RESUMEN
INTRODUCTION: A key mechanism of lithium is the inhibition of glycogen synthase kinase-3ß (GSK3ß) and activation of mammalian target of rapamycin (mTOR), two contributors to insulin signaling. We explored the relationship between these markers and clinical response to lithium in bipolar disorder (BD). METHODS: Thirty-four subjects with BD who had been taking lithium for ≥2 years and had a maintenance lithium Alda score defined as either high (≥7; n = 20) or low (≤2; n = 14) were included in the study. Baseline protein expression of GSK3ß and mTOR (total and phosphorylated (p)) was obtained from a buffy coat. Peripheral blood mononuclear cells (PBMCs) from a subset of each group (n = 11) were stimulated with insulin (10 µg) and change in protein expression was determined using Western blot. RESULTS: In buffy coat samples, significantly higher levels of pmTOR were present in subjects with an Alda score ≤2 (lithium non-responsive), relative to those with scores ≥7 (lithium-responsive). No differences were observed for pGSK3ß. In contrast, functional PBMC responses to 5 min of insulin stimulation demonstrated robust increases in pGSK3ß (87.05 ± 43.41%) and pmTOR (105.7 ± 66.48%) in the lithium responsive group only. This contrasted observed decreases in pGSK3ß (34.08 ± 16.12%) and pmTOR (37.84 ± 14.39%) 5 mins post-insulin in non-responders. CONCLUSIONS: Dynamic increases in pmTOR and pGSK3ß post-insulin stimulation may reflect an immunometabolic state that facilitates lithium response. Further prospective analyses are needed to replicate and extend these preliminary findings and further investigate the role of insulin signaling in lithium response in BD.
Asunto(s)
Trastorno Bipolar , Litio , Trastorno Bipolar/tratamiento farmacológico , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasa 3 beta , Humanos , Insulina , Leucocitos Mononucleares/metabolismo , Litio/farmacología , Litio/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In patients with metastatic melanoma, high blood levels of galectin-9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin-9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin-9 with macrophages. We aimed to determine whether galectin-9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor-supportive. It was determined that incubation of CD68+ macrophages with galectin-9 or anti-CD206 blocked target binding and that both CD206 and galectin-9 were detected by immunoprecipitation of cell lysates. CD206 and galectin-9 had a binding affinity of 2.8 × 10-7 m. Galectin-9 causes CD206+ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP-1), but less macrophage-derived chemokine (MDC). Galectin-9 had no effect on classical monocyte subsets, but caused expansion of the non-classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin-9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin-9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti-tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Galectinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Lectinas de Unión a Manosa/metabolismo , Melanoma/metabolismo , Receptores de Superficie Celular/metabolismo , Neoplasias Cutáneas/metabolismo , Microambiente Tumoral , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Quimiocina CCL2/metabolismo , Quimiocina CCL22/metabolismo , Femenino , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Macrófagos/patología , Masculino , Receptor de Manosa , Melanoma/secundario , Persona de Mediana Edad , Neovascularización Patológica , Fenotipo , Unión Proteica , Transducción de Señal , Neoplasias Cutáneas/patología , Células THP-1 , Adulto JovenRESUMEN
Mutations in the tumor suppressors BRCA1 and BRCA2, which encode proteins that are key participants in homologous recombination (HR) repair, occur in â¼20% of high grade serous ovarian cancers. Although only 20% of these tumors have mutations in BRCA1 and BRCA2, nearly 50% of these tumors have defects in HR. Notably, however, the underlying genetic defects that give rise to HR defects in the absence of BRCA1 and BRCA2 mutations have not been fully elucidated. Here we show that the recurrent somatic CDK12 mutations identified in ovarian cancers impair the catalytic activity of this kinase, which is involved in the transcription of a subset of genes, including BRCA1 and other DNA repair genes. Furthermore, we show that disabling CDK12 function in ovarian cancer cells reduces BRCA1 levels, disrupts HR repair, and sensitizes these cells to the cross-linking agents melphalan and cisplatin and to the poly(ADP-ribose) polymerase (PARP) inhibitor veliparib (ABT-888). Taken together, these findings suggest that many CDK12 mutations are an unrecognized cause of HR defects in ovarian cancers.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Recombinación Homóloga/genética , Mutación , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Biocatálisis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Reactivos de Enlaces Cruzados/farmacología , Quinasas Ciclina-Dependientes/deficiencia , Inhibidores Enzimáticos/farmacología , Femenino , Recombinación Homóloga/efectos de los fármacos , Humanos , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genéticaRESUMEN
Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB-deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB-deficient cells and tissues from CypB-knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.
