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1.
BMC Bioinformatics ; 24(1): 252, 2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37322439

RESUMO

BACKGROUND: Bioinformatics capability to analyze spatio-temporal dynamics of gene expression is essential in understanding animal development. Animal cells are spatially organized as functional tissues where cellular gene expression data contain information that governs morphogenesis during the developmental process. Although several computational tissue reconstruction methods using transcriptomics data have been proposed, those methods have been ineffective in arranging cells in their correct positions in tissues or organs unless spatial information is explicitly provided. RESULTS: This study demonstrates stochastic self-organizing map clustering with Markov chain Monte Carlo calculations for optimizing informative genes effectively reconstruct any spatio-temporal topology of cells from their transcriptome profiles with only a coarse topological guideline. The method, eSPRESSO (enhanced SPatial REconstruction by Stochastic Self-Organizing Map), provides a powerful in silico spatio-temporal tissue reconstruction capability, as confirmed by using human embryonic heart and mouse embryo, brain, embryonic heart, and liver lobule with generally high reproducibility (average max. accuracy = 92.0%), while revealing topologically informative genes, or spatial discriminator genes. Furthermore, eSPRESSO was used for temporal analysis of human pancreatic organoids to infer rational developmental trajectories with several candidate 'temporal' discriminator genes responsible for various cell type differentiations. CONCLUSIONS: eSPRESSO provides a novel strategy for analyzing mechanisms underlying the spatio-temporal formation of cellular organizations.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Humanos , Animais , Camundongos , Reprodutibilidade dos Testes , Encéfalo , Análise por Conglomerados , Análise Espaço-Temporal
2.
iScience ; 25(7): 104538, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35754715

RESUMO

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90-1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82-0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

3.
Biomaterials ; 284: 121491, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35395453

RESUMO

The loss of nucleus pulposus (NP) precedes the intervertebral disk (IVD) degeneration that causes back pain. Here, we demonstrate that the implantation of human iPS cell-derived cartilaginous tissue (hiPS-Cart) restores this loss by replacing lost NP spatially and functionally. NP cells consist of notochordal NP cells and chondrocyte-like NP cells. Single cell RNA sequencing (scRNA-seq) analysis revealed that cells in hiPS-Cart corresponded to chondrocyte-like NP cells but not to notochordal NP cells. The implantation of hiPS-Cart into a nuclectomized space of IVD in nude rats prevented the degeneration of the IVD and preserved its mechanical properties. hiPS-Cart survived and occupied the nuclectomized space for at least six months after implantation, indicating spatial and functional replacement of lost NP by hiPS-Cart. Further scRNA-seq analysis revealed that hiPS-Cart cells changed their profile after implantation, differentiating into two lineages that are metabolically distinct from each other. However, post-implanted hiPS-Cart cells corresponded to chondrocyte-like NP cells only and did not develop into notochordal NP cells, suggesting that chondrocyte-like NP cells are nearly sufficient for NP function. The data collectively indicate that hiPS-Cart is a candidate implant for regenerating NP spatially and functionally and preventing IVD degeneration.


Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração do Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Animais , Cartilagem , Humanos , Degeneração do Disco Intervertebral/terapia , Ratos , Regeneração
4.
iScience ; 24(7): 102783, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34308289

RESUMO

Despite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency.

5.
J Toxicol Sci ; 46(3): 131-142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33642519

RESUMO

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.


Assuntos
Diferenciação Celular , Resistência a Medicamentos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Amiodarona/toxicidade , Animais , Aspirina/toxicidade , Atorvastatina/toxicidade , Linhagem Celular , Clotrimazol/toxicidade , Resistência a Múltiplos Medicamentos , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia
6.
Biochem Biophys Res Commun ; 520(1): 159-165, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31582216

