RESUMEN
Preventing clinical drug-induced liver injury (DILI) remains a major challenge, because DILI develops via multifactorial mechanisms. Immune and inflammatory reactions are considered important mechanisms of DILI; however, biomarkers from in vitro systems using immune cells have not been comprehensively studied. The aims of this study were (1) to identify promising biomarker genes for predicting DILI in an in vitro coculture model of peripheral blood mononuclear cells (PBMCs) with a human liver cell line, and (2) to evaluate these genes as predictors of DILI using a panel of drugs with different clinical DILI risk. Transcriptome-wide analysis of PBMCs cocultured with HepG2 or differentiated HepaRG cells that were treated with several drugs revealed an appropriate separation of DILI-positive and DILI-negative drugs, from which 12 putative biomarker genes were selected. To evaluate the predictive performance of these genes, PBMCs cocultured with HepG2 cells were exposed to 77 different drugs, and gene expression levels in PBMCs were determined. The MET proto-oncogene receptor tyrosine kinase (MET) showed the highest area under the receiver-operating characteristic curve (AUC) value of 0.81 among the 12 genes with a high sensitivity/specificity (85/66%). However, a stepwise logistic regression model using the 12 identified genes showed the highest AUC value of 0.94 with a high sensitivity/specificity (93/86%). Taken together, we established a coculture system using PBMCs and HepG2 cells and selected biomarkers that can predict DILI risk. The established model would be useful in detecting the DILI potential of compounds, in particular those that involve an immune mechanism.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hepatocitos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Técnicas de Cocultivo , Perfilación de la Expresión Génica , Marcadores Genéticos , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proto-Oncogenes Mas , Medición de RiesgoRESUMEN
Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14(+)CD16(+) monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1(op)/Csf1(op)) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.
Asunto(s)
Aspartato Aminotransferasas/sangre , Creatina Quinasa/sangre , Macrófagos del Hígado/patología , Hígado/enzimología , Músculo Esquelético/enzimología , Osteopetrosis/patología , Animales , Anticuerpos Monoclonales/farmacología , Conservadores de la Densidad Ósea/farmacología , Ácido Clodrónico/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Hígado/lesiones , Hígado/patología , Macaca fascicularis , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor Estimulante de Colonias de Macrófagos/fisiología , Masculino , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/patología , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Osteopetrosis/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de IgG/metabolismoRESUMEN
Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.
Asunto(s)
Células Epiteliales Alveolares/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Modelos Biológicos , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenocarcinoma del Pulmón/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/ultraestructura , Células Cultivadas , Evaluación Preclínica de Medicamentos , Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína C Asociada a Surfactante Pulmonar/metabolismoRESUMEN
Tofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis. Tofacitinib preferentially inhibits receptor signaling through JAK3 and JAK1, relative to JAK2. In the 2-year rat carcinogenicity study, there were tofacitinib, dose-related increases in the incidences of testicular Leydig cell hyperplasia and benign adenomas in male rats, and decreased incidences of mammary tumors and duct dilatation/galactocele in female rats. Such findings in rats are typical of agents, such as dopamine agonists, which decrease prolactin (PRL) activity. Since prolactin signals through the JAK2 pathway, we hypothesized that these findings were off-target effects due to inhibition of PRL signaling via JAK2. The studies reported here were designed to investigate the interruption of PRL signaling pathways in Leydig cells. In isolated primary rat Leydig cells, PRL increased phosphorylated Signal Transducer and Activator of Transcription-5 protein, and mRNA levels for luteinizing hormone receptor. Tofacitinib, at concentrations observed in the rat carcinogenicity study, dose-dependently inhibited these effects. These observations illustrate a novel mechanism, the inhibition of prolactin signaling by which modulation of JAK activity can modulate PRL signaling pathways to induce Leydig cell tumors in rats. Since human Leydig cells lack this PRL dependence for normal function, these rodent tumors do not indicate a health risk to human patients.
Asunto(s)
Adenoma/patología , Hiperplasia/inducido químicamente , Células Intersticiales del Testículo/efectos de los fármacos , Piperidinas/farmacología , Prolactina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Células Intersticiales del Testículo/metabolismo , Masculino , Ratas , Testículo/patologíaRESUMEN
Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.
