scholarly journals Hip Is a Pro-survival Substrate of Granzyme B

2007 ◽  
Vol 282 (38) ◽  
pp. 27865-27874 ◽  
Author(s):  
Daniel R. Hostetter ◽  
Carly R. K. Loeb ◽  
Feixia Chu ◽  
Charles S. Craik
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Cell Line ◽  
Cleavage Site ◽  
Mutational Analysis ◽  
Nk Cell ◽  
Granzyme B ◽  
Independent Manner ◽  
Interacting Protein

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip. Because the role of Hip in apoptosis is unknown, its proteolysis by GrB was used as a basis to test whether it has anti-apoptotic activity. Previous work on Hip was limited to in vitro characterization; therefore, it was important to demonstrate Hip cleavage in a physiological context and to show its relevance to natural killer (NK) cell-mediated death. Hip is cleaved at both GrB cleavage sites during NK-mediated cell death in a caspase-independent manner, and its cleavage is due solely to GrB and not other granule components. Furthermore, Hip is not cleaved upon stimulation of the Fas receptor in the Jurkat T-cell line, suggesting that Hip is a substrate unique to GrB. RNA interference-mediated reduction of Hip within the K562 cell line rendered the cells more susceptible to NK cell-mediated lysis, indicating that proteolysis by GrB of Hip contributes to death induction. The small effect of RNA interference-mediated Hip deficiency on cytotoxicity is in agreement with the inherent redundancy of NK cell-mediated cell death. The identification of additional members of the chaperone superfamily as GrB substrates and the validation of Hip as an anti-apoptotic protein contribute to understanding the interplay between stress response and apoptosis.

Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 920-920
Author(s):  
Todd A. Fehniger ◽  
Sheng F. Cai ◽  
Xuefang Cao ◽  
Andrew J. Bredemeyer ◽  
Rachel M. Presti ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Target Cell ◽  
Post Infection ◽  
Nk Cell ◽  
Granzyme B ◽  
Poly I:C ◽  
Wild Type

Abstract NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P<0.01) and perforin −/− (P<0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P<0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses. Figure Figure

scholarly journals IMMU-47. PROTEIN PHOSPHATASE 2A INHIBITION IN NK CELL THERAPY FOR TREATMENT OF MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii115-ii115
Author(s):  
Rongze Olivia Lu ◽  
Winson Ho ◽  
Brandon Chiou
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Protein Phosphatase ◽  
Nk Cell ◽  
Protein Phosphatase 2A ◽  
T Cell Response ◽  
Intrinsic Activity ◽  
Cd107a Expression ◽  
Phosphatase 2A

Abstract Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.

scholarly journals Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato
Keyword(s):  
Cell Line ◽  
Low Cost ◽  
Neural Cell ◽  
New Drugs ◽  
High Yield ◽  
Time Saving ◽  
Interacting Protein ◽  
Neuronal Marker ◽  
Differentiation Status

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

1996 ◽  
Vol 24 (4) ◽  
pp. 581-587
Author(s):  
Cristiana Zanetti ◽  
Arrnalaura Stammati ◽  
Orazio Sapora ◽  
Flavia Zucco
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
In Vitro Model ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Type ◽  
Vitro Model ◽  
Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

scholarly journals A COMPARATIVE ANTICANCER STUDY OF BIOSYNTHESIZED NANOPARTICLES, DOXORUBICIN & NANO-DOX CONJUGATE AGAINST CERVICAL CANCER HELA CELL LINE

2020 ◽  
pp. 4-7
Author(s):  
M. R. Kamala Priya ◽  
Priya R. Iyer
Keyword(s):  
Cervical Cancer ◽  
Gold Nanoparticles ◽  
Cell Death ◽  
Cancer Therapy ◽  
Hela Cell ◽  
Cell Line ◽  
In Vitro Cytotoxicity ◽  
Hela Cell Line ◽  
Vero Cell Line

Doxorubicin is the most common chemotherapy drug used in cancer therapy. Its usage is associated with various side-effects. In order to overcome the challenges in Doxorubicin administration, the present study has focussed on synthesizing a drug conjugate with biosynthesized gold nanoparticles and doxorubicin. The gold nanoparticles were biosynthesized using green extracts of medicinal plants with potential anticancer activities. The nanoparticle that possesses anticancer activity was conjugated with the drug for a combinatorial effect of the nanoparticles and the drug. The in vitro cytotoxicity was checked in Vero cell line through MTT assay. The in vitro anti proliferative effects were screened against cervical cancer in HeLa cell line. Fluorescence activated cell sorting analysis was carried out to detect the difference between live and dead cell populations. The preliminary confirmation was carried out in UV-VIS spectrophotometer. The morphological characterization was carried out by SEM and stability by Zeta potential. The IC50 of the nanocompounds demonstrated anti-proliferative activity against cervical cancer similar to the chemotherapeutic drug, Doxorubicin; additionally in a much lesser concentration of the drug. The in vitro cytotoxicity exhibited high viability of cells in Vero cell line with minimum viability of 80% in all the tested concentrations. There was a synergistic effect of the nanoparticles along with the drug; thereby an enhanced therapeutic efficiency was achieved. FACS analysis showed 36% of cell death in Dox treated HeLa cells whereas 96% of cell death in Nano-Dox treated HeLa cells. Nano-Dox conjugate has demonstrated high anticancer effects than drug alone Doxorubicin. Further biosynthesized nanomaterials based drug formulation can be developed as a potential strategy in cancer therapy.

