RESUMEN
Huntington's disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories.
Asunto(s)
Enfermedad de Huntington , Animales , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Péptidos/genética , Péptidos/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
1alpha,25-Dihydroxyvitamin D3 (1,25D3) exhibits antitumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 before cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 before cisplatin compared with either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin but increased by 1,25D3 in SCC cells. Knocking down p73 by small interfering RNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin antitumor activity in vitro and in vivo in a SCC model system possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Vitamina D/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Proteínas Nucleares/genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética , Vitamina D/farmacologíaRESUMEN
The expression of chemokine receptors and chemokine production by adult human non-transformed astrocytes, grade III astrocytoma and grade IV glioblastoma tumour cell lines were determined. Here, we show an increased expression of CXCR3 and CXCR4, and a decreased expression of CXCR1 and CCR4 by glioma cells compared to adult human astrocytes. Glioma cells showed increased production of CXCL10, whereas production of other chemokines was decreased (CXCL8, CCL2, CCL5, and CCL22). CXCL10 induced an ERK1/2-dependent increase in [(3)H] thymidine uptake. These results suggest that expression of chemokine receptor/ligand pairs such as CXCR3/CXCL10 have an important role in the proliferation of glioma cells.
Asunto(s)
Proliferación Celular , Quimiocina CXCL10/biosíntesis , Quimiocinas/biosíntesis , Glioma/metabolismo , Receptores de Quimiocina/biosíntesis , Astrocitos/metabolismo , Western Blotting , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Citometría de Flujo , Expresión Génica , Humanos , ARN Mensajero , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The 1alpha,25-dihydroxy-vitamin D(3) (1alpha,25(OH)(2)D(3)) mediated gene transcription in primary cultures of human prostate cells was analyzed using an adenoviral luciferase expression reporter under the control of the 25-hydroxy-vitamin D(3)-24-hydroxylase (CYP24) gene promoter. Stromal cells isolated from benign and malignant associated stroma (BAS and CAS) of a human clinical sample have been determined to contain similar levels of functional 1alpha,25(OH)(2)D(3) receptor (VDR). However, VDR-mediated reporter activity of the luciferase reporter has been found to be limited 7-9-fold in CAS compared to 14-16-fold in BAS. Chromatin immunoprecipitation (ChIP) assays indicate that in the absence of added ligand VDR interact with the silencing mediator for retinoid and thyroid hormone (SMRT) corepressor in both cell types, with higher recruitment in CAS as compared to BAS cells. In the presence of added ligand, VDR in CAS cells exhibited decreased ligand-inducible DNA binding activity, altered recruitment of coregulators SRC-1 and CBP, and increased recruitment of SMRT corepressor, as compared to BAS. Additionally, overexpression of wild-type VDR recovered VDR-mediated transaction of CYP24 luciferase reporter. These results indicate that VDR structure/function and coregulator recruitment to 1alpha,25(OH)(2)D(3) regulated genes is altered in the CaP stroma microenvironment.
Asunto(s)
Neoplasias de la Próstata/metabolismo , Receptores de Calcitriol/metabolismo , Células del Estroma/metabolismo , Transcripción Genética/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Células del Estroma/patología , Células Tumorales CultivadasRESUMEN
Calcitriol (1,25-dihydroxycholecalciferol), the active form of Vitamin D, is anti-proliferative in tumor cells and tumor-derived endothelial cells (TDEC). However, endothelial cells isolated from normal tissues as cell lines or freshly isolated cells or from implanted Matrigel plugs (MDEC) are relatively resistant. Both TDEC and MDEC express similar amounts of Vitamin D receptor (VDR) protein. Although the VDR from TDEC has higher binding affinity for calcitriol than those from MDEC, VDR in both cell types translocates to the nucleus and transactivates the 24-hydroxylase promoter-luciferase construct. Calcitriol selectively inhibits the growth of TDEC but not MDEC by inducing G(0)/G(1) cell cycle arrest and by promoting apoptosis. This selectivity appears to be related to 24-hydroxylase (CYP24) expression. Calcitriol significantly induced CYP24 expression in MDEC but not in TDEC and inhibition of CYP24 activity in MDEC restores their sensitivity to calcitriol. These findings indicate that the induction of CYP24 expression differs in endothelial cells isolated from different microenvironments (TDEC versus MDEC) and that this distinction contributes to selective calcitriol-mediated growth inhibition in these cell types.
Asunto(s)
Calcitriol/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias/patología , Receptores de Calcitriol/metabolismo , Esteroide Hidroxilasas/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
BACKGROUND: Huntington's disease (HD) is an autosomal dominant neurodegenerative condition caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Optimizing peripheral quantification of huntingtin throughout the course of HD is valuable not only to illuminate the natural history and pathogenesis of disease, but also to detect peripheral effects of drugs in clinical trial. RATIONALE: We previously demonstrated that mutant HTT (mHTT) was significantly elevated in purified HD patient leukocytes compared with controls and that these levels track disease progression. Our present study investigates whether the same result can be achieved with a simpler and more scalable collection technique that is more suitable for clinical trials. METHODS: We collected whole blood at 133 patient visits in two sample sets and generated peripheral blood mononuclear cells (PBMCs). Levels of mHTT, as well as N-, and C-terminal and mid-region huntingtin were measured in the PBMCs using ELISA-based Meso Scale Discovery (MSD) electrochemiluminescence immunoassay platforms, and we evaluated the relationship between different HTT species, disease stage, and brain atrophy on magnetic resonance imaging. CONCLUSIONS: The assays were sensitive and accurate. We confirm our previous findings that mHTT increases with advancing disease stage in patient PBMCs, this time using a simple collection protocol and scalable assay.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteína Huntingtina/sangre , Enfermedad de Huntington/sangre , Leucocitos/metabolismo , Encéfalo/diagnóstico por imagen , Estudios Transversales , Electroquímica , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/diagnóstico por imagen , Luminiscencia , MutaciónRESUMEN
It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.
Asunto(s)
Astrocitos/inmunología , Encefalopatías/inmunología , Encéfalo/inmunología , División Celular/inmunología , Quimiotaxis/inmunología , Microglía/inmunología , Receptores de Quimiocina/metabolismo , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Encefalopatías/patología , Encefalopatías/fisiopatología , División Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL10 , Quimiocinas CXC/farmacología , Quimiotaxis/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Microglía/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Quimiocina/efectos de los fármacos , Receptores de Quimiocina/genética , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
Huntington's disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Enfermedad de Huntington/metabolismo , Receptor trkB/agonistas , Receptor trkB/metabolismo , Animales , Anticuerpos Monoclonales/química , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Gemcitabine is the standard care chemotherapeutic agent to treat pancreatic cancer. Previously we demonstrated that calcitriol (1, 25-dihydroxycholecalciferol) has significant anti-proliferative effects in vitro and in vivo in multiple tumor models and enhances the activity of a variety of chemotherapeutic agents. We therefore investigated whether calcitriol could potentiate the cytotoxic activity of gemcitabine in the human pancreatic cancer Capan-1 model system. Isobologram analysis revealed that calcitriol and gemcitabine had synergistic antiproliferative effect over a wide range of drug concentrations. Calcitriol did not reduce the cytidine deaminase activity in Capan-1 tumors nor in the livers of Capan-1 tumor bearing mice. Calcitriol and gemcitabine combination promoted apoptosis in Capan-1 cells compared with either agent alone. The combination treatment also increased the activation of caspases-8, -9, -6 and -3 in Capan-1 cells. This result was confirmed by substrate-based caspase activity assay. Akt phosphorylation was reduced by calcitriol and gemcitabine combination treatment compared to single agent treatment. However, ERK1/2 phosphorylation was not modulated by either agent alone or by the combination. Tumor regrowth delay studies showed that calcitriol in combination with gemcitabine resulted in a significant reduction of Capan-1 tumor volume compared to single agent treatment. Our study suggests that calcitriol and gemcitabine in combination promotes caspase-dependent apoptosis, which may contribute to increased anti-tumor activity compared to either agent alone.
Asunto(s)
Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Calcitriol/farmacología , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/patología , Animales , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Ratones , Neoplasias Pancreáticas/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The most active metabolite of vitamin D, calcitriol, is growth inhibitory for various tumor types in vitro and in vivo and inhibits the growth of endothelial cells freshly isolated from tumors [tumor-derived endothelial cells (TDEC)]. We compared the effects of calcitriol on Matrigel-derived endothelial cells (MDEC) and TDEC isolated from Matrigel plugs and squamous cell carcinoma tumors, respectively. TDEC and MDEC expressed vitamin D receptor (VDR) and responded to calcitriol by increasing VDR protein expression. Although no mutations were found in VDR from either cell type, Scatchard plot analysis revealed a higher ligand-binding affinity in TDEC (K(d), 0.26 nmol/L) than MDEC (K(d), 0.65 nmol/L). The VDR signaling axis in both cells was intact as shown using nuclear translocation and 24-hydroxylase promoter-luciferase reporter assays. However, unlike TDEC, MDEC were resistant to calcitriol-induced growth inhibition. Calcitriol (10 nmol/L) resulted in a 12.3% growth inhibition of MDEC compared with 47% in TDEC. In TDEC, calcitriol resulted in induction of G(0)/G(1) arrest (10.75%) and reduction of S-phase cells (6.8%) with induction of p27 and down-regulation of p21 protein expression. Apoptotic effects, determined by Annexin V staining were also observed in calcitriol-treated TDEC (38.6%). Calcitriol caused reduced expression of p-Erk and p-Akt and an increase of poly(ADP-ribose) polymerase and caspase-3 cleavage in TDEC. By contrast, none of these effects on cell cycle or apoptosis were seen in calcitriol-treated MDEC. These results show that TDEC were more sensitive than MDEC to the antiproliferative effects of calcitriol despite apparently normal VDR content and structure of signaling axis in both cell types.
Asunto(s)
Calcitriol/farmacología , Carcinoma de Células Escamosas/irrigación sanguínea , División Celular/efectos de los fármacos , Endotelio Vascular/patología , Animales , Carcinoma de Células Escamosas/patología , Ciclo Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Núcleo Celular/fisiología , Colágeno , Cartilla de ADN , Combinación de Medicamentos , Endotelio Vascular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Laminina , Ratones , Ratones Endogámicos C3H , Invasividad Neoplásica/prevención & control , Proteoglicanos , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Calcitriol (1,25-dihydroxycholecalciferol) has antiproliferative and/or proapoptotic effects on many cell types and the glucocorticoid dexamethasone enhances these effects. We have shown that calcitriol modulates several key signaling proteins involved in differentiation, proliferation and apoptosis in tumor-derived murine endothelial cells (TDEC) and that these effects were not seen with endothelial cells isolated similarly from normal tissues. In the present study, TDEC and mouse embryonic yolk sac endothelial cells (MYSEC) were treated with calcitriol and followed over time for an effect. MYSEC were utilized as 'normal' control endothelial cells because they were more primitive, being isolated from a highly neovascular tissue, and had a similar morphology without the stimulus of the tumor microenvironment. The vitamin D receptor (VDR) is present in TDEC and MYSEC, and was upregulated in calcitriol-treated TDEC and MYSEC; dexamethasone further increased VDR expression following 48 h of treatment. The modulatory effects on signaling proteins were maximal by treatment for 48 h; phospho-Erk, phospho-Akt, p21 and bcl-2 were decreased in treated TDEC with the induction of p27 but there were no effects on MYSEC. After 48 h increased apoptosis was seen in treated TDEC by annexin V labeling with caspase-3 cleavage and decreased levels of poly(ADP-ribose) polymerase, but no effects were seen in MYSEC. Cell cycle analysis showed increased G(0)/G(1) arrest and an increase in the apoptotic sub-G(1) peak in treated TDEC but similar effects were not seen in MYSEC following 48-hour treatment. Proliferation assays were utilized and TDEC demonstrated decreased proliferation compared to normal endothelial cells at 48 h. To determine whether or not the VDR signaling was impaired in MYSEC, we performed the 24-hydroxylase (CYP24) promoter-luciferase reporter assay. CYP24 is a key enzyme involved in the breakdown of vitamin D. VDR signaling was intact in both cell types and calcitriol induced CYP24 mRNA expression in MYSEC but not in TDEC. Taken together, despite similar levels of VDR expression and intact signaling in both cell types, calcitriol selectively inhibits proliferation and induces apoptosis in TDEC with no effect on MYSEC. Thus calcitriol exerts differential effects on TDEC compared to normal cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Calcitriol/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Dexametasona/farmacología , Células Endoteliales/efectos de los fármacos , Receptores de Calcitriol/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Calcitriol/uso terapéutico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular/efectos de los fármacos , Dexametasona/uso terapéutico , Células Endoteliales/metabolismo , Inducción Enzimática/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos C3H , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroide Hidroxilasas/biosíntesis , Factores de Tiempo , Vitamina D3 24-Hidroxilasa , Saco Vitelino/efectos de los fármacos , Saco Vitelino/embriologíaRESUMEN
We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.