scholarly journals A High-Throughput Phenotypic Screen of Cytotoxic T Lymphocyte Lytic Granule Exocytosis Reveals Candidate Immunosuppressants

2014 ◽  
Vol 20 (3) ◽  
pp. 359-371 ◽  
Author(s):  
Ziyan Zhao ◽  
Mark K. Haynes ◽  
Oleg Ursu ◽  
Bruce S. Edwards ◽  
Larry A. Sklar ◽  
...  
Keyword(s):  
T Lymphocyte ◽  
High Throughput ◽  
Cytotoxic T Lymphocyte ◽  
Mitogen Activated Protein Kinase ◽  
Granule Exocytosis ◽  
Calcium Dependent ◽  
Extracellular Signal ◽  
Phenotypic Screen ◽  
Mitogen Activated Protein ◽  
Lytic Granule

We screened the National Institutes of Health’s Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose–response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

scholarly journals Flow Cytometry Enables a High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis

2012 ◽  
Vol 18 (4) ◽  
pp. 420-429 ◽  
Author(s):  
Amy E. Florian ◽  
Christopher K. Lepensky ◽  
Ohyun Kwon ◽  
Mark K. Haynes ◽  
Larry A. Sklar ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Cytotoxic T Lymphocyte ◽  
Biological Activities ◽  
Fluorescent Antibody ◽  
Cytotoxic T Cell ◽  
Granule Exocytosis ◽  
Mitogen Activated Protein ◽  
Intracellular Ca ◽  
Lytic Granule

We developed a homogeneous phenotypic fluorescence end-point assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 µM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 µM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the mitogen-activated protein (MAP) kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.

scholarly journals Development of an Enhanced Phenotypic Screen of Cytotoxic T-Lymphocyte Lytic Granule Exocytosis Suitable for Use with Synthetic Compound and Natural Product Collections

2016 ◽  
Vol 21 (6) ◽  
pp. 556-566 ◽  
Author(s):  
Ziyan Zhao ◽  
James A. deMayo ◽  
Ashley M. West ◽  
Marcy J. Balunas ◽  
Adam Zweifach
Keyword(s):  
Small Molecules ◽  
Natural Product ◽  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Synthetic Compound ◽  
Synthetic Compounds ◽  
Associated Bacteria ◽  
Granule Exocytosis ◽  
Phenotypic Screen ◽  
Lytic Granule

We previously developed an assay of cytotoxic T-lymphocyte lytic granule exocytosis based on externalization of LAMP-1/CD107A using nonphysiological stimuli to generate maximal levels of exocytosis. Here, we used polystyrene beads coated with anti-CD3 antibodies to stimulate cells. Light scatter let us distinguish cells that contacted beads from cells that had not, allowing comparison of signaling events and exocytosis from stimulated and unstimulated cells in one sample. Bead stimulation resulted in submaximal exocytosis, making it possible to detect compounds that either augment or inhibit lytic granule exocytosis. Coupled with the assay’s ability to distinguish responses in cells that have and have not contacted a stimulatory bead, it is possible to detect three kinds of compounds: inhibitors, stimulators, which cause exocytosis, and augmenters, which enhance receptor-stimulated exocytosis. To validate the assay, we screened a set of synthetic compounds identified using our previous assay and a library of 320 extracts prepared from tunicate-associated bacteria. One of the extracts augmented exocytosis threefold. Activity-guided fractionation and structure elucidation revealed that this compound is the known PKC activator teleocidin A-1. We conclude that our modified assay is suitable for screening synthetic compound plates and natural product collections, and will be useful for identifying immunologically active small molecules.

scholarly journals Calcineurin Activation Is Only One Calcium-dependent Step in Cytotoxic T Lymphocyte Granule Exocytosis

2007 ◽  
Vol 282 (25) ◽  
pp. 18009-18017 ◽  
Author(s):  
Michael J. Grybko ◽  
Jakub P. Bartnik ◽  
Georjeana A. Wurth ◽  
Arun T. Pores-Fernando ◽  
Adam Zweifach
Keyword(s):  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Granule Exocytosis ◽  
Calcium Dependent

scholarly journals A mutation in Irak2c identifies IRAK-2 as a central component of the TLR regulatory network of wild-derived mice

2009 ◽  
Vol 206 (7) ◽  
pp. 1615-1631 ◽  
Author(s):  
James R. Conner ◽  
Irina I. Smirnova ◽  
Alexander Poltorak
Keyword(s):  
Late Phase ◽  
Model Organism ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Model Organisms ◽  
Central Component ◽  
Iκb Kinase ◽  
Extracellular Signal ◽  
Phenotypic Screen ◽  
Mitogen Activated Protein

In a phenotypic screen of the wild-derived mouse strain MOLF/Ei, we describe an earlier and more potent toll-like receptor (TLR)–mediated induction of IL-6 transcription compared with the classical inbred strain C57BL/6J. The phenotype correlated with increased activity of the IκB kinase axis as well as p38, but not extracellular signal-regulated kinase or c-Jun N-terminal kinase, mitogen-activated protein kinase (MAPK) phosphorylation. The trait was mapped to the Why1 locus, which contains Irak2, a gene previously implicated as sustaining the late phase of TLR responses. In the MOLF/Ei TLR signaling network, IRAK-2 promotes early nuclear factor κB (NF-κB) activity and is essential for the activation of p38 MAPK. We identify a deletion in the MOLF/Ei promoter of the inhibitory Irak2c gene, leading to an increased ratio of pro- to antiinflammatory IRAK-2 isoforms. These findings demonstrate that IRAK-2 is an essential component of the early TLR response in MOLF/Ei mice and show a distinct pathway of p38 and NF-κB activation in this model organism. In addition, they demonstrate that studies in evolutionarily divergent model organisms are essential to complete dissection of signal transduction pathways.

