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1.
J Biol Chem ; 295(50): 17071-17082, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33023909

RESUMEN

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Acilación , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosforilación , Serina , Molécula de Interacción Estromal 1/genética
2.
Dev Growth Differ ; 63(2): 116-126, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33540477

RESUMEN

Recently, the fields of embryology, developmental biology, stem cell biology, and cell reprogramming, have intersected with synthetic embryo systems (SESs) from cultured cells. Among such SESs, several approaches have engaged early-embryo-like cells, cells with atypical potency, or assembled traditional in vitro stem cell populations with synergy, to advance life discovery systems that may yield emergent knowledge and biotechnical advance. Such models center on the competent generation of blastocyst-like and post-implantation embryo-like forms. Our group, and several others have recently pioneered unique SES strategies covering a broad spectrum of key early embryo-like developmental stages and features to seed an emerging SES field. Herein, we provide a comprehensive perspective of synthetic embryology and the powerful promise that excites us.


Asunto(s)
Técnicas de Cultivo de Célula , Embrión de Mamíferos/citología , Modelos Biológicos , Animales , Diferenciación Celular , Humanos
3.
J Biol Chem ; 294(51): 19577-19588, 2019 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-31723030

RESUMEN

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.


Asunto(s)
Muerte Celular , Regulación Enzimológica de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Quinasas Asociadas a rho/metabolismo , Amidas/farmacología , Diferenciación Celular , Membrana Celular/metabolismo , Supervivencia Celular , Citosol/metabolismo , Endodermo/citología , Humanos , Concentración de Iones de Hidrógeno , Mesodermo/citología , Metilaminas/farmacología , Necrosis , Fosforilación , Piridinas/farmacología
4.
Proc Natl Acad Sci U S A ; 113(44): 12478-12483, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27738243

RESUMEN

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling.


Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Factor Inhibidor de Leucemia/farmacología , Lisofosfolípidos/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Ácido Ascórbico/farmacología , Línea Celular , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones Endogámicos C57BL , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Vitaminas/farmacología
5.
Proc Natl Acad Sci U S A ; 113(46): 13057-13062, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27794120

RESUMEN

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miositis Osificante , Proteínas Smad/metabolismo , Receptores de Activinas Tipo I/genética , Adolescente , Adulto , Animales , Línea Celular , Reprogramación Celular , Senescencia Celular , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Femenino , Humanos , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Mutación , Miositis Osificante/genética , Transducción de Señal
6.
Proc Natl Acad Sci U S A ; 112(15): 4666-71, 2015 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-25825768

RESUMEN

NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.


Asunto(s)
Proliferación Celular/genética , Reprogramación Celular/genética , Proteínas de Homeodominio/genética , Mutación , Células Madre Pluripotentes/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Conformación de Ácido Nucleico , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Transfección
7.
Oncol Lett ; 26(6): 520, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37927418

RESUMEN

Gefitinib is a key drug used in the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib therapy is superior to conventional chemotherapy for the progression-free survival rate of patients with EGFR mutations. However, 10-26% of patients develop grade 3 or higher hepatotoxicity during gefitinib treatment; therefore, the development of preclinical tests for hepatotoxicity prior to clinical use is desirable. The present study evaluated the use of induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iPSC-heps), as a platform for preclinical test development. Patient-derived iPSCs were generated by reprogramming peripheral blood mononuclear cells obtained from two groups of gefitinib-treated patients with severe hepatotoxicity [toxicity group (T group)] or mild hepatotoxicity [no clinical toxicity group (N group)]. To examine the hepatotoxicity, the iPSCs from both T and N groups were differentiated into hepatocytes to obtain iPSC-heps. Differentiation was confirmed by measuring the expression levels of hepatocyte markers, such as albumin or α-fetoprotein, via western blotting and quantitative PCR analyses. Cytotoxicity in iPSCs and iPSC-heps after gefitinib treatment was evaluated using a lactate dehydrogenase release assay. The gefitinib-induced cytotoxicity in iPSCs from the T group was significantly higher than that from the N group, whereas there were no significant differences between the groups of iPSC-heps. These results suggested that using iPSCs in preclinical assessment may be a good indicator for the prediction of gefitinib-induced cytotoxicity in clinical use.

