inGal-1 expression was that T-cells became more sensitive to exGal-1 added either as a soluble protein or bound to the surface of a Gal-1-secreting effector cell. This was also verified when the susceptibility of activated T-cells from wild type or Gal-1 knockout mice to Gal-1-induced apoptosis were compared. Murine T-cells expressing Gal-1 were more sensitive to the cytotoxicity of the exGal-1 than their Gal-1 knockout counterparts. We also conducted a study with activated T-cells from patients with systemic lupus erythematosus (SLE), a disease in which dysregulated T-cell apoptosis has been well described. SLE T-cells expressed lower amounts of Gal-1 than healthy T-cells and were less sensitive to exGal-1. These results suggested a novel role of inGal-1 in T-cells as a regulator of T-cell response to exGal-1, and its likely contribution to the mechanism in T-cell apoptosis deficiency in lupus.


Assuntos
Galectina 1/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Estudos de Casos e Controles , Linhagem Celular , Galectina 1/genética , Galectina 1/farmacologia , Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Células Jurkat , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Linfócitos T/efeitos dos fármacos
14.
Stem Cells Dev ; 24(18): 2171-80, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26153898

RESUMO

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2≥MSC5>MSC4>MSC3 >> MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN-γ and TNF-α resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions.


Assuntos
Adipogenia/fisiologia , Citocinas/imunologia , Tolerância Imunológica/imunologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Dinoprostona/imunologia , Inflamação/imunologia , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologia
15.
Mol Med Rep ; 12(3): 4610-1619, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26126392

RESUMO

Translational research in radiation oncology is important for the detection of adverse radiation effects, cellular responses, and radiation modifications, and may help to improve the outcome of radiation therapy in patients with cancer. The present study aimed to optimize and validate a real­time label­free assay for the dynamic monitoring of cellular responses to ionizing radiation. The xCELLigence system is an impedance­based platform that provides continuous information on alterations in cell size, shape, adhesion, proliferation, and survival. In the present study, various malignant human primary fibroblast cells (U251, GBM2, MCF7, A549, HT­29) were exposed to 0, 5 and 10 Gy of Cobalt60 radiation. As well as the xCELLigence system, cell survival and proliferation was evaluated using the following conventional end­point cell­based methods: Clonogenic, MTS, and lactate dehydrogenase assays, and apoptosis was detected by fluorescence­activated cell sorting. The effects of ionizing radiation were detected for each cell line using impedance monitoring. The real­time data correlated with the colony forming assay results. At low cell densities (1,000­2,000 cells/well) the impedance­based method was more accurate at monitoring dose­dependent changes in the malignant human primary fibroblast cell lines, as compared with the end­point assays. The results of the present study demonstrated that the xCELLigence system may be a reliable and rapid diagnostic method for the monitoring of dynamic cell behavior following radiation. In addition, the xCELLigence system may be used to investigate the cellular mechanisms underlying the radiation response, as well as the time­dependent effects of radiation on cell proliferation and viability.


Assuntos
Apoptose/efeitos da radiação , Bioensaio/instrumentação , Fibroblastos/efeitos da radiação , Raios gama , Adesão Celular/efeitos da radiação , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Forma Celular/efeitos da radiação , Tamanho Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Impedância Elétrica , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , L-Lactato Desidrogenase/metabolismo , Especificidade de Órgãos , Cultura Primária de Células , Fatores de Tempo
16.
Immunol Lett ; 82(1-2): 149-54, 2002 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-12008046

RESUMO

Galectin-1 (Gal-1) is an endogenous mammalian S-type lectin with highly pleiotropic effect on different tissues. The viability of the lymphoid cells is reduced by gal-1 by triggering apoptosis, however, the mechanism of the gal-1 induced apoptosis is still under investigation. The receptor tyrosine phosphatase, CD45, a heavily glycosylated cell surface molecule binds to gal-1 with high affinity, however, its contribution to the gal-1 induced apoptosis is still controversial. In this study we show that galectin-1 binds to cells deficient for CD45, although CD45 is one of the galectin-1-binding cell surface proteins on T cells. Moreover, the CD45 deficient Jurkat variant, J45.01 responds readily with tyrosine phosphorylation and subsequent apoptosis to galectin-1 treatment in a similar degree as its wild type counterpart, Jurkat does. These results strongly indicate that CD45 is not the receptor via gal-1 mediates the apoptotic signal into the cells as it was suggested in previous studies.


