RESUMO
Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Animais , Glucose/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Células-Tronco Pluripotentes/metabolismoRESUMO
Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell-cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.
Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Sefarose/química , Esferoides Celulares/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura/instrumentação , Desenho de Equipamento , Humanos , Hidrogéis/química , Células-Tronco Mesenquimais/citologiaRESUMO
TNF-related apoptosis-inducing ligand (TRAIL) kills tumor cells selectively. We asked how emerging tumor cells escape elimination by TRAIL and how tumor-specific killing by TRAIL could then be restored. We found that TRAIL expression is consistently downregulated in HRAS(G12V)-transformed cells in stepwise tumorigenesis models derived from four different tissues due to DNA hypermethylation of CpG clusters within the TRAIL promoter. Decitabine de-silenced TRAIL, which remained inducible by interferon, while induction of TRAIL by blocking the HRAS(G12V)-activated mitogen-activated protein kinase pathway was subordinated to epigenetic silencing. Decitabine induced apoptosis through upregulation of endogenous TRAIL in cooperation with favorable regulation of key players acting in TRAIL-mediated apoptosis. Apoptosis induction by exogenously added TRAIL was largely increased by decitabine. In vivo treatment of xenografted human HRAS(G12V)-transformed human epithelial kidney or syngenic mice tumors by decitabine blocked tumor growth induced TRAIL expression and apoptosis. Our results emphasize the potential of decitabine to enhance TRAIL-induced apoptosis in tumors and thus provide a rationale for combination therapies with decitabine to increase tumor-selective apoptosis.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Transformação Celular Neoplásica/genética , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Transformada , Linhagem Celular Tumoral , Decitabina , Regulação para Baixo/genética , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties.
Assuntos
Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reguladores , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. It is still unclear, to what extend the escape of emerging cancer cells from recognition and elimination by the immune system is determined by similar mechanisms. We compared the transcriptomes of HCT116 colorectal cancer cells deficient in DNA methyltransferases (DNMTs) and of cells, in which the RAS pathway as the major growth-promoting signaling system is blocked by inhibition of MAPK. We identified the MHC Class I genes HLA-A1/A2 and the ULBP2 gene encoding 1 of the 8 known ligands of the activating NK receptor NKG2D among a cluster of immune genes up-regulated under the conditions of both DNMT-deficiency and MEK-inhibition. Bisulphite sequencing analyses of HCT116 with DNMT deficiency or after MEK-inhibition showed that de-methylation of the ULPB2 promoter correlated with its enhanced surface expression. The HLA-A promoters were not methylated indicating that components of the HLA assembly machinery were also suppressed in DNMT-deficient and MEK-inhibited cells. Increased HLA-A2 surface expression was correlated with enhanced recognition and lysis by A2-specific CTL. On the contrary, elevated ULBP2 expression was not reflected by enhanced recognition and lysis by NK cells. Cosuppression of HLA Class I and NKG2D ligands and genes encoding peptide transporters or proteasomal genes mediates a strong functional link between RAS activation, DNMT activity and disruption of the antigen presenting system controlling immune recognition in colorectal cancer cells.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/imunologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Antígeno HLA-A2/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas ras/metabolismo , Benzenossulfonatos/farmacologia , Butadienos/farmacologia , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferase 1 , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células Matadoras Naturais/imunologia , Niacinamida/análogos & derivados , Nitrilas/farmacologia , Compostos de Fenilureia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras) , Piridinas/farmacologia , Sorafenibe , DNA Metiltransferase 3BRESUMO
Ion channels are at present the third biggest target class in drug discovery. Primary research is continually uncovering potential new ion channel targets in indications such as cancer, diabetes and respiratory diseases, as well as the more established fields of pain, cardiovascular disease, and neurological disorders. Despite the physiological significance and therapeutic relevance in a wide variety of biological systems, ion channels still remain under exploited as drug targets. This is to a large extent resulting from the historical lack of screening technologies to provide the throughput and quality of data required to support medicinal chemistry. Although technical challenges still lie ahead, this historic bottleneck in ion channel drug discovery is now being overcome by novel technologies that can be integrated into lead generation stages of ion channel drug discovery to allow the development of novel therapeutic agents. This review describes the variety of technologies available for ion channel screening and discusses the opportunities these technologies provide. The challenges that remain to be addressed are highlighted.
Assuntos
Avaliação Pré-Clínica de Medicamentos/tendências , Canais Iônicos/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Eletrofisiologia , Corantes Fluorescentes , HumanosRESUMO
There is little information available concerning the link between the ryanodine (RY) receptors and the downstream Ca(2+) signaling events in beta-cells. In fura-2 loaded INS-1E cells, activation of RY receptors by 9-methyl 5,7-dibromoeudistomin D (MBED) caused a rapid rise of [Ca(2+)]i followed by a plateau and repetitive [Ca(2+)]i spikes on the plateau. The [Ca(2+)]i plateau was abolished by omission of extracellular Ca(2+) and by SKF 96365. In the presence of SKF 96365, MBED produced a transient increase of [Ca(2+)]i, which was abolished by thapsigargin. Activation of RY receptors caused Ca(2+) entry even when the ER Ca(2+) pool was depleted by thapsigargin. The [Ca(2+)]i plateau was not inhibited by nimodipine or ruthenium red, but was inhibited by membrane depolarization, La(3+), Gd(3+), niflumic acid, and 2-aminoethoxydiphenyl borate, agents that inhibit the transient receptor potential channels. The [Ca(2+)]i spikes were inhibited by nimodipine and ryanodine, indicating that they were due to Ca(2+) influx through the voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR). Activation of RY receptors depolarized membrane potential as measured by patch clamp. Thus, activation of RY receptors leads to coherent changes in Ca(2+) signaling, which includes activation of TRP-like channels, membrane depolarization, activation of the voltage-gated Ca(2+) channels and CICR.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Linhagem Celular Tumoral , Insulinoma/química , Insulinoma/metabolismo , Insulinoma/patologia , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Técnicas de Patch-Clamp/métodos , Ratos , Ratos WistarRESUMO
To understand the relationship between oncogenic signaling and the reprogramming of gene expression, we performed transcriptional profiling in rat ovarian surface epithelial cells (ROSE), in which neoplastic transformation is driven by a mutated KRAS oncogene. We identified >200 genes whose expression was elevated or reduced following permanent KRAS expression. Deregulated KRAS-responsive genes encode transcriptional regulators, signaling effectors, proteases, extracellular matrix and adhesion proteins, transformation-suppressing proteins and negative growth regulators. Many of them have not been previously identified in cells expressing oncogenic RAS genes or in other well-studied models of oncogenic signaling. The number of critical genes related to the execution of anchorage-independent proliferation and epithelial-mesenchymal transition was narrowed down to 79 by selectively inhibiting the mitogen-activated protein kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3K) pathways. Blocking MAPK/ERK-signaling caused reversion to the normal epithelial phenotype in conjunction with the reversal of deregulated target transcription to pretransformation levels. In addition, silencing of the overexpressed transcriptional regulator Fra-1 by RNA interference resulted in growth reduction, suggesting that this factor partially contributes to, but is not sufficient for the proliferative capacity of KRAS-transformed epithelial cells.
Assuntos
Transformação Celular Neoplásica/genética , Genes ras , Ovário/patologia , Transcrição Gênica , Animais , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno , Morfolinas/farmacologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-raf/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Transdução de SinaisRESUMO
Insulin-like Growth Factor (IGF)-II is frequently overexpressed in experimental and human hepatocellular carcinomas (HCCs) and has been correlated with increased tumor growth. We have analyzed, whether IGF-II affects chemotherapy response and apoptosis in human liver tumor cells. Three liver tumor cell lines highly expressed IGF-II and supported their growth in an autocrine manner by secreting excessive amounts of IGF-II. Neutralization of IGF-II significantly increased response to the chemotherapeutic agents cisplatin and etoposide especially at lower, cytostatic doses. While blocking of IGF-II did not increase spontaneous cell death in exponentially growing cultures, increased cell death was found under conditions of confluent growth and chemotherapy. Thus in HCC cells, IGF-II is a relevant protumorigenic growth factor that significantly reduces susceptibility to apoptosis and chemotherapeutic treatment. Therefore interference with IGF-II activity may improve response of HCCs to otherwise inefficient chemotherapeutic agents.