RESUMO

Cell adhesion is mediated by adhesion molecules, but also regulated by adhesion inhibitory molecules. Molecules such as leukocyte sialomucin and phosphorylated-Ezrin/Radixin/Moesin (ERM) inhibit cell-substratum adhesion. Here we show that these adhesion inhibitory molecules also inhibit aggregate formation of adherent cells in suspension culture. Expression of sialomucin, CD43 or CD34, inhibited formation of packed aggregates in HEK293T cells. Deletion mutant analysis and enzymatic cleavage indicated the significance of the extracellular sialomucin domain for this inhibition. Meanwhile, phosphorylated-ERM were decreased coincidently with aggregate formation. Combined with the inhibition of aggregate formation by the expression of phospho-mimetic Moesin mutant (Moesin-T558D), phosphorylated-ERM are inhibitors for aggregate formation. Increase of phosphorylated-ERM by CD43 and sialomucin-dependence of Moesin-T558D's inhibition indicate that sialomucin and phosphorylated-ERM collaborate to inhibit aggregate formation. Because aggregate formation of HEK293T cells is mediated by N-cadherin, sialomucin and phosphorylated-ERM inhibit cadherin-mediated cell-cell adhesion. Thus, sialomucin and phosphorylated-ERM are inhibitors for both cell-cell adhesion and cell-substratum adhesion, and regulation of these inhibitory molecules is essential for cell adhesion.


Assuntos
Caderinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Leucossialina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sialomucinas/farmacologia , Antígenos CD34/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Sialoglicoproteínas/metabolismo
7.
Sci Rep ; 9(1): 12597, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467377

RESUMO

Deciphering the key mechanisms of morphogenesis during embryonic development is crucial to understanding the guiding principles of the body plan and promote applications in biomedical research fields. Although several computational tissue reconstruction methods using cellular gene expression data have been proposed, those methods are insufficient with regard to arranging cells in their correct positions in tissues or organs unless spatial information is explicitly provided. Here, we report SPRESSO, a new in silico three-dimensional (3D) tissue reconstruction method using stochastic self-organizing map (stochastic-SOM) clustering, to estimate the spatial domains of cells in tissues or organs from only their gene expression profiles. With only five gene sets defined by Gene Ontology (GO), we successfully demonstrated the reconstruction of a four-domain structure of mid-gastrula mouse embryo (E7.0) with high reproducibility (success rate = 99%). Interestingly, the five GOs contain 20 genes, most of which are related to differentiation and morphogenesis, such as activin A receptor and Wnt family member genes. Further analysis indicated that Id2 is the most influential gene contributing to the reconstruction. SPRESSO may provide novel and better insights on the mechanisms of 3D structure formation of living tissues via informative genes playing a role as spatial discriminators.


Assuntos
Simulação por Computador , Gástrula/crescimento & desenvolvimento , Morfogênese , Animais , Sequência de Bases , Gástrula/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Camundongos , Modelos Biológicos , Processos Estocásticos
9.
Yakugaku Zasshi ; 138(6): 815-822, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29863053

RESUMO

 Toxicity prediction based on stem cells and tissue derived from stem cells plays a very important role in the fields of biomedicine and pharmacology. Here we report on qRT-PCR data obtained by exposing 20 compounds to human embryonic stem (ES) cells. The data are intended to improve toxicity prediction, per category, of various compounds through the use of support vector machines, and by applying gene networks. The accuracy of our system was 97.5-100% in three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs), and non-genotoxic carcinogens (NGCs). We predicted that two uncategorized compounds (bisphenol-A and permethrin) should be classified as follows: bisphenol-A as a non-genotoxic carcinogen, and permethrin as a neurotoxin. These predictions are supported by recent reports, and as such constitute a good outcome. Our results include two important features: 1) The accuracy of prediction was higher when machine learning was carried out using gene networks and activity, rather than the normal quantitative structure-activity relationship (QSAR); and 2) By using undifferentiated ES cells, the late effect of chemical substances was predicted. From these results, we succeeded in constructing a highly effective and highly accurate system to predict the toxicity of compounds using stem cells.


Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Máquina de Vetores de Suporte , Testes de Toxicidade/métodos , Compostos Benzidrílicos/toxicidade , Carcinógenos/toxicidade , Humanos , Neurotoxinas/toxicidade , Permetrina/toxicidade , Fenóis/toxicidade , Relação Quantitativa Estrutura-Atividade
10.
Nucleic Acids Res ; 44(12): 5515-28, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27207879

RESUMO

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development.