Assuntos
Apoptose , Hemaglutininas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Endocitose , Galectina 1 , Galectinas , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/fisiologia , Fosforilação , Fosfotirosina/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Células Tumorais Cultivadas
17.
Immunol Lett ; 91(1): 17-21, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14757365

RESUMO

Lysophospholipids, particularly lysophosphatidylcholine (lyso-PC), have been implicated in modulating T cell functions at the sites of inflammation and atherosclerosis. Although the chemotactic and immunomodulatory effects are well documented, the exact signaling pathway of lyso-PC action is poorly defined. In this work, we studied the earliest biochemical events in T cells triggered by lyso-PC. A marked and immediate tyrosine phosphorylation was induced in the leukemic T cell line, Jurkat. Phosphorylation of cellular substrates included src family kinase, p56(lck) and syk family kinase, ZAP70. The lyso-PC induced tyrosine phosphorylation was largely dependent on the presence of functional p56(lck). Tyrosine phosphorylation was followed by the elevation of intracellular Ca(2+) concentration. The magnitude of the mobilization of the intracellular Ca(2+) was similar in the absence of the p56(lck) activity in JCaM1.6 cells as in Jurkat cells, however, it was slightly but reproducibly delayed compared to that in the wild type cells. Inhibition of the Ser/Thr kinases and tyrosine kinases with staurosporine and genistein, respectively, decreased the rise in the intracellular Ca(2+) content. Moreover, pertussis toxin completely blocked the Ca(2+) signal supporting the role of the G-protein coupled LPC receptor in this event.


Assuntos
Cálcio/metabolismo , Lisofosfatidilcolinas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T/enzimologia , Humanos , Células Jurkat , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo
18.
Int J Oncol ; 20(3): 563-70, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11836569

RESUMO

The fermented wheat germ extract (code name: MSC, trade name: Avemar), with standardized benzoquinone content has been shown to inhibit tumor propagation and metastases formation in vivo. The aim of this study was to understand the molecular and cellular mechanisms of the anti-tumor effect of MSC. Therefore, we have designed in vitro model experiments using T and B tumor lymphocytic cell lines. Tyrosine phosphorylation of intracellular proteins and elevation of the intracellular Ca2+ concentration were examined using immunoblotting with anti-phosphotyrosine antibody and cytofluorimetry by means of Ca2+ sensitive fluorescence dyes, Fluo-3AM and FuraRed-AM, respectively. Apoptosis was measured with cytofluorimetry by staining the DNA with propidium iodide and detecting the cell population. The level of the cell surface MHC class I molecules was analysed with indirect immunofluorescence on cytofluorimeter using a monoclonal antibody to the non-polymorphic region of the human MHC class I. MSC stimulated tyrosine phosphorylation of intracellular proteins and the influx of extracellular Ca2+ resulted in elevation of intracellular Ca2+ concentration. Prominent apoptosis of 20-40% was detected upon 24 h of MSC treatment of the cell lines. As a result of the MSC treatment, the amount of the cell surface MHC class I proteins was downregulated by 70-85% compared to the non-stimulated control. MSC did not induce a similar degree of apoptosis in healthy peripheral blood mononuclear cells. Inhibition of the cellular tyrosine phosphatase activity or Ca2+ influx resulted in the opposite effect increasing or diminishing the Avemar induced apoptosis as well as the MHC class I downregulation, respectively. A benzoquinone component (2,6-dimethoxi-p-benzoquinone) in MSC induced similar apoptosis and downregulation of the MHC class I molecules in the tumor T and B cell lines to that of MSC. These results suggest that MSC acts on lymphoid tumor cells by reducing MHC class I expression and selectively promoting apoptosis of tumor cells on a tyrosine phosphorylation and Ca2+ influx dependent way. One of the components in MSC, 2,6-dimethoxi-p-benzoquinone was shown to be an important factor in MSC mediated cell response.