scholarly journals Cytoprotective potential of anti-ischemic drugs against chemotherapy-induced cardiotoxicity in H9c2 myoblast cell line

2013 ◽  
Vol 63 (4) ◽  
pp. 493-503 ◽  
Author(s):  
Tiam Feridooni ◽  
Chris Mac Donald ◽  
Di Shao ◽  
Pollen Yeung ◽  
Remigius U. Agu
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cancer Drug ◽  
H9c2 Cell ◽  
Drug Induced ◽  
Cardiac Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
H9c2 Cell Line

Abstract To investigate potential prevention or attenuation of anti- cancer drug induced cardiotoxicity using anti-ischemic drugs, a rat myoblast (H9c2) cell line was used as our in vitro cardiac model. Irinotecan and doxorubicin were found to be cytotoxic for the H9c2 cell line with IC50 of 30.69 ± 6.20 and 20.94 ± 6.05 mmol L-1, respectively. 5-Flurouracil and cladribine were not cytotoxic and thus IC50 could not be calculated. When 100 mmol L-1 doxorubicin was incubated for 72 hours with 50 mmol L-1 diltiazem, 100 mmol L-1 dexrazoxane and 100 mmol L-1 losartan, respectively, there was a 58.7 ± 10.2, 52.2 ± 11.7 and 44.7 ± 5.4 % reduction in cell death. When 200 mmol L-1 irinotecan was incubated for 72 hours with 100 mmol L-1 dexrazoxane, losartan and diltiazem, respectively, a 27.7 ± 6.9, 25.6 ± 5.1, and 19.1 ± 2.3 % reduction in cell death was observed. Our data suggests that losartan and diltiazem were as effective as dexrazoxane in protecting the cells against irinotecan- and doxorubicin-induced cell toxicity. These findings offer potential uses of anti- -ischemic drugs for ablation of cytotoxicity in response to mitochondrial injury, thereby improving patient outcomes and reducing health-care costs.

scholarly journals Drosophila Homeodomain-Interacting Protein Kinase (Hipk) Phosphorylates the Homeodomain Proteins Homeobrain, Empty Spiracles, and Muscle Segment Homeobox

2019 ◽  
Vol 20 (8) ◽  
pp. 1931
Author(s):  
Eva Louise Steinmetz ◽  
Denise Nicole Dewald ◽  
Nadine Luxem ◽  
Uwe Walldorf
Keyword(s):  
Nervous System ◽  
Protein Kinase ◽  
Mutational Analysis ◽  
Bimolecular Fluorescence Complementation ◽  
Physical Interaction ◽  
Homeodomain Proteins ◽  
Interacting Protein ◽  
Enzyme Substrate

The Drosophila homeodomain-interacting protein kinase (Hipk) is the fly representative of the well-conserved group of HIPKs in vertebrates. It was initially found through its characteristic interactions with homeodomain proteins. Hipk is involved in a variety of important developmental processes, such as the development of the eye or the nervous system. In the present study, we set Hipk and the Drosophila homeodomain proteins Homeobrain (Hbn), Empty spiracles (Ems), and Muscle segment homeobox (Msh) in an enzyme-substrate relationship. These homeoproteins are transcription factors that function during Drosophila neurogenesis and are, at least in part, conserved in vertebrates. We reveal a physical interaction between Hipk and the three homeodomain proteins in vivo using bimolecular fluorescence complementation (BiFC). In the course of in vitro phosphorylation analysis and subsequent mutational analysis we mapped several Hipk phosphorylation sites of Hbn, Ems, and Msh. The phosphorylation of Hbn, Ems, and Msh may provide further insight into the function of Hipk during development of the Drosophila nervous system.

scholarly journals NK-cell activation is associated with increased HIV transcriptional activity following allogeneic hematopoietic cell transplantation

2018 ◽  
Vol 2 (12) ◽  
pp. 1412-1416
Author(s):  
Louise E. Hogan ◽  
Christian Körner ◽  
Kristen Hobbs ◽  
Camille R. Simoneau ◽  
Cassandra Thanh ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcriptional Activity ◽  
Cell Activation ◽  
Hematopoietic Cell Transplantation ◽  
Nk Cell ◽  
Killer Cell ◽  
Graft Versus Host ◽  
Key Points ◽  
Hiv 1

Key Points Graft-versus-host effects may lead to HIV-1 reactivation and cell death of infected pre-HCT CD4+ T cells. Natural killer cell activation correlates with in vitro HIV-1 transcriptional activity in the setting of HCT.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

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