scholarly journals Visualizing cell death in live retina: Using calpain activity detection as a biomarker for retinal degeneration

2021 ◽  
Author(s):  
Soumaya Belhadj ◽  
Nina Hermann ◽  
Gustav Christensen ◽  
Torsten Strasser ◽  
François Paquet-Durand
Keyword(s):  
Cell Death ◽  
Retinal Degeneration ◽  
Imaging Techniques ◽  
Death Process ◽  
Specific Substrate ◽  
Activity Detection ◽  
Wild Type ◽  
Calpain Activity ◽  
Calpain 2

AbstractCalpains are a family of calcium-activated proteases involved in numerous disorders. Notably, previous studies have shown that calpain activity was substantially increased in various models for inherited retinal degeneration (RD). In the present study, we tested the capacity of the t-BOC-Leu-Met-CMAC calpain-specific substrate to detect calpain activity in living retina, in organotypic retinal explant cultures derived from wild-type mice, as well as from rd1 and RhoP23H/+ RD-mutant mice. Test conditions were refined until the calpain substrate readily detected large numbers of cells in the photoreceptor layer of RD retina but not in wild-type retina. At the same time, the calpain substrate was not obviously toxic to photoreceptor cells. Comparison of calpain activity with an immunostaining for activated calpain-2 furthermore suggested that individual calpain isoforms may be active in distinct temporal stages of photoreceptor cell death. Notably, calpain-2 activity may be a relatively short-lived event, occurring only towards the end of the cell death process. Finally, our results support the development of calpain activity detection as a novel in vivo biomarker for RD, suitable for combination with non-invasive imaging techniques.

scholarly journals APR-246 induces early cell death by ferroptosis in acute myeloid leukemia.

Haematologica ◽  
2021 ◽  
Author(s):  
Rudy Birsen ◽  
Clement Larrue ◽  
Justine Decroocq ◽  
Natacha Johnson ◽  
Nathan Guiraud ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Lipid Peroxides ◽  
Death Process ◽  
Glutathione Biosynthesis ◽  
Acute Myeloid

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia. APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

Abstract 165: The 18 kDa FGF-2 Prevents the Doxorubicin-induced Cardiac Damage and Amp-activated Kinase Activation, in vitro and in vivo

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Navid Koleini ◽  
Jon Jon Santiago ◽  
Barbara E Nickel ◽  
Robert Fandrich ◽  
Davinder S Jassal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Death ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Wild Type ◽  
Rat Cardiomyocytes ◽  
Ventricular Ejection Fraction ◽  
Compound C

Introduction: Protection of the heart from chemotherapeutic (Doxorubicin, DOX) drug-induced toxicity is a desirable goal, to limit side effects of cancer treatments. DOX toxicity has been linked to the activation (phosphorylation) of the AMP-activated kinase, AMPK. The 18 kDa low molecular weight isoform of fibroblast growth factor 2 (Lo-FGF-2) is a known cardioprotective and cytoprotective agent. In this study we have tested the ability of Lo-FGF-2 to protect from DOX-induced damage in rat cardiomyocytes in vitro, and in transgenic mouse models in vivo, in relation to AMPK activation. Methods: Rat neonatal cardiomyocytes in culture were exposed to DOX (0.5 μM) in the presence or absence of pre-treatment Lo-FGF-2 (10 ng/ml). Compound C was used to block phosphorylation (activity) of AMPK. Levels of cell viability/death (using Calcein-AM/Propidium iodide assay), phospho -and total AMPK, and apoptotic markers such as active caspase 3 were analyzed. In addition, transgenic mice expressing only Lo-FGF2, and wild type mice, expressing both high molecular weight (Hi-FGF2) as well as Lo-FGF2 were subjected to DOX injection (20 mg/kg, intraperitoneal); echocardiography was used to examine cardiac function at baseline and at 10 days post-DOX. Results: DOX-induced cell death of cardiomyocytes in culture was maximal at 24 hours post-DOX coinciding with significantly increased in activated (phosphorylated) AMPK. Compound C attenuated DOX-induced cardiomyocyte loss. Pre-incubation with Lo-FGF-2 decreased DOX induced cell death, and also attenuated the phosphorylation of AMPK post-DOX. Relative levels of phospho-AMPK were lower in the hearts of Lo-FGF2-expressing male mice compared to wild type. DOX-induced loss of contractile function (left ventricular ejection fraction and endocardial velocity) was negligible in Lo-FGF2-expressing mice but significant in wild type mice. Conclusion: Lo-FGF-2 protects the heart from DOX-induced damage in vitro and in vivo, by a mechanism likely involving an attenuation of AMPK activity.

RelB silencing to reverse tamoxifen resistance by regulating GPx4 and ferroptosis in breast cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e12513-e12513
Author(s):  
Zhi Xu ◽  
Jinhai Tang
Keyword(s):  
Breast Cancer ◽  
Lipid Peroxidation ◽  
Cell Death ◽  
Cell Lines ◽  
Apoptotic Cell ◽  
Morphological Changes ◽  
Death Process ◽  
Enhancer Element ◽  
High Level

e12513 Background: Tamoxifen(Tam), as an essential therapeutic treatment of estrogen receptor(ER)-positive breast cancer(BCa), has been available for the past three decades. However, the induction of Tam resistance during therapy has indicated a significant challenge with regards to this agent. Tam could increase oxidative stress and induce cell death by regulating reactive oxygen species(ROS). Ferroptosis, a cell death process driven by the accumulation of iron-dependent lipid peroxides, has been induced by inactivation/depletion of glutathione peroxidases(GPxs). Our previous studies found that the expression level of RelB gene, a member of NF-κB family, is negatively correlated with ER targeted by Tam in BCa. Methods: The RelB level of BCa tumor tissues and the corresponding cell lines were examined by immunoblotting and western blot. The effects of Tam on cell viability were determined using colony survival and MTT assay. The ROS and oxygen consumption rates(OCR) were measured using specific ROS detection probes and a Seahorse XF96 Analyzer, respectively. The lipid peroxidation level of cells was analyzed by immunofluorescence assay. The morphological changes of mitochondria were observed by transmission electron microscope. RelB binding to the NF-κB intronic enhancer region of the human GPx4 gene was determined using a ChIP assay. Accordingly, the effect of RelB on BCa Tam resistance was further validated using BCa mice xenograft models. Results: RelB was uniquely expressed at the high level in Tam resistance BCa tissues and cell lines. Down-regulation of RelB based on a CRISPR/Cas9 system remarkably sensitized resistance BCa cells to Tam. Treatment with SN52, a RelB inhibitor, illuminated the role of RelB in Tam-treated BCa cells. The high level of ROS and declination of mitochondrial respiration which induced by Tam were inhibited in resistance cells. Tam enhanced lipid peroxidation with concomitant non-apoptotic cell death, which are negatively regulated by GPx4 activity. In addition to GPx4 knockdown, deferoxamine was able to rescue Tam-induced cell death in BCa cells, verifying that Tam induces cell death partially through ferroptosis. Importantly, RelB upregulates GPx4 expression through binding to an NF-κB enhancer element located at the 5’-flanking region. Consistently, in vivo functional validation confirmed that RelB inhibition not only impairs tumor growth, but also inhibits Tam resistance in nude mice. Conclusions: RelB could inhibit ferroptosis which induced by hydroxyl radicals accumulation through upregulating GPx4 in BCa.

scholarly journals Inflammation, necrosis, and the kinase RIP3 are key mediators of AAG-dependent alkylation-induced retinal degeneration

2019 ◽  
Vol 12 (568) ◽  
pp. eaau9216 ◽  
Author(s):  
Mariacarmela Allocca ◽  
Joshua J. Corrigan ◽  
Aprotim Mazumder ◽  