8.
Dev Cell ; 58(16): 1477-1488.e5, 2023 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-37354899

RESUMEN

Biological patterning events that occur early in development establish proper tissue morphogenesis. Identifying the mechanisms that guide these patterning events is necessary in order to understand the molecular drivers of development and disease and to build tissues in vitro. In this study, we use an in vitro model of gastrulation to study the role of tight junctions and apical/basolateral polarity in modulating bone morphogenic protein-4 (BMP4) signaling and gastrulation-associated patterning in colonies of human pluripotent stem cells (hPSCs). Disrupting tight junctions via knockdown (KD) of the scaffolding tight junction protein-1 (TJP1, also known as ZO1) allows BMP4 to robustly and ubiquitously activate pSMAD1/5 signaling over time, resulting in loss of the patterning phenotype and marked differentiation bias of pluripotent stem cells to primordial germ cell-like cells (PGCLCs). These findings give important insights into how signaling events are regulated and lead to spatial emergence of diverse cell types in vitro.


Asunto(s)
Gastrulación , Células Madre Pluripotentes , Humanos , Linaje de la Célula , Gastrulación/fisiología , Diferenciación Celular , Células Germinativas , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo
9.
Heliyon ; 8(12): e12009, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36506411

RESUMEN

Non-thermal atmospheric-pressure plasma has been used for biological applications, including sterilization and stimulation of cell growth and differentiation. Here, we demonstrate that plasma exposure influences the differentiation pattern of human induced pluripotent stem cells (hiPSCs). We treated hiPSCs with dielectric barrier-discharge air plasma and found an exposure dose that does not kill hiPSCs. Immunohistochemical staining for E-CADHERIN showed that the exposure affected cell-cell attachment and doubled the average size of the hiPSCs. Analysis of mRNAs in embryoid bodies (EBs) from plasma-treated hiPSCs revealed repression of ectoderm genes, including WNT1, and increased expression of mesoderm genes. Importantly, hiPSCs deficient in DNA repair only displayed minimal damage after plasma exposure. Collectively, our results suggest that plasma treatment can be another tool for directing the fate of pluripotent stem cells without disrupting their genomic integrity.

10.
Stem Cell Reports ; 16(5): 1197-1209, 2021 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-33891866

RESUMEN

Recently, a new wave of synthetic embryo systems (SESs) has been established from cultured cells for efficient and ethical embryonic development research. We recently reported our epiblast stem cell (EPISC) reprogramming SES that generates numerous blastocyst (BC)-like hemispheres (BCLH) with pluripotent and extraembryonic cell features detected by microscopy. Here, we further explored the system over key time points with single-cell RNA-sequencing analysis. We found broad induction of the 2C-like reporter MERVL and RNA velocities diverging to three major cell populations with gene expression profiles resembling those of pluripotent epiblast, primitive endoderm, and trophectoderm. Enrichment of those three induced BC-like cell fates involved key gene-regulatory networks, zygotic genome activation-related genes, and specific RNA splicing, and many cells closely resembled in silico models. This analysis confirms the induction of extraembryonic cell populations during EPISC reprogramming. We anticipate that our unique BCLH SES and rich dataset may uncover new facets of cell potency, improve developmental biology, and advance biomedicine.


Asunto(s)
Blastocisto/citología , Reprogramación Celular , Implantación del Embrión , Células Madre Embrionarias/citología , Estratos Germinativos/citología , Animales , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Implantación del Embrión/efectos de los fármacos , Implantación del Embrión/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Factor Inhibidor de Leucemia/farmacología , Ratones Endogámicos C57BL , Modelos Biológicos , ARN/metabolismo , Factores de Tiempo
11.
Bone ; 153: 116129, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34311122

