RESUMEN
Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate® Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate® Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.
Asunto(s)
Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Animales , Técnicas de Cocultivo , Hepatocitos , HumanosRESUMEN
A growing body of work suggests that astrocytomas and glioblastoma multiforme will require carefully tailored, molecularly targeted therapy for successful treatment. Recent efforts to comprehensively identify mutations and gene expression changes in glioblastoma have shown that mutation of NF1 is a common alteration in human glioblastoma. We have developed and characterized a panel of 14 tumor lines from grades II through IV astrocytomas developed from our Nf1-/+;Trp53-/+cis mouse model and have used this panel to characterize signal transduction pathways and inhibitors that are candidate therapeutic targets for astrocytoma and glioblastoma. We show that these tumors express platelet-derived growth factor receptor-α, epidermal growth factor receptor, and their respective ligands to varying degrees. We find that both the MEK and PI3K signaling pathways downstream of epidermal growth factor receptor and platelet-derived growth factor receptor-α are necessary for full proliferation of astrocytoma cells; however, inhibition of the PI3K pathway is more effective than inhibition of MEK at blocking cell growth. We have examined inhibitors of the PI3K/Akt/mTOR signaling pathway and find that PI-103 and TCN show particular promise for inhibiting growth in Nf1 and Trp53 mutant astrocytoma cells.
Asunto(s)
Acenaftenos/farmacología , Astrocitoma/tratamiento farmacológico , Astrocitoma/metabolismo , Proliferación Celular , Furanos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Ribonucleótidos/farmacología , Animales , Astrocitoma/patología , Western Blotting , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Adhesión Celular , Movimiento Celular , Receptores ErbB/metabolismo , Femenino , Genes de Neurofibromatosis 1/fisiología , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Mouse models of human cancer have played a vital role in understanding tumorigenesis and answering experimental questions that other systems cannot address. Advances continue to be made that allow better understanding of the mechanisms of tumor development, and therefore the identification of better therapeutic and diagnostic strategies. We review major advances that have been made in modeling cancer in the mouse and specific areas of research that have been explored with mouse models. For example, although there are differences between mice and humans, new models are able to more accurately model sporadic human cancers by specifically controlling timing and location of mutations, even within single cells. As hypotheses are developed in human and cell culture systems, engineered mice provide the most tractable and accurate test of their validity in vivo. For example, largely through the use of these models, the microenvironment has been established to play a critical role in tumorigenesis, since tumor development and the interaction with surrounding stroma can be studied as both evolve. These mouse models have specifically fueled our understanding of cancer initiation, immune system roles, tumor angiogenesis, invasion, and metastasis, and the relevance of molecular diversity observed among human cancers. Currently, these models are being designed to facilitate in vivo imaging to track both primary and metastatic tumor development from much earlier stages than previously possible. Finally, the approaches developed in this field to achieve basic understanding are emerging as effective tools to guide much needed development of treatment strategies, diagnostic strategies, and patient stratification strategies in clinical research.
Asunto(s)
Modelos Animales de Enfermedad , Ingeniería Genética , Neoplasias Experimentales/diagnóstico , Neoplasias Experimentales/terapia , Animales , Investigación Biomédica , Humanos , RatonesRESUMEN
Neurofibomatosis (NF1) patients are susceptible to multiple tumors of the nervous system including neurofibromas, optic glioma, malignant peripheral nerve sheath tumors (MPNSTs), and astrocytoma. The Nf1+/-;Trp53+/- (NPcis) mouse model of NF1 spontaneously develops astrocytoma and MPNSTs that are very similar to human NF1 tumors. To use this model for testing potential therapeutics, we have developed systems that take advantage of bioluminescent reporters of tumor growth. We have generated E2F1 promoter-driving luciferase (ELUX) reporter mice to detect proliferating tumors in NPcis mice in vivo using bioluminescence. The power of this system is that it enables looking at tumor evolution and detecting spontaneous tumors at early stages of development as they evolve within their natural haploinsufficient microenvironment. This system can be used to identify tumors at different stages of tumorigenesis and to examine where spontaneous NF1 tumors initiate. The ability to rapidly screen multiple animals at a time increases the potential for use of this model in preclinical trials. This model will be valuable for the characterization of spontaneous NF1 tumors and will be important for studying the treatment and prevention of NF1 tumors in vivo.
Asunto(s)
Modelos Animales de Enfermedad , Mediciones Luminiscentes/métodos , Neurofibromatosis/patología , Animales , Astrocitoma/patología , Citomegalovirus/genética , Genes p53 , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Vaina del Nervio/patología , Neurofibromatosis/terapia , Regiones Promotoras GenéticasRESUMEN
Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma, there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective antitumor agents. The authors have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential antitumor treatments that specifically target astrocytoma.
Asunto(s)
Antineoplásicos , Astrocitoma/tratamiento farmacológico , Citostáticos/uso terapéutico , Citotoxinas/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Camptotecina/uso terapéutico , Humanos , Nocodazol/uso terapéutico , Reproducibilidad de los ResultadosRESUMEN
The mechanisms underlying responses to drugs of abuse have been widely investigated; however, less is known about pathways normally protective against the development of drug reinforcement. These pathways are also important since they may regulate individual differences in vulnerability to addiction. The neuropeptide galanin and its binding sites are expressed in brain areas important for drug reward. Previous studies have shown that centrally infused galanin attenuates morphine place preference and peripheral injection of galnon, a galanin agonist, decreases opiate withdrawal signs. The current studies in galanin knockout (GKO) mice examined the hypothesis that galanin is an endogenous negative regulator of opiate reward and identified downstream signaling pathways regulated by galanin. We show that GKO mice demonstrate increased locomotor activation following morphine administration, which is inhibited by acute administration of galnon. GKO mice also show enhanced morphine place preference, supporting the idea that galanin normally antagonizes opiate reward. In addition, morphine-induced ERK1/2 phosphorylation was increased in the VTA of both wild-type and GKO mice, but only the GKO mice showed increases in ERK1/2 and CREB phosphorylation in the amygdala or nucleus accumbens. Furthermore, a single systemic injection of galnon in GKO mice was sufficient to reverse some of the biochemical changes brought about by morphine administration. These data suggest that galanin normally attenuates behavioral and neurochemical effects of opiates; thus, galanin agonists may represent a new class of therapeutic targets for opiate addiction.
Asunto(s)
Conducta Animal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Galanina/fisiología , Dependencia de Morfina/genética , Dependencia de Morfina/psicología , Recompensa , Animales , Western Blotting , Condicionamiento Operante/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Galanina/genética , Genotipo , Ratones , Ratones Noqueados , Morfina/farmacología , Dependencia de Morfina/fisiopatología , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Narcóticos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.
Asunto(s)
Expresión Génica , Neurofibromina 1/genética , Animales , Astrocitoma/genética , Mapeo Cromosómico , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 19 , Modelos Animales de Enfermedad , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Variación Genética , Heterocigoto , Humanos , Ratones , Neurofibromatosis 1/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias del Sistema Nervioso Periférico/genéticaRESUMEN
The neuropeptide galanin is widely distributed in the central nervous system and plays a role in a number of processes in the adult brain. Galanin also has neurotrophic effects in the developing nervous system and after nerve injury. The current study investigated the mechanism by which galanin promotes neurite outgrowth in the neuronal cell line PC12 and in neurospheres derived from adult hippocampal progenitor cells. We demonstrated that galanin can induce extracellular signal-related kinase (ERK) phosphorylation transiently in a concentration-dependent manner in neurons. Galanin-like peptide, which is thought to signal primarily through the GalR2 receptor subtype, induced ERK phosphorylation with similar kinetics to galanin. In functional studies, the ability of galanin and galanin-like peptide to induce neurite outgrowth was dependent on activation of both protein kinase C and ERK. This study identified a novel physiological role for galanin-induced ERK phosphorylation and identified ERK and protein kinase C as important signaling components in the galanin-mediated modulation of neurite outgrowth.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Péptido Similar a Galanina/farmacología , Galanina/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Proteína Quinasa C/fisiología , Animales , Western Blotting/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neuronas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptores de Galanina/genética , Receptores de Galanina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Células Madre/efectos de los fármacosRESUMEN
Repeated morphine administration leads to molecular alterations of the neural circuitry in the locus coeruleus and nucleus accumbens. These changes include increased activity of several components of the cAMP signaling pathway that are thought to be associated with psychological and somatic signs of opiate withdrawal. The neuropeptide galanin has been shown to attenuate cAMP signaling in multiple cell types. The current study demonstrates that acute galanin treatment blocks the consequences of increased cAMP signaling following chronic opiate administration and withdrawal in Cath.a cells and primary cultures of striatal neurons as measured by phosphorylation of the transcription factor cAMP regulatory element-binding protein (CREB). In addition, galanin-mediated attenuation of CREB phosphorylation is independent of galanin-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in Cath.a cells. These data suggest that galanin receptors may serve as an additional potential therapeutic target for the treatment of opiate withdrawal.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Cuerpo Estriado/fisiología , Galanina/farmacología , Morfina/farmacología , Naloxona/farmacología , Neuronas/fisiología , Animales , Proteína de Unión a CREB/genética , Línea Celular , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , AMP Cíclico/fisiología , Cartilla de ADN , Cinética , Ratones , Narcóticos/farmacología , Neuronas/efectos de los fármacos , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
Galanin and its receptors are expressed in brain areas associated with opiate reinforcement and withdrawal. An emerging body of data suggests that galanin can attenuate the neurochemical, physiological and behavioral signs of opiate reinforcement and withdrawal. Experiments in transgenic mice overexpressing galanin and knockout mice lacking the peptide support a role for endogenous galanin in modulating the actions of opiates on brain regions associated with reinforcement and withdrawal. These studies suggest that galanin receptor agonists could be useful therapeutic agents to combat opiate addiction. Further, genetic variation in the genes encoding galanin and its receptors could be associated with altered susceptibility to opiate dependence.
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Galanina/genética , Galanina/metabolismo , Trastornos Relacionados con Opioides/fisiopatología , Síndrome de Abstinencia a Sustancias/fisiopatología , Animales , Galanina/uso terapéutico , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Trastornos Relacionados con Opioides/tratamiento farmacológico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológicoRESUMEN
The galanin receptors GalR1, GalR2 and GalR3 are widely expressed throughout the mouse brain and are enriched in catecholaminergic nuclei. Here, we show that GalR1 protein levels are regulated by neuronal activity and changes in cAMP levels. GalR1, but not GalR2 or GalR3, is specifically up-regulated in the LC-like Cath.a cell line in a cAMP-dependent manner. GalR1 protein and mRNA levels are also up-regulated in the LC of galanin knockout mice, whereas GalR2 and GalR3 are not. Lack of galanin-maintained cAMP tone in the galanin knockout mouse appears to result in a loss of negative feedback resulting in increased levels of CREB phosphorylation and increased GalR1 expression. These findings suggest that changes in levels of GalR1 may play an important role in modulating signaling events and neuroplasticity underlying physiological functions of the LC.
Asunto(s)
AMP Cíclico/fisiología , Galanina/metabolismo , Locus Coeruleus/metabolismo , Receptor de Galanina Tipo 1/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Retroalimentación Fisiológica , Galanina/genética , Masculino , Ratones , Ratones Noqueados/genética , Fosforilación , ARN Mensajero/metabolismo , Receptor de Galanina Tipo 1/genética , Receptor de Galanina Tipo 2/metabolismo , Receptor de Galanina Tipo 3/metabolismoRESUMEN
The distribution of immunoreactivity for the three identified neuropeptide galanin receptors, GalR1, GalR2, and GalR3, was determined in areas of the mouse brain involved in drug addiction, including the ventral tegmental area (VTA), substantia nigra (SN), nucleus accumbens (NA), and locus coeruleus (LC). All three galanin receptors are found in the VTA, SN, NA, and LC; however, GalR1 protein is most highly represented in the VTA, NA, and SN, suggesting that GalR1 may play a predominant role in galanin-mediated regulation of dopamine neurotransmission. GalR1 and GalR3 protein levels are high in the LC, suggesting that these isoforms may be important for galanin-mediated regulation of noradrenergic transmission during opiate withdrawal. Although the distribution of GalR1, GalR2, and GalR3 largely recapitulates the pattern of galanin binding throughout the brain, some discrepancies exist, suggesting that another galanin receptor(s) may be present in some brain areas. Overall, GalR1, GalR2, and GalR3 are distributed widely throughout the brain, correlate with widespread galanin binding, and colocalize with tyrosine hydroxylase in catecholaminergic brain areas.