Asunto(s)
Ciclofilinas/deficiencia , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/patología , Animales , Tamaño Corporal , Enfermedades Óseas Metabólicas/complicaciones , Enfermedades Óseas Metabólicas/patología , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Ciclofilinas/metabolismo , Proteínas de la Matriz Extracelular , Células HeLa , Humanos , Cifosis/complicaciones , Cifosis/patología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Chaperonas Moleculares , Osteogénesis Imperfecta/complicaciones , Fenotipo , Unión Proteica , Transporte de Proteínas , Proteínas/metabolismo , Proteoglicanos/metabolismo , Anomalías Cutáneas/patologíaRESUMEN
Calcium-modulating cyclophilin ligand (CAML) is a ubiquitous protein that has been implicated in signaling from the cell surface receptor TACI in lymphocytes, although its role and mechanism of action are unknown. To study its function in the mouse, we disrupted the CAML gene and found it to be required for early embryonic development, but not for cellular viability. CAML-deficient cells have severely impaired proliferative responses to the epidermal growth factor (EGF). Although EGF-induced activation of signaling intermediates and internalization of the EGF receptor (EGFR) are normal in the absence of CAML, the recycling of internalized receptors to the plasma membrane is defective, leading to its reduced surface accumulation. We demonstrate that CAML normally associates directly with the kinase domain of the EGFR in a ligand-dependent manner. These data implicate CAML in EGFR signaling and suggest that it may play a role in receptor recycling during long-term proliferative responses to EGF.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Portadoras/genética , Línea Celular , Colorantes Fluorescentes/metabolismo , Humanos , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Unión Proteica , Transporte de Proteínas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Rodaminas/metabolismoRESUMEN
Lithium has been shown to have some therapeutic efficacy as an adjunctive treatment for intractable forms of major depression. Activation of mammalian target of rapamycin (mTOR) and inhibition of glycogen synthase kinase-3ß (GSK3ß) have been implicated in its putative mechanisms of action. These proteins are integral components of the insulin signaling pathway, which may serve as a critical co-regulator of drug action. Utilizing an animal model of tricyclic antidepressant resistance, we investigated the relationship between insulin signaling and antidepressant response to lithium augmentation. Pre-treatment with adrenocorticotropic hormone (ACTH 100 µg/day i.p.) for 14 days effectively blocked the immobility-reducing effects of an acute dose of imipramine (10 mg/kg i.p.) in the forced swim test (FST). Lithium augmentation (100 mg/kg i.p.) rescued the antidepressant-like effects of imipramine in this model. Total and phosphorylated (p) levels of protein kinase B (Akt), mTOR, and GSK3ß protein were quantified in the infralimbic cortex (ILPFC) following FST stress via Western blot. Levels of mTOR and pmTOR were further quantified in isolated peripheral blood mononuclear cells (PBMCs) following insulin stimulation (10 mg/mL for 5 min) via ELISA. Elevated levels of phosphorylated insulin signaling proteins were present in the ILPFC of ACTH-pretreated animals that received both imipramine and lithium, together with a concurrent increase in mTOR activation in PBMCs. Large correlations were observed between immobility time and insulin-stimulated mTOR levels in PBMCs. We propose that PBMC insulin challenge may be a useful probe for predicting antidepressant response to lithium administration, and potentially other therapies acting via similar mechanisms of action.