Asunto(s)
Anticarcinógenos/uso terapéutico , Neoplasias Pulmonares/prevención & control , Tretinoina/uso terapéutico , Enzimas Activadoras de Ubiquitina/genética , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Enzimas Activadoras de Ubiquitina/análisis , Enzimas Activadoras de Ubiquitina/fisiologíaRESUMEN
Retinoids, the natural and synthetic derivatives of vitamin A, have a role in cancer treatment and prevention. There is a need to reveal mechanisms that account for retinoid response or resistance. This study identified candidate all-trans-retinoic acid (RA) target genes linked to growth suppression in BEAS-2B human bronchial epithelial cells. Microarray analyses were performed using Affymetrix arrays. A total of 11 RA-induced species were validated by reverse transcription polymerase chain reaction (RT-PCR), Western or Northern analyses. Three of these species were novel candidate RA-target genes in human bronchial epithelial cells. These included: placental bone morphogenetic protein (PLAB), polyamine oxidase isoform 1 (PAOh1) and E74-like factor 3 (ELF3). Expression patterns were studied in RA-resistant BEAS-2B-R1 cells. In BEAS-2B-R1 cells, RA dysregulated the expression of the putative lymphocyte G0/G1 switch gene (G0S2), heme oxygenase 1 (HMOX1), tumor necrosis factor-alpha-induced protein 2 (TNFAIP2), inhibitor of DNA binding 1(Id1), fos-like antigen 1 (FOSL1), transglutaminase 2 (TGM2), asparagine synthetase (ASNS), PLAB, PAOh1 and ELF3, while prominent induction of insulin-like growth-factor-binding protein 6 (IGFBP6) still occurred. In summary, this study identified 11 candidate RA-target genes in human bronchial epithelial cells including three novel species. Expression studies in BEAS-2B-R1 cells indicated that several were directly implicated in RA signaling, since their aberrant expression was linked to RA resistance of human bronchial epithelial cells.
Asunto(s)
Bronquios/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Tretinoina/farmacología , Northern Blotting , Western Blotting , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/metabolismo , Medio de Cultivo Libre de Suero/farmacología , Resistencia a Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Endocitosis/efectos de los fármacos , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos/toxicidad , Transcriptoma/efectos de los fármacos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Clatrina/metabolismo , Endocitosis/genética , Perfilación de la Expresión Génica , Inyecciones Subcutáneas , Hígado/patología , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismoRESUMEN
Pretreatment of mice with the peroxisome proliferator clofibrate (CFB) protects against acetaminophen (APAP)-induced hepatotoxicity. Previous studies have shown that activation of the nuclear peroxisome proliferator activated receptor-alpha (PPARalpha) is required for this effect. The present study utilizes gene expression profile analysis to identify potential pathways contributing to PPARalpha-mediated hepatoprotection. Gene expression profiles were compared between wild type and PPARalpha-null mice pretreated with vehicle or CFB (500 mg/kg, i.p., daily for 10 days) and then challenged with APAP (400 mg/kg, p.o.). Total hepatic RNA was isolated 4 h after APAP treatment and hybridized to Affymetrix Mouse Genome MGU74 v2.0 GeneChips. Gene expression analysis was performed utilizing GeneSpring software. Our analysis identified 53 genes of interest including vanin-1, cell cycle regulators, lipid-metabolizing enzymes, and aldehyde dehydrogenase 2, an acetaminophen binding protein. Vanin-1 could be important for CFB-mediated hepatoprotection because this protein is involved in the synthesis of cysteamine and cystamine. These are potent antioxidants capable of ameliorating APAP toxicity in rodents and humans. HPLC-ESI/MS/MS analysis of liver extracts indicates that enhanced vanin-1 gene expression results in elevated cystamine levels, which could be mechanistically associated with CFB-mediated hepatoprotection.
Asunto(s)
Clofibrato/farmacología , Perfilación de la Expresión Génica/métodos , Hígado/efectos de los fármacos , PPAR alfa/genética , Acetaminofén/administración & dosificación , Acetaminofén/toxicidad , Acil-CoA Oxidasa/genética , Acil-CoA Oxidasa/metabolismo , Amidohidrolasas , Animales , Anticolesterolemiantes/farmacología , Anticolesterolemiantes/uso terapéutico , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas , Clofibrato/uso terapéutico , Análisis por Conglomerados , Cistamina/química , Cistamina/metabolismo , Cisteamina/química , Cisteamina/metabolismo , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Proteínas Ligadas a GPI , Hígado/metabolismo , Hígado/patología , Hepatopatías/genética , Hepatopatías/prevención & control , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , PPAR alfa/metabolismo , Panteteína/química , Panteteína/metabolismo , Ácido Pantoténico/química , Ácido Pantoténico/metabolismo , Proliferadores de Peroxisomas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection. Expression of Nur77 in thymocytes and cell lines leads to apoptosis through a mechanism that remains unclear. In some cell lines, Nur77 was reported to act through a transcription-independent mechanism involving translocation to mitochondria, leading to cytochrome c release. However, we show here that Nur77-mediated apoptosis in thymocytes does not involve cytoplasmic cytochrome c release and cannot be rescued by Bcl-2. Microarray analysis shows that Nur77 induces many genes, including two novel genes (NDG1, NDG2) and known apoptotic genes FasL and TRAIL. Characterization of NDG1 and NDG2 indicates that NDG1 initiates a novel apoptotic pathway in a Bcl-2-independent manner. Thus Nur77-mediated apoptosis in T cells involves Bcl-2 independent transcriptional activation of several known and novel apoptotic pathways.