Assuntos
Apoptose , Linfócitos B/citologia , Regulação para Baixo , Genes MHC Classe I/genética , Lectinas/farmacologia , Extratos Vegetais/farmacologia , Linfócitos T/citologia , Triticum/química , Benzoquinonas/farmacologia , Cálcio/metabolismo , Divisão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/biossíntese , Leucócitos Mononucleares/metabolismo , Fenótipo , Fosforilação , Lectinas de Plantas , Fatores de Tempo , Células Tumorais Cultivadas
19.
Mol Immunol ; 57(2): 302-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24231767

RESUMO

One prominent immunoregulatory function of galectin-1 (Gal-1), a ß-galactoside binding mammalian lectin, is induction of apoptosis in activated T-cells by a process depending on the activity of Src family tyrosine kinase, Lck. Although the requirement for Lck in Gal-1 induced T-cell death and the ability of Gal-1 to affect the membrane localization of extracellular Gal-1-binding proteins have been well documented, the consequence of the complex and related reorganization of extra- and intracellular signaling components upon Gal-1 treatment of T-cells has not yet been revealed. Therefore, we have analyzed the plasma membrane movement of Lck upon Gal-1 triggered signaling, and the significance of this event in Gal-1 induced T-cell death. Non-receptor tyrosine kinase, Lck primarily localized in the synapse of tumor cell-T-cell during 15 min of the established direct cell contact. Later, after 30 min, a lateral segregation of Lck from the cell synapse was observed. The migration of Lck to the opposite of the cell contact apparently depended on the expression and cell surface presentation of Gal-1 on the effector (tumor) cells and was accompanied by phosphorylation on the negative regulatory tyrosine residue, Tyr505. Receptor tyrosine phosphatase, CD45 played crucial role in this event since CD45 deficiency or inhibition of its phosphatase activity resulted in the failure of Lck membrane movement. Level of the Gal-1-binding glycolipid GM1 ganglioside also essentially regulated Lck localization. Segregation of Lck and Gal-1 induced apoptosis was diminished in T-cells with low GM1 expression compared to T-cells with high GM1. Our results show that spatial regulation of Lck by CD45 and GM1 ganglioside determines the outcome of apoptotic response to Gal-1 and this local regulation may occur only upon intimate effector (Gal-1 expressing) cell-T-cell attachment.


Assuntos
Apoptose/imunologia , Gangliosídeo G(M1)/metabolismo , Galectina 1/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/antagonistas & inibidores , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Fosforilação , Transdução de Sinais/imunologia
20.
Stem Cells Dev ; 21(5): 814-28, 2012 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-22149974

RESUMO

Although mesenchymal stem cells (MSCs) of distinct tissue origin have a large number of similarities and differences, it has not been determined so far whether tissue-resident MSCs are the progenies of one ancestor cell lineage or the results of parallel cell developmental events. Here we compared the expression levels of 177 genes in murine MSCs derived from adult and juvenile bone marrow and adult adipose tissue, as well as juvenile spleen, thymus, and aorta wall by quantitative real-time polymerase chain reaction and the results were partially validated at protein level. All MSC lines uniformly expressed a large set of genes including well-known mesenchymal markers, such as α-smooth muscle actin, collagen type I α-chain, GATA6, Mohawk, and vimentin. In contrast, pluripotency genes and the early mesodermal marker T-gene were not expressed. On the other hand, different MSC lines consistently expressed distinct patterns of Hox genes determining the positional identity of a given cell population. Moreover, MSCs of different origin expressed a few other transcription factors also reflecting their topological identity and so the body segment or organ to which they normally contributed in vivo: (1) thymus-derived cells specifically expressed Tbx5 and Pitx2; (2) spleen-derived MSCs were characterized with Tlx1 and Nkx2.5; (3) Pitx1 designated femoral bone marrow cells and (4) En2 appeared in aorta wall-derived MSCs. Thus, MSCs exhibited topographic identity and memory even after long-term cultivation in vitro. On the basis of these results, we suggest that postnatal MSCs isolated from different anatomical sites descend from precursor cells developing in the postsegmentation mesoderm.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mesoderma/citologia , Tecido Adiposo/citologia , Tecido Adiposo/crescimento & desenvolvimento , Animais , Aorta/citologia , Aorta/crescimento & desenvolvimento , Western Blotting , Células da Medula Óssea/citologia , Linhagem da Célula/genética , Células Cultivadas , Análise por Conglomerados , Citometria de Fluxo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/crescimento & desenvolvimento , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimento , Fatores de Tempo
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