Asunto(s)
Antidepresivos Tricíclicos/farmacología , Imipramina/farmacología , Leucocitos Mononucleares/metabolismo , Litio/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Depresión/tratamiento farmacológico , Depresión/fisiopatología , Insulina/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Fosforilación , Distribución Aleatoria , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , NataciónRESUMEN
Epithelial tumors including melanoma often first metastasize to regional, sentinel lymph nodes (SLN). Thus, the presence of SLN metastases is a critical prognostic factor of survival. Prior to metastasis, accumulating evidence suggests the SLN is immunologically compromised; however, the process by which pre-metastatic niche formation occurs remains unknown. In this prospective study, freshly dissected, afferent lymphatic fluid was obtained during SLN biopsy in three patients with primary cutaneous melanoma. Lymphatic extracellular vesicles (L-EV) were visualized by transmission electron microscopy and proteomic cargo profiled by mass spectrometry. Flow cytometry assessed L-EV effects on autologous dendritic cell maturation in vitro. Immunogold electron microscopy and immunohistochemistry visualized expression of EV cargo within the primary tumor and SLN. Lymphatic extracellular vesicles from each afferent lymphatic channel demonstrated inhibition of autologous dendritic cell maturation. Proteomic profiling identified 81 peptides shared among the L-EV preparations including a signature of 18 immune-modulating proteins including previously established inhibitor of dendritic cell maturation, S100A9. Immunohistochemistry and immunogold electron microscopy confirmed S100A9 tracking along the lymphatic path, from keratinocytes in the primary tumor to sub-capsular macrophages in the SLN. Our findings suggest L-EV cargo may serve as early mediators of tumor-induced immune subversion in regional lymph nodes, by preceding malignant cells and trafficking within the lymphatic vasculature to harbor the first pre-metastatic niche.
RESUMEN
We developed a nano-antibody targeted chemotherapy (nATC) delivery strategy in which tumor specific and clinically relevant antibodies (rituximab, anti-CD20) are non-covalently bound to the albumin scaffold of nab-paclitaxel (ABX). We define the nanoparticle formed when the 2 drugs are bound (AR160). The newly created nATC retains the cytotoxicity of ABX and CD20 affinity of rituximab in vitro. We describe the binding characteristics of the ABX and rituximab in AR160 using peptide mapping/Biacore approach. Flow-based methods, including ImageStream and nanoparticle tracking, were used to characterize the AR160 particles in vitro. A mouse model of human B-cell lymphoma was utilized to test in vivo efficacy of AR160 therapy, which suggested improved tumor targeting (biodistribution) as the most likely mechanism of AR160 therapeutic superiority over ABX or rituximab alone. These data suggest a novel platform for nATC delivery using a slight modification of existing cancer drugs with significantly improved treatment efficacy.
Asunto(s)
Antígenos CD20/metabolismo , Antineoplásicos Fitogénicos/administración & dosificación , Linfoma de Células B/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Nanopartículas/administración & dosificación , Paclitaxel/administración & dosificación , Rituximab/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Resultado del TratamientoRESUMEN
High frequency deep brain stimulation (DBS) of the lateral habenula (LHb) reduces symptoms of depression in severely treatment-resistant individuals. Despite the observed therapeutic effects, the molecular underpinnings of DBS are poorly understood. This study investigated the efficacy of high frequency LHb DBS (130Hz; 200µA; 90µs) in an animal model of tricyclic antidepressant resistance. Further, we reported DBS mediated changes in Ca(2+)/calmodulin-dependent protein kinase (CaMKIIα/ß), glycogen synthase kinase 3 (GSK3α/ß) and AMP-activated protein kinase (AMPK) both locally and in the infralimbic cortex (IL). Protein expressions were then correlated to immobility time during the forced swim test (FST). Antidepressant actions were quantified via FST. Treatment groups comprised of animals treated with adrenocorticotropic hormone alone (ACTH; 100µg/day, 14days, n=7), ACTH with active DBS (n=7), sham DBS (n=8), surgery only (n=8) or control (n=8). Active DBS significantly reduced immobility in ACTH-treated animals (p<0.05). For this group, western blot results demonstrated phosphorylation status of LHb CaMKIIα/ß and GSK3α/ß significantly correlated to immobility time in the FST. Concurrently, we observed phosphorylation status of CaMKIIα/ß, GSK3α/ß, and AMPK in the IL to be negatively correlated with antidepressant actions of DBS. These findings suggest that activity dependent phosphorylation of CaMKIIα/ß, and GSK3α/ß in the LHb together with the downregulation of CaMKIIα/ß, GSK3α/ß, and AMPK in the IL, contribute to the antidepressant actions of DBS.