Assuntos
Carcinógenos/toxicidade , Dano ao DNA/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Neurotoxinas/toxicidade , Compostos Benzidrílicos/toxicidade , Biologia Computacional , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Permetrina/toxicidade , Fenóis/toxicidade , Relação Quantitativa Estrutura-Atividade , Máquina de Vetores de Suporte
11.
Cancer Sci ; 107(5): 619-28, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26892864

RESUMO

An increasingly accepted concept is that the progression of colorectal cancer is accompanied by epithelial-mesenchymal transition (EMT). In our study, in order to characterize the properties of EMT in 16 colorectal cancer cell lines, the cells were first orthotopically implanted into nude mice, and the tumors in vivo, as well as cells cultured in vitro, were immunostained for EMT markers. The immunostaining revealed that seven of the cells had an epithelial phenotype with a high expression of E-cadherin, whereas other cells showed opposite patterns, such as a high expression of vimentin (CX-1, COLO205, CloneA, HCT116, and SW48). Among the cells expressing vimentin, some expressed vimentin in the orthotopic tumors but not in the cultured cells (SW480, SW620, and COLO320). We evaluated these findings in combination with microarray analyses, and selected five genes: CHST11, SERPINI1, AGR2, FBP1, and FOXA1. Next, we downregulated the expression of SERPINI1 with siRNA in the cells, the results of which showed reverse-EMT changes at the protein level and in the cellular morphology. Along with immunohistochemical analyses, we confirmed the effect of the intracellular and secreted SERPINI1 protein of SW620 cells, which supported the importance of SERPINI1 in EMT. The development of therapeutic strategies targeting EMT is ongoing, including methods targeting the transforming growth factor-ß signaling pathway as well as the Wnt pathway. SERPINI1 is an important regulator of EMT. Our findings help to elucidate the signaling pathways of EMT, hopefully clarifying therapeutic pathways as well.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Modelos Biológicos , Transplante de Neoplasias , Neuropeptídeos/metabolismo , Serpinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Serpinas/deficiência , Serpinas/genética , Sulfotransferases/deficiência , Sulfotransferases/genética , Sulfotransferases/metabolismo , Vimentina/metabolismo , Via de Sinalização Wnt , Neuroserpina
13.
Oncol Rep ; 28(5): 1585-90, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22923031

RESUMO

Valproic acid, a histone deacetylase inhibitor, increases the expression of cell surface MHC class I-related chain molecules (MICs) A and B (MICA and B) in osteosarcoma cells and decreases their secretion of soluble MICA and MICB, which are produced by the proteolytic cleavage of cell surface MICs. Osteosarcoma cells have been reported to produce high levels of matrix metalloproteinase (MMP)-2 and -9. In this study, we investigated the involvement of MMP-2 and -9 in the inhibitory action of valproic acid (VPA) on the proteolytic cleavage of cell surface MICs using the U-2 OS and SaOS-2 osteosarcoma cell lines. VPA caused a marked decrease in the expression of MMP-9 mRNA in the U-2 OS and SaOS-2 cells and in the expression of MMP-2 mRNA in the U-2 OS cells, but only a slight decrease in the expression of MMP-2 mRNA in the SaOS-2 cells. The transfection of small interfering RNA (siRNA) for MMP-9 decreased the secretion of soluble MICA and MICB by both U-2 OS and SaOS-2 cells, but that of siRNA for MMP-2 did not. The present study therefore demonstrates that the downregulation of MMP-9 mRNA by VPA plays a role in the inhibitory action of VPA on the secretion of soluble MICA and MICB in osteosarcoma cells.


Assuntos
Antígenos de Histocompatibilidade Classe I/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Osteossarcoma/genética , Ácido Valproico/farmacologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Regulação para Baixo , Humanos , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Membrana/biossíntese , Osteossarcoma/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno
14.
Int J Oncol ; 41(1): 83-91, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22576685

RESUMO

We investigated the effects of valproic acid (VPA), a histone deacetylase inhibitor, in combination with hydralazine, a DNA methylation inhibitor, on the expression of cell-surface Fas and MHC-class I-related chain molecules A and B (MICA and B), the ligands of NKG2D which is an activating receptor of NK cells, and on production of their soluble forms in HOS, U-2 OS and SaOS-2 human osteosarcoma cell lines. We also examined the susceptibility of these cells to Fas- and NK cell-mediated cell death. VPA did not increase the expression of Fas on the surface of osteosarcoma cells, while hydralazine did, and the combination of VPA with hydralazine increased the expression of cell-surface Fas. In contrast, the combination of VPA with hydralazine did not increase the production of soluble Fas by osteosarcoma cells. Both VPA and hydralazine increased the expression of cell-surface MICA and B in osteosarcoma cells, and their combination induced a greater increase in their expression. VPA inhibited the production of both soluble MICA and MICB by osteosarcoma cells while hydralazine produced no effect. Both VPA and hydralazine enhanced the susceptibility of osteosarcoma cells to Fas- and NK cell-mediated cell death and the combination of VPA with hydralazine further enhanced the effects. The present results suggest that combined administration of VPA and hydrazine is valuable for enhancing the therapeutic effects of immunotherapy for osteosarcomas.


