RESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for both malignant and nonmalignant hematologic disorders. However, graft-versus-host disease (GVHD) and malignant relapse limit its therapeutic success. We previously demonstrated that the blockade of interferon-gamma receptor (IFNGR) signaling in donor T cells resulted in a reduction in GVHD while preserving graft-versus-leukemia (GVL) effects. However, the underlying molecular mechanisms remain inconclusive. In this study, we found that S100A9 is a novel GVHD suppressor upregulated when IFNGR is blocked in T cells. Both Ifngr1-/- and S100a9-overexpressing T cells significantly reduced GVHD without compromising GVL, altering donor T-cell trafficking to GVHD target organs in our mouse model of allo-HSCT. In addition, in vivo administration of recombinant murine S100A9 proteins prolongs the overall survival of recipient mice. Furthermore, in vivo administration of anti-human IFNGRα neutralizing antibody (αhGR-Nab) significantly upregulates the expression of S100A9 in human T cells and improved GVHD in our mouse model of xenogeneic human peripheral blood mononuclear cell transplantation. Consistent with S100a9-overexpressing T cells in our allo-HSCT model, αhGR-Nab reduced human T-cell trafficking to the GVHD target organs. Taken together, S100A9, a downstream molecule suppressed by IFNGR signaling, functions as a novel GVHD suppressor without compromising GVL.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Ratones , Humanos , Animales , Trasplante Homólogo , Leucocitos Mononucleares/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Linfocitos T , Proteínas Recombinantes/metabolismo , Efecto Injerto vs Leucemia , Calgranulina BRESUMEN
Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Pirazoles , Trasplante Homólogo , Animales , Ratones , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/metabolismo , Azetidinas/farmacología , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de las Cinasas Janus/farmacología , Ratones Endogámicos C57BL , Purinas/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
S100A8 and S100A9 are multifunctional proteins that can initiate various signaling pathways and modulate cell function both inside and outside immune cells, depending on their receptors, mediators, and molecular environment. They have been reported as dysregulated genes and proteins in a wide range of cancers, including hematologic malignancies, from diagnosis to response to therapy. The role of S100A8 and S100A9 in hematologic malignancies is highlighted due to their ability to work together or as antagonists to modify cell phenotype, including viability, differentiation, chemosensitivity, trafficking, and transcription strategies, which can lead to an oncogenic phase or reduced symptoms. In this review article, we discuss the critical roles of S100A8, S100A9, and calprotectin (heterodimer or heterotetramer forms of S100A8 and S100A9) in forming and promoting the malignant bone marrow microenvironment. We also focus on their potential roles as biomarkers and therapeutic targets in various stages of hematologic malignancies from diagnosis to treatment.
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Calgranulina A , Calgranulina B , Neoplasias Hematológicas , Humanos , Diferenciación Celular , Neoplasias Hematológicas/tratamiento farmacológico , Complejo de Antígeno L1 de Leucocito , Microambiente TumoralRESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main therapeutic strategy for patients with both malignant and nonmalignant disorders. The therapeutic benefits of allo-HSCT in malignant disorders are primarily derived from the graft-versus-leukemia (GvL) effect, in which T cells in the donor graft recognize and eradicate residual malignant cells. However, the same donor T cells can also recognize normal host tissues as foreign, leading to the development of graft-versus-host disease (GvHD), which is difficult to separate from GvL and is the most frequent and serious complication following allo-HSCT. Inhibition of donor T cell toxicity helps in reducing GvHD but also restricts GvL activity. Therefore, developing a novel therapeutic strategy that selectively suppresses GvHD without affecting GvL is essential. Recent studies have shown that inhibition of histone deacetylases (HDACs) not only inhibits the growth of tumor cells but also regulates the cytotoxic activity of T cells. Here, we compile the known therapeutic potential of HDAC inhibitors in preventing several stages of GvHD pathogenesis. Furthermore, we will also review the current clinical features of HDAC inhibitors in preventing and treating GvHD as well as maintaining GvL.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Efecto Injerto vs Leucemia/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias/terapia , Ensayos Clínicos como Asunto , Enfermedad Injerto contra Huésped/enzimología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Linfocitos T Citotóxicos/efectos de los fármacos , Trasplante HomólogoRESUMEN
Purpose To demonstrate that positron emission tomography (PET) with fluorine 18 (18F) fluorthanatrace (FTT) depicts activated poly (adenosine diphosphate-ribose)polymerase (PARP) expression and is feasible for clinical trial evaluation. Materials and Methods All studies were conducted prospectively from February 2012 through July 2015 under protocols approved by the local animal studies committee and institutional review board. The area under the receiver operating characteristic curve (AUC, in g/mL· min) for 18F-FTT was assessed in normal mouse organs before and after treatment with olaparib (n = 14), a PARP inhibitor, or iniparib (n = 11), which has no PARP inhibitory activity. Murine biodistribution studies were performed to support human translational studies. Eight human subjects with cancer and eight healthy volunteers underwent imaging to verify the human radiation dosimetry of 18F-FTT. The Wilcoxon signed rank test was used to assess for differences among treatment groups for the mouse studies. Results In mice, olaparib, but not iniparib, significantly reduced the 18F-FTT AUC in the spine (median difference before and after treatment and interquartile range [IQR]: -17 g/mL· min and 10 g/mL · min, respectively [P = .0001], for olaparib and -3 g/mL · min and 13 g/mL · min [P = .70] for iniparib) and in nodes (median difference and interquartile range [IQR] before and after treatment: -23 g/mL · min and 13 g/mL · min [P = .0001] for olaparib; -9 g/mL · min and 17 g/mL · min [P = .05] for iniparib). The effective dose was estimated at 6.9 mSv for a 370-MBq 18F-FTT dose in humans. In humans, the organs with the highest uptake on images were the spleen and pancreas. Among five subjects with measurable tumors, increased 18F-FTT uptake was seen in one subject with pancreatic adenocarcinoma and another with liver cancer. Conclusion The results suggest that 18F-FTT uptake reflects PARP expression and that its radiation dosimetry profile is compatible with those of agents currently in clinical use. © RSNA, 2016 Online supplemental material is available for this article.