RESUMEN

Macrophages play crucial roles in many human disease processes. However, obtaining large numbers of primary cells for study is often difficult. We describe 2D and 3D methods for directing human induced pluripotent stem cells (hiPSCs) into macrophages (iMACs). iMACs generated in 2D culture showed functional similarities to human primary monocyte-derived M2-like macrophages, and could be successfully polarized into a M1-like phenotype. Both M1- and M2-like iMACs showed phagocytic activity and reactivity to endogenous or exogenous stimuli. In contrast, iMACs generated by a 3D culture system showed mixed M1- and M2-like functional characteristics. 2D-iMACs from patients with fibrodysplasia ossificans progressiva (FOP), an inherited disease with progressive heterotopic ossification driven by inflammation, showed prolonged inflammatory cytokine production and higher Activin A production after M1-like polarization, resulting in dampened responses to additional LPS stimulation. These results demonstrate a simple and robust way of creating hiPSC-derived M1- and M2-like macrophage lineages, while identifying macrophages as a source of Activin A that may drive heterotopic ossification in FOP.


Asunto(s)
Células Madre Pluripotentes Inducidas , Miositis Osificante , Osificación Heterotópica , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/metabolismo , Transducción de Señal
12.
Cell Transplant ; 29: 963689720970456, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33349053

RESUMEN

Miscarriage is the most common complication of pregnancy, and about 1% of pregnant women suffer a recurrence. Using a widely used mouse miscarriage model, we previously showed that intravenous injection of bone marrow (BM)-derived endothelial progenitor cells (EPCs) may prevent miscarriage. However, preparing enough BM-derived EPCs to treat a patient might be problematic. Here, we demonstrated the generation of mouse pluripotent stem cells (PSCs), propagation of sufficient PSC-derived cells with endothelial potential (PSC-EPs), and intravenous injection of the PSC-EPs into the mouse miscarriage model. We found that the injection prevented miscarriage. Three-dimensional reconstruction images of the decidua after tissue cleaning revealed robust fetomaternal neovascularization induced by the PSC-EP injection. Additionally, the injected PSC-EPs directly formed spiral arteries. These findings suggest that intravenous injection of PSC-EPs could become a promising remedy for recurrent miscarriage.


Asunto(s)
Aborto Habitual/prevención & control , Células Madre Pluripotentes/citología , Animales , Células Progenitoras Endoteliales/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Microscopía Fluorescente , Neovascularización Fisiológica/fisiología
13.
Stem Cell Reports ; 13(3): 485-498, 2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-31402336

RESUMEN

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.


Asunto(s)
Blastocisto/metabolismo , Células Madre Pluripotentes/citología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Blastocisto/citología , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario , Femenino , Genes Reporteros , Ratones , Ratones Endogámicos ICR , Células Madre Pluripotentes/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Útero/patología , Proteínas Señalizadoras YAP
15.
FEBS Lett ; 580(25): 5836-44, 2006 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-17027978

RESUMEN

Jab1 overexpression is observed in many human cancers, but its physiological significance remains to be investigated. We reduced the level of Jab1 expression in pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 by the RNA interference and found that Jab1-knockdown resulted in impaired cell proliferation and enhanced apoptosis regardless of the genotype of the tumor suppressor p53. This growth inhibition was rescued by the introduction of siRNA-resistant mouse Jab1 cDNA. Jab1-knocked-down cells expressed a higher level of c-myc, and additional depletion of c-myc rescued cells from Jab1-knockdown-mediated growth suppression. Thus, Jab1 overexpression contributes to pancreatic cancer cell proliferation and survival. Jab1 could be a novel target in cancer therapy.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Complejo del Señalosoma COP9 , Línea Celular Tumoral , Proliferación Celular , ADN de Neoplasias/genética , Fase G1 , Expresión Génica , Genes myc , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/genética
16.
FEBS Lett ; 579(5): 1047-54, 2005 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-15710389

RESUMEN

Jab1 interacts with a variety of cell cycle and signal transduction regulators to control cell proliferation, differentiation, and tumorigenesis. In this study, we employed a non-denaturing gel electrophoresis method to separate different Jab1-containing complexes, the COP9 signalosome complex and the small Jab1-containing subcomplex. The formation of the small Jab1 complex was dependent on a low cell density and anchorage to a solid support, and enhanced during the early G1 phase of the cell cycle, which was abrogated in ras-transformed cells. The small Jab1-containing subcomplex may be a novel mediator of anchorage and cell-cell contact-dependent signal transduction.