Asunto(s)
Estimulación Encefálica Profunda/métodos , Trastorno Depresivo Resistente al Tratamiento/terapia , Habénula/fisiología , Lóbulo Límbico/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Hormona Adrenocorticotrópica/uso terapéutico , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Glucógeno Sintasa Quinasa 3/metabolismo , Pérdida de Tono Postural/efectos de los fármacos , Pérdida de Tono Postural/fisiología , Lóbulo Límbico/efectos de los fármacos , Masculino , Fosforilación , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Natación/psicologíaRESUMEN
Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Calor , Complejos Multiproteicos/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Western Blotting , Carcinoma Hepatocelular/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Proteína Oncogénica v-akt/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Transducción de Señal/genética , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Ketamine, N-methyl-d-aspartate (NMDA) receptor antagonist and anti-inflammatory agent, has rapid therapeutic effects in a subset of patients with more intractable forms of depression. Irregular proinflammatory cytokine and acute-reactive protein levels have been reported in clinical and preclinical depression research. We explored the association between the rapid antidepressant-like effects of ketamine and peripheral proinflammatory profile in a model of antidepressant-resistance. Male Wistar rats were pre-treated with ACTH-(1-24) 100µg/d or saline (0.9%) for 14d. Antidepressant-like effects were assessed with the forced swim test (FST). Ketamine (10mg/kg) significantly reduced immobility duration in saline-pretreated control animals. In contrast, a divergent response was observed in ACTH-pretreated antidepressant resistant animals, with 50% responders and 50% non-responders. Plasma samples were analyzed via enzyme-linked immunosorbent assay (ELISA) for interleukin 6 (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP). Levels of CRP and TNFα differentiated ketamine responders and non-responders.
Asunto(s)
Antidepresivos/uso terapéutico , Proteína C-Reactiva/metabolismo , Citocinas/metabolismo , Depresión/tratamiento farmacológico , Depresión/metabolismo , Ketamina/uso terapéutico , Hormona Adrenocorticotrópica/farmacología , Análisis de Varianza , Animales , Antidepresivos/farmacología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Conducta Exploratoria/efectos de los fármacos , Pérdida de Tono Postural/efectos de los fármacos , Ketamina/farmacología , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Wistar , NataciónRESUMEN
INTRODUCTION: Pulmonary carcinoid tumors account for approximately 5% of all lung malignancies in adults, and comprise 30% of all carcinoid tumors. There are limited reagents available to study these rare tumors, and consequently no major advances have been made for patient treatment. We report the generation and characterization of human pulmonary carcinoid tumor cell lines to study underlying biology, and to provide models for testing novel chemotherapeutic agents. METHODS: Tissue was harvested from three patients with primary pulmonary typical carcinoid tumors undergoing surgical resection. The tumor was dissociated and plated onto dishes in culture media. The established cell lines were characterized by immunohistochemistry, Western blotting, and cell proliferation assays. Tumorigenicity was confirmed by soft agar growth and the ability to form tumors in a mouse xenograft model. Exome and RNA sequencing of patient tumor samples and cell lines was performed using standard protocols. RESULTS: Three typical carcinoid tumor lines grew as adherent monolayers in vitro, expressed neuroendocrine markers consistent with the primary tumor, and formed colonies in soft agar. A single cell line produced lung tumors in nude mice after intravenous injection. Exome and RNA sequencing of this cell line showed lineage relationship with the primary tumor, and demonstrated mutations in a number of genes related to neuronal differentiation. CONCLUSION: Three human pulmonary typical carcinoid tumor cell lines have been generated and characterized as a tool for studying the biology and novel treatment approaches for these rare tumors.
Asunto(s)
Tumor Carcinoide/genética , Neoplasias Pulmonares/genética , Adulto , Anciano , Animales , Tumor Carcinoide/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones DesnudosRESUMEN
PURPOSE: To investigate the potential role for CD44(+) and CD90(+) hepatocellular carcinoma (HCC) cellular subpopulations in biological response to thermal ablation-induced heat stress. METHODS: This study was approved by the institutional animal care committee. The N1S1 rat HCC cell line was subjected to sublethal heat stress (45 °C) or control (37 °C) for 10 min, costained with fluorescent-labeled antibodies against CD44, CD90, and 7-AAD after a 48-h recovery and analyzed by flow cytometry to assess the percentage of live CD44(+) and CD90(+) HCC cells (n = 4). Experiments were repeated with pretreatment of N1S1 cells with a dose titration of the dual PI3K-mTOR inhibitor BEZ235 or vehicle control (n = 3). Rats bearing orthotopic N1S1 tumors were subjected to ultrasound-guided partial laser ablation (n = 5) or sham ablation (n = 3), euthanized 24 h after ablation, and liver/tumor analyzed for immunohistochemical staining of CD44 and CD90. Differences between groups were compared with an unpaired t test. RESULTS: Sublethal heat stress induced a significant increase in the relative proportion of live CD44(+) and CD90(+) HCC cells compared to the control group: CD44(+)CD90(-) (5.3-fold; p = 0.0001), CD44(-)CD90(+) (2.4-fold; p = 0.003), and CD44(+)CD90(+) (22.0-fold; p < 0.03). Inhibition of PI3K-mTOR prevented heat stress-induced enrichment of the population of live CD44(+) HCC cells (p < 0.01), but not CD90(+) cells (p > 0.10). Immunohistochemical analysis demonstrated preferential localization of clusters of CD44(+) cells at both the tumor margin and ablation margin. CONCLUSION: These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.