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Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/metabolismo , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Tomografía de Emisión de Positrones/métodos , Adulto , Animales , Benzamidas/farmacología , Biomarcadores Farmacológicos/metabolismo , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Femenino , Radioisótopos de Flúor , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Estudios Prospectivos , RadiometríaRESUMEN
A gram-negative bacterium, designated FJY1(T), was isolated from a soil sample obtained from a university campus in South Korea. Examination showed that FJY1(T) was red-pigmented, aerobic, motile, and composed of nonspore-forming rods. This strain grew in a temperature range of 15-37 °C and a pH range of 7-9. Based on 16S rRNA gene sequence similarity, strain FJY1(T) was most closely related to Rufibacter roseus H359(T) and Rufibacter tibetensis 1351(T), with sequence similarities of 95.98 and 95.46 %, respectively. Its major cellular fatty acids were iso-C15:0 (18.16 %) and summed feature 4 (C17:1 iso I and C17:1 anteiso B; 15.17 %). The DNA G+C content of FJY1(T) was 49.7 mol%; its predominant quinone was MK-7; and its major polar lipid was phosphatidylethanolamine. Phylogenetic analysis and analysis of its physiological and biochemical characteristics indicated that this isolate constituted a novel species, for which we propose the name Rufibacter soli sp. nov., with the type strain FJY1(T) (=KCTC 42815(T) = JCM 31024(T)).
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Cytophagaceae/aislamiento & purificación , Microbiología del Suelo , Composición de Base , Cytophagaceae/clasificación , Cytophagaceae/genética , Cytophagaceae/metabolismo , ADN Bacteriano/genética , ADN Ribosómico/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Filogenia , ARN Ribosómico 16S/genética , República de CoreaRESUMEN
Solid organ transplant represents a potentially lifesaving procedure for patients suffering from end-stage heart, lung, liver, and kidney failure. However, rejection remains a significant source of morbidity and immunosuppressive medications have significant toxicities. Janus kinase (JAK) inhibitors are effective immunosuppressants in autoimmune diseases and graft versus host disease after allogeneic hematopoietic cell transplantation. Here we examine the role of JAK inhibition in preclinical fully major histocompatibility mismatched skin and heart allograft models. Baricitinib combined with cyclosporine A (CsA) preserved fully major histocompatibility mismatched skin grafts for the entirety of a 111-day experimental period. In baricitinib plus CsA treated mice, circulating CD4+T-bet+ T cells, CD8+T-bet+ T cells, and CD4+FOXP3+ regulatory T cells were reduced. Single cell RNA sequencing revealed a unique expression profile in immune cells in the skin of baricitinib plus CsA treated mice, including decreased inflammatory neutrophils and increased CCR2- macrophages. In a fully major histocompatibility mismatched mismatched heart allograft model, baricitinib plus CsA prevented graft rejection for the entire 28-day treatment period compared with 9 days in controls. Our findings establish that the combination of baricitinib and CsA prevents rejection in allogeneic skin and heart graft models and supports the study of JAK inhibitors in human solid organ transplantation.
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Ciclosporina , Trasplante de Corazón , Humanos , Animales , Ratones , Ciclosporina/uso terapéutico , Rechazo de Injerto/prevención & control , Trasplante de Corazón/efectos adversos , SulfonamidasRESUMEN
Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.
Asunto(s)
Azetidinas/farmacología , Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de las Cinasas Janus/farmacología , Purinas/farmacología , Pirazoles/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Aloinjertos , Animales , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Efecto Injerto vs Leucemia/efectos de los fármacos , Efecto Injerto vs Leucemia/genética , Efecto Injerto vs Leucemia/inmunología , Interleucina-15/genética , Interleucina-15/inmunología , Janus Quinasa 1/genética , Janus Quinasa 1/inmunología , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.
RESUMEN
Asian dust or yellow sand events in East Asia are a major issue of environmental contamination and human health, causing increasing concern. A high amount of dust particles, especially called as particulate matter 10 (PM10), is transported by the wind from the arid and semi-arid tracks to the Korean peninsula, bringing a bacterial population that alters the terrestrial and atmospheric microbial communities. In this study, we aimed to explore the bacterial populations of Asian dust samples collected during November-December 2014. The dust samples were collected using the impinger method, and the hypervariable regions of the 16S rRNA gene were amplified using PCR followed by pyrosequencing. Analysis of the sequencing data were performed using Mothur software. The data showed that the number of operational taxonomic units and diversity index during Asian dust events were higher than those during non-Asian dust events. At the phylum level, the proportions of Proteobacteria, Actinobacteria, and Firmicutes were different between Asian dust and non-Asian dust samples. At the genus level, the proportions of the genus Bacillus (6.9%), Arthrobacter (3.6%), Blastocatella (2%), Planomicrobium (1.4%) were increased during Asian dust compared to those in non-Asian dust samples. This study showed that the significant relationship between bacterial populations of Asian dust samples and non-Asian dust samples in Korea, which could significantly affect the microbial population in the environment.
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Microbiología del Aire , Contaminantes Atmosféricos/análisis , Polvo/análisis , Material Particulado/análisis , Monitoreo del Ambiente/métodos , Humanos , Metagenómica , ARN Ribosómico 16S/genética , SeúlAsunto(s)
Azetidinas/farmacología , Receptores ErbB/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Intestinos/efectos de los fármacos , Janus Quinasa 3/antagonistas & inhibidores , Purinas/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Linfocitos T Reguladores/inmunología , Animales , Receptores ErbB/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Intestinos/lesiones , Intestinos/patología , RatonesRESUMEN
During Asian dust events, a relatively high concentration of particulate matter is transported by wind from arid and semi-arid regions, such as the Gobi and Taklamakan deserts, to nearby countries, including China, Korea, and Japan. The dust particles contain various microorganisms, which can affect human health as well as the environmental microbe population. In the current study, we investigated the characteristics of the airborne bacterial community during Asian dust events between February and March 2015 in South Korea. Bacterial diversity indexes such as operational taxonomic units, Chao1 and Inverse Simpson index were increased, along with total 16S rRNA gene copy number during Asian dust events. The bacterial community structure during Asian dust events was clearly distinguishable from that during non-Asian dust days. The genera Bacillus and Modestobacter were increased 3.9- and 2.7-fold, respectively, while Escherichia-Shigella was decreased by 89.8%. A non-metric multidimensional scaling plot with metadata analysis revealed association of particulate matter concentration, but not temperature, humidity or wind speed, with bacterial community structure, suggesting that the newly transported dust particles contain various microorganisms that influence the airborne bacterial environment.
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Microbiología del Aire , Contaminantes Atmosféricos/análisis , Polvo/análisis , Material Particulado/análisis , Viento , Bacterias/clasificación , Bacterias/genética , China , Monitoreo del Ambiente/métodos , Humanos , Japón , Filogenia , ARN Ribosómico 16S/genética , República de Corea , Estaciones del Año , Análisis de Secuencia de ADNRESUMEN
We present a plasmonic-based strategy for the colourimetric and spectroscopic differentiation of various organic acids produced by bacteria. The strategy is based on our discovery that particular concentrations of dl-lactic, acetic, and butyric acids induce different assembly structures, colours, and optical spectra of gold nanoparticles. We selected wild-type (K-12 W3110) and genetically-engineered (JHL61) Escherichia coli (E. coli) that are known to primarily produce acetic and butyric acid, respectively. Different assembly structures and optical properties of gold nanoparticles were observed when different organic acids, obtained after the removal of acid-producing bacteria, were mixed with gold nanoparticles. Moreover, at moderate cell concentrations of K-12 W3110 E. coli, which produce sufficient amounts of acetic acid to induce the assembly of gold nanoparticles, a direct estimate of the number of bacteria was possible based on time-course colour change observations of gold nanoparticle aqueous suspensions. The plasmonic-based colourimetric and spectroscopic methods described here may enable onsite testing for the identification of organic acids produced by bacteria and the estimation of bacterial numbers, which have applications in health and environmental sciences.
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Acetatos/metabolismo , Butiratos/metabolismo , Escherichia coli/metabolismo , Oro/metabolismo , Ácido Láctico/biosíntesis , Nanopartículas del Metal/química , Acetatos/análisis , Butiratos/análisis , Colorimetría , Ingeniería Genética , Oro/química , Ácido Láctico/análisis , Fotograbar , Espectrofotometría UltravioletaRESUMEN
We present a simple and industrially accessible method of producing liquid crystalline lipid nanoparticles with various internal structures based on phytantriol, Pluronic F127, and vitamin E acetate. Bilayer vesicles were produced when an ethanolic solution dissolving the lipid components was mixed with deionized water. After the evaporation of ethanol from the aqueous mixture, vesicles were transformed into lipid-filled liquid crystalline nanoparticles with well-defined internal structures such as hexagonal lattices (mostly inverted cubic Pn3m), lined or coiled pattern (inverted hexagonal H2), and disordered structure (inverse microemulsion, L2), depending on the compositions. Further studies suggested that their internal structures were also affected by temperature. The internal structures were characterized from cryo-TEM and small-angle X-ray scattering results. Microcalorimetry studies were performed to investigate the degree of molecular ordering/crystallinity of lipid components within the nanostructures. From the comparative studies, we demonstrated the present method could produce the lipid nanoparticles with similar characteristics to those made from a conventional method. More importantly, the production only requires simple tools for mixing and ethanol evaporation and it is possible to produce 10 kg or so per batch of aqueous lipid nanoparticles dispersions, enabling the large-scale production of the liquid crystalline nanoparticles for various biomedical applications.
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Lípidos/química , Cristales Líquidos/química , Nanoestructuras/química , Alcoholes Grasos/química , Nanopartículas/química , Poloxámero/química , Dispersión del Ángulo Pequeño , Temperatura , Vitamina E/química , Difracción de Rayos XRESUMEN
In head and neck squamous cell cancer (HNSCC), four intrinsic subtypes (or groups) have been identified, and each one possesses a unique biology that will require specific treatment strategies. We previously reported that mesenchymal (group 2) tumors exhibit reduced levels of Trop2 expression. In this study, we investigated the functional role of Trop2 in HNSCC and find that loss results in autocrine activation of the EGFR family member ErbB3 via neuregulin-1. Trop2 localizes to both the cell surface and cytosol of HNSCC cells and forms a complex with neuregulin-1, which is predominantly cytosolic. Inactivation of Trop2 increases the concentration of neuregulin-1 at the cell surface where it is cleaved to activate ErbB3. In primary HNSCC, detection of ErbB3 activation was limited to Trop2 negative tumors. An analysis of the Cancer Genome Atlas (TCGA) HNSCC dataset confirms enrichment for ErbB3 activity in mesenchymal tumors. Notably, Trop2 loss triggers sensitivity to anti-ErbB3 antibodies, which results in reduced proliferation and tumorigenic growth of Trop2 negative HNSCC cancer cells. These results uncover a molecular mechanism by which tumor cells control the amount of cell-surface neuregulin-1 available for cleavage and ErbB3 activation. Moreover, we demonstrate that Trop2 is a potential surrogate biomarker to identify tumors with ErbB3 activation and may therefore respond to anti-ErbB3 therapeutics.
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Antígenos de Neoplasias/genética , Carcinoma de Células Escamosas/genética , Moléculas de Adhesión Celular/genética , Neoplasias de Cabeza y Cuello/genética , Neurregulina-1/metabolismo , Receptor ErbB-3/metabolismo , Animales , Anticuerpos Antineoplásicos/inmunología , Carcinoma de Células Escamosas/clasificación , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Activación Enzimática/genética , Femenino , Neoplasias de Cabeza y Cuello/clasificación , Neoplasias de Cabeza y Cuello/patología , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Interferencia de ARN , ARN Interferente Pequeño , Receptor ErbB-3/inmunología , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello , Trasplante HeterólogoRESUMEN
Notch signaling regulates many cellular processes during development and adult tissue renewal. Upon ligand binding, Notch receptors undergo ectodomain shedding followed by γ-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA binding protein CSL [CBF1/RBPjκ/Su(H)/Lag1] to activate gene expression. Mammalian cells contain four Notch receptors that can have both redundant and specific activities. To monitor activation of specific Notch paralogs in live cells and in real time, we developed luciferase complementation imaging (LCI) reporters for NICD-CSL association and validated them as a specific, robust, and sensitive assay system that enables structure-function and pharmacodynamic analyses. Detailed kinetic analyses of various mechanistic aspects of Notch signaling, including nuclear translocation and inhibition of the activities of γ-secretase and ADAM metalloproteases, as well as agonist- and ligand-dependent activation, were conducted in live cells. These experiments showed that Notch-LCI is an effective approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.