Assuntos
Antineoplásicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Hidralazina/farmacologia , Células Matadoras Naturais/fisiologia , Ácido Valproico/farmacologia , Receptor fas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Sinergismo Farmacológico , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Osteossarcoma , Regiões Promotoras Genéticas , Evasão Tumoral/efeitos dos fármacos , Receptor fas/imunologia
15.
Toxicol Lett ; 212(1): 1-10, 2012 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-22555245

RESUMO

The establishment of more efficient in vitro approaches has been widely acknowledged as a critical need for toxicity testing. In this study, we examined the effects of methylmercury (MeHg), which is a well-known developmental neurotoxicant, in two neuronal differentiation systems of mouse and human embryonic stem cells (mESCs and hESCs, respectively). Embryoid bodies were generated from gathering of mESCs and hESCs using a micro-device and seeded onto ornithine-laminin-coated plates to promote proliferation and neuronal differentiation. The cells were exposed to MeHg from the start of neuronal induction until the termination of cultures, and significant reductions of mESCs and hESCs were observed in the cell viability assays at 1,10,100 and 1000nM, respectively. Although the mESC derivatives were more sensitive than the hESC derivatives to MeHg exposure in terms of cell viability, the morphological evaluation demonstrated that the neurite length and branch points of hESC derivatives were more susceptible to a low concentration of MeHg. Then, the mRNA levels of differentiation markers were examined using quantitative RT-PCR analysis and the interactions between MeHg exposure and gene expression levels were visualized using a network model based on a Bayesian algorithm. The Bayesian network analysis showed that a MeHg-node was located on the highest hierarchy in the hESC derivatives, but not in the mESC derivatives, suggesting that MeHg directly affect differentiation marker genes in hESCs. Taken together, effects of MeHg were observed in our neuronal differentiation systems of mESCs and hESCs using a combination of morphological and molecular markers. Our study provided possible, but limited, evidences that human ESC models might be more sensitive in particular endpoints in response to MeHg exposure than that in mouse ESC models. Further investigations that expand on the findings of the present paper may solve problems that occur when the outcomes from laboratory animals are extrapolated for human risk evaluation.


Assuntos
Células-Tronco Embrionárias/efeitos dos fármacos , Compostos de Metilmercúrio/toxicidade , Neurônios/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Neurônios/citologia , Neurônios/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real
16.
Cell Adh Migr ; 5(2): 119-32, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21045567

RESUMO

CD43/sialophorin/leukosialin, a common leukocyte antigen, is known as an inhibitor for cell adhesion. The ectodomain of CD43 is considered as a molecular barrier for cell adhesion, while the cytoplasmic domain has a binding site for Ezrin/Radixin/Moesin (ERM). We found expression of CD43 induced cell rounding, inhibition of cell re-attachment, augmentation of microvilli, and phosphorylation of ERM in HEK293T cells. Mutant studies revealed the ectodomain of CD43, but not the intracellular domain, essential and sufficient for all these phenomena. We also found that forced cell detachment by itself induced phosphorylation of ERM in HEK293T cells. Taken together, these findings indicate that inhibition of cell adhesion by the ectodomain of CD43 induces phosphorylation of ERM, microvilli formation, and eventual cell rounding. Furthermore, our study suggests a novel possibility that cell detachment itself induces activation of ERM and modification of cell shape.