Ecdysone-inducible expression of oncogenic Ha-Ras in NIH 3T3 cells leads to transient nuclear localization of activated extracellular signal-regulated kinase regulated by mitogen-activated protein kinase phosphatase-1

2002 ◽  
Vol 362 (2) ◽  
pp. 305-315 ◽  
Author(s):  
David PLOWS ◽  
Paraskevi BRIASSOULI ◽  
Carolyn OWEN ◽  
Vassilis ZOUMPOURLIS ◽  
Michelle D. GARRETT ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Calcium Dependent ◽  
Transient Nature ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The Ras family of GTP-binding proteins are key transducers of extracellular signals, particularly through the mitogen-activated protein kinase (MAPK) pathway. Constitutively active forms of Ras are found in a variety of tumours, suggesting an important role for this pathway in cancer. Here we report that initial cellular exposure to oncogenic Ras chronically activated the MAPK pathway in the cytoplasm, but transiently activated the same pathway in the nucleus. Nuclear-activated extracellular signal-regulated kinase (ERK) was rapidly dephosphorylated, with consequent short-term activation of the Elk-1 transcription factor and expression of the c-fos gene. Additional experiments suggested that the regulatory mechanism involved requires the calcium-dependent protein phosphotyrosine phosphatase MAPK phosphatase-1 (MKP-1). This is the first report on the ability of Ras, in the absence of growth factors, to transiently activate the MAPK pathway in the nucleus and show an involvement of MKP-1 in nuclear ERK2 regulation. In addition we show that transient activation of the MAPK pathway is sufficient to drive chronic cell-cycle progression. We conclude that, whereas the MAPK pathway is necessary to initiate cellular proliferation and transformation, the transient nature of the MAPK pathway activation suggests the involvement of additional signalling pathway(s) regulated by Ras.

scholarly journals Role of Calcium Influx in Cytotoxic T Lymphocyte Lytic Granule Exocytosis during Target Cell Killing

Immunity ◽  
2001 ◽  
Vol 15 (5) ◽  
pp. 847-859 ◽  
Author(s):  
Taras A. Lyubchenko ◽  
Georjeana A. Wurth ◽  
Adam Zweifach
Keyword(s):  
T Lymphocyte ◽  
Target Cell ◽  
Cytotoxic T Lymphocyte ◽  
Calcium Influx ◽  
Cell Killing ◽  
Granule Exocytosis ◽  
Lytic Granule

scholarly journals Bromamine T (BAT) Exerts Stronger Anti-Cancer Properties than Taurine (Tau)

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 182
Author(s):  
Stella Baliou ◽  
Maria Goulielmaki ◽  
Petros Ioannou ◽  
Christina Cheimonidi ◽  
Ioannis P. Trougakos ◽  
...  
Keyword(s):  
Cytotoxic Effect ◽  
Human Colon ◽  
Mitogen Activated Protein Kinase ◽  
Mitochondrial Apoptosis ◽  
Therapeutic Modalities ◽  
Cancer Pathogenesis ◽  
Extracellular Signal ◽  
Anti Cancer ◽  
Mitogen Activated Protein

Background: Taurine (Tau) ameliorates cancer pathogenesis. Researchers have focused on the functional properties of bromamine T (BAT), a stable active bromine molecule. Both N-bromotaurine (TauNHBr) and BAT exert potent anti-inflammatory properties, but the landscape remains obscure concerning the anti-cancer effect of BAT. Methods: We used Crystal Violet, colony formation, flow cytometry and Western blot experiments to evaluate the effect of BAT and Tau on the apoptosis and autophagy of cancer cells. Xenograft experiments were used to determine the in vivo cytotoxicity of either agent. Results: We demonstrated that both BAT and Tau inhibited the growth of human colon, breast, cervical and skin cancer cell lines. Among them, BAT exerted the greatest cytotoxic effect on both RKO and MDA-MB-468 cells. In particular, BAT increased the phosphorylation of c-Jun N-terminal kinases (JNK½), p38 mitogen-activated protein kinase (MAPK), and extracellular-signal-regulated kinases (ERK½), thereby inducing mitochondrial apoptosis and autophagy in RKO cells. In contrast, Tau exerted its cytotoxic effect by upregulating JNK½ forms, thus triggering mitochondrial apoptosis in RKO cells. Accordingly, colon cancer growth was impaired in vivo. Conclusions: BAT and Tau exerted their anti-tumor properties through the induction of (i) mitochondrial apoptosis, (ii) the MAPK family, and iii) autophagy, providing novel anti-cancer therapeutic modalities.

Sign in / Sign up

Export Citation Format

Share Document