Asunto(s)
Ciclo Celular , Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas ras/metabolismo , Animales , Complejo del Señalosoma COP9 , Adhesión Celular , Recuento de Células , Línea Celular , Inhibición de Contacto , Péptidos y Proteínas de Señalización Intracelular , Ratones , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas , Unión Proteica , Proteínas ras/genética
17.
Curr Protoc Hum Genet ; 87: 21.2.1-21.2.21, 2015 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-26439714

RESUMEN

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1.


Asunto(s)
Técnicas de Reprogramación Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Plásmidos/genética , Técnicas de Cultivo de Célula , Separación Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Transfección
18.
Oncol Rep ; 11(2): 277-84, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14719054

RESUMEN

p27Kip1 belongs to the family of small polypeptides collectively termed cyclin-dependent kinase inhibitors, which negatively regulate the cell cycle progression. In various human cancers, the reduced p27Kip1 expression correlates well with poor prognosis. Recently, Jab1/CSN5, the fifth component of the COP9 signalosome complex, was found to specifically translocate p27Kip1 from the nucleus to the cytoplasm, and reduce the protein level of p27Kip1 by accelerating its degradation. In this study, we investigated the expression of p27Kip1 and Jab1 in 61 cases with pancreatic ductal adenocarcinoma. The p27Kip1 expression was positive in 41% (25/61) of the tumors. Of the 25 positive tumors, 12 cases had p27Kip1 positive expression mainly in the nucleus of the tumor cells, while 13 cases had p27Kip1 in the cytoplasm as well as in the nucleus. Among a variety of clinicopathological factors, only tumor status was inversely correlated with p27Kip1 expression (p=0.019). The Jab1 expression was detected both in the nucleus and the cytoplasm in almost all pancreatic cancer cells. The intensity of Jab1 expression in tumor cells, especially in the cytoplasm, was much stronger than in normal pancreatic ductal epithelial cells. The patients with positive p27Kip1 expression had significantly better prognosis than ones with negative p27Kip1 expression (p=0.008). Furthermore, 12 patients with exclusively nuclear p27Kip1 expression, but not 13 patients with both nuclear and cytoplasmic p27Kip1 expression, had significantly better prognosis than 36 patients with negative p27Kip1 expression (p=0.009). In multivariate survival analysis, localized p27Kip1 expression was an independent prognostic factor (p=0.016). The results of our study suggested that the mislocalization as well as the downregulation of p27Kip1 had significant prognostic value in pancreatic cancer and that Jab1 might play an important role in carcinogenesis of pancreatic cancer. Cell cycle control targeting p27Kip1 might be a promising future therapeutic modality against pancreatic cancer.


Asunto(s)
Adenocarcinoma/patología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Neoplasias Pancreáticas/patología , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Complejo del Señalosoma COP9 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Genes Supresores de Tumor , Humanos , Péptidos y Proteínas de Señalización Intracelular , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/cirugía , Péptido Hidrolasas , Modelos de Riesgos Proporcionales , Transporte de Proteínas , Análisis de Supervivencia
19.
Stem Cell Reports ; 3(2): 269-81, 2014 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-25254341

RESUMEN

Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca(2+)]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca(2+)]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca(2+). Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca(2+)]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na(+)-Ca(2+) exchange. These results suggest that LQTS mutations act partly on cytosolic Ca(2+) cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site.


Asunto(s)
Arritmias Cardíacas/patología , Calcio/metabolismo , Células Madre Pluripotentes Inducidas/citología , Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/metabolismo , Cafeína/farmacología , Diferenciación Celular , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Genotipo , Humanos , Recién Nacido , Persona de Mediana Edad , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Nifedipino/farmacología , Técnicas de Placa-Clamp , Fenotipo , Polimorfismo de Nucleótido Simple , Adulto Joven
20.
Cell Stem Cell ; 11(1): 91-9, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22770243

RESUMEN

Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Inactivación del Cromosoma X/genética , Diferenciación Celular/genética , Línea Celular , Cromosomas Humanos X/genética , Células Nutrientes/citología , Células Nutrientes/metabolismo , Femenino , Regulación de la Expresión Génica , Genes Ligados a X , Humanos , Células Madre Pluripotentes Inducidas/citología , Análisis de Secuencia de ADN
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