Assuntos
Células Epiteliais/metabolismo , Leucossialina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Clonagem Molecular , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/citologia , Expressão Gênica , Células HEK293 , Humanos , Leucócitos/metabolismo , Leucossialina/genética , Leucossialina/farmacologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/fisiologia , Mutação , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia
17.
Oncol Rep ; 24(6): 1621-7, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21042760

RESUMO

MHC class I-related chain molecules A and B (MICA and B) expressed on the cell-surface of tumor cells are ligands for an activating receptor, NKG2D, expressed on natural killer (NK) cells and stimulate the NK cell-mediated cytotoxicity. On the other hand, the soluble form of MICA and B produced by proteolytic cleavage of cell-surface MIC interferes with NK cell-mediated cytotoxicity. We investigated effect of sodium valproate (VPA), a histone deacetylase inhibitor, on the production of cell-surface and soluble MICA and B and NK cell-mediated cytotoxicity in four human osteosarcoma cells. VPA at 0.5 and 1.0 mM induced acetylation of histones bound to MICA and B gene promoters, increased cell-surface but not soluble MICA and B, and augmented the susceptibility of osteosarcoma cells to NK cell-mediated cytotoxicity. The present results indicate that VPA sensitizes human osteosarcoma cells to cytotoxicity of NK cells.


Assuntos
Neoplasias Ósseas/terapia , Antígenos de Histocompatibilidade Classe I/genética , Imunidade Celular/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Osteossarcoma/terapia , Ácido Valproico/farmacologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Terapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imunoterapia/métodos , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Osteossarcoma/genética , Osteossarcoma/imunologia , Osteossarcoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Solubilidade , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
18.
J Cancer Res Clin Oncol ; 135(7): 879-89, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19066961

RESUMO

PURPOSE: Effects of valproic acid (VPA), a histone deacetylase inhibitor, on the susceptibility to cell death induced by agonistic anti-Fas antibody were examined using four human osteosarcoma cell lines. METHOD: Cell growth, secretion of soluble Fas, expression of cell-surface Fas, and sensitivity to Fas-mediated cell death were examined using cell proliferation assay, flow cytometry, enzyme-linked immunosorbent assay, and agonistic anti-Fas antibody, respectively. RESULTS: VPA suppressed the growth of all the four osteosarcoma cell lines and the secretion of soluble Fas from these cells. VPA showed no or slight suppressive effect on the expression of cell-surface Fas in the four osteosarcoma cell lines, but increased the sensitivity of three of four osteosarcoma cell lines to Fas-mediated cell death. CONCLUSION: VPA enhances the susceptibility of human osteosarcoma cells to Fas-ligand-induced cell death by decreasing the secretion of soluble Fas and increasing the sensitivity to Fas-mediated cell death.


Assuntos
Anticorpos/farmacologia , Neoplasias Ósseas/metabolismo , Osteossarcoma/metabolismo , Ácido Valproico/farmacologia , Receptor fas/metabolismo , Neoplasias Ósseas/patologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Osteossarcoma/patologia , Solubilidade , Células Tumorais Cultivadas , Receptor fas/imunologia
19.
Exp Cell Res ; 313(16): 3472-85, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17698061

RESUMO

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.


Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Proteínas de Ligação a DNA/ultraestrutura , Endossomos/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/química , Transporte Proteico , Proteínas Qa-SNARE/metabolismo , Interferência de RNA , Ratos , Fatores de Transcrição/ultraestrutura
20.
Biochem Biophys Res Commun ; 361(4): 1038-43, 2007 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-17679140

RESUMO

In the present study, we investigated if Cl(-) regulates the proliferation of the MC3T3-E1 osteoblastic cells. The proliferation of MC3T3-E1 osteoblastic cells was diminished by lowering the extracellular Cl(-) concentration ([Cl(-)](o)) in the culture medium. The lowered in [Cl(-)](o) increased the periods of the G(0)/G(1) and the G(2)/M phases in cell cycle. We further studied the effects of [Cl(-)](o) on the key enzymes, Rb and cdc2, playing key roles in checking points of the G(0)/G(1) and the G(2)/M phases in cell cycle. The lowered in [Cl(-)](o) diminished the active forms of enzymes, Rb and cdc2. We further found that the action of lowered [Cl(-)](o) on the cell proliferation, the cell cycle, Rb and cdc2 was abolished by the presence of 2mM glutamine, but not by that of pyruvate as another Krebs cycle substrate. Taken together, these observations indicate here for the first time that Cl(-) modulates Rb and cdc2, enhancing the proliferation of the MC3T3-E1 osteoblastic cells.


Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular , Cloretos/fisiologia , Osteoblastos/enzimologia , Proteína do Retinoblastoma/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Camundongos , Osteoblastos/citologia
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