Asunto(s)
Carcinoma Hepatocelular/cirugía , Calor , Terapia por Láser/métodos , Neoplasias Hepáticas Experimentales/cirugía , Células Madre/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo/métodos , Técnicas In Vitro , Neoplasias Hepáticas Experimentales/metabolismo , Proyectos Piloto , RatasRESUMEN
Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.
Asunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/genética , Proteína BRCA2/genética , Bencimidazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cisplatino/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Immunoblotting , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosforilación/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pirazinas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonas/farmacología , Topotecan/farmacología , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismo , GemcitabinaRESUMEN
OBJECTIVES: The objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. MATERIALS AND METHODS: This study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging-guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. RESULTS: The MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with the percentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. CONCLUSIONS: Bioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.
Asunto(s)
Terapia por Láser/métodos , Mediciones Luminiscentes/métodos , Imagen por Resonancia Magnética/métodos , Neoplasias Experimentales/patología , Neoplasias Experimentales/cirugía , Cirugía Asistida por Computador/métodos , Animales , Línea Celular Tumoral , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heme oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-kappaB binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-kappaB, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-kappaB-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1.
Asunto(s)
Quimiocina CCL2/biosíntesis , Hemo-Oxigenasa 1/biosíntesis , Fallo Renal Crónico/fisiopatología , Riñón/enzimología , Albúmina Sérica Bovina/farmacología , Animales , Butadienos/farmacología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , FN-kappa B/metabolismo , Nitrilos/farmacología , Ratas , Regulación hacia ArribaRESUMEN
B-lymphocyte homeostasis and function are regulated by complementary actions of the TNFR family members TACI, BCMA, and BAFF-R, which are expressed by mature B cells. How these receptors are differentially activated is not entirely understood, because the primary ligand BAFF binds to all three. We searched for alternative ligands for TACI using recombinant TACI-Fc fusion protein as a probe and identified syndecan-2 as a new binding partner. TACI binding appears to require heparan sulfate posttranslational modifications of syndecan-2, because free heparin or pretreatment with heparitinase blocked the interaction. Syndecan-2 bound TACI but bound neither BAFF-R nor BCMA. Transfected cells expressing syndecan-2 activated signaling through TACI, as indicated by an NFAT-specific reporter. Syndecan-1 and syndecan-4 were also able to induce TACI signaling in a similar manner. This is the first identification of ligands that selectively activate TACI without simultaneously triggering BCMA or BAFF-R. This finding may help explain the alternative outcomes of signaling from this family of receptors in B cells.
Asunto(s)
Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Membrana/inmunología , Proteoglicanos/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Transducción de Señal/inmunología , Receptor del Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Linfocitos B/metabolismo , Expresión Génica/genética , Expresión Génica/inmunología , Humanos , Células Jurkat , Activación de Linfocitos/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/biosíntesis , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/inmunología , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Receptores del Factor de Necrosis Tumoral/biosíntesis , Transducción de Señal/genética , Sindecano-1 , Sindecano-2 , Sindecano-4 , Sindecanos , Transfección , Proteína Activadora Transmembrana y Interactiva del CAMLRESUMEN
Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed protein implicated in T cell signaling, although its mechanism and physiologic role in the immune system are unknown. We show here that CAML is essential for peripheral T cell development. Inactivation of CAML in mouse thymocytes lowered the numbers of double-positive and single-positive thymocytes, concomitant with reduced positive and enhanced negative selection. We found that CAML interacts with p56Lck and appears to regulate subcellular localization of the kinase in both resting and T cell receptor (TCR)-stimulated cells. CAML-deficient cells displayed enhanced p56lck and ZAP-70 phosphorylation and increased IL2 production and cell death after TCR stimulation, suggesting that CAML may act as a negative regulator of p56lck. Our data establish a novel role for CAML as an essential mediator of T cell survival during thymopoiesis and indicate that its loss deregulates p56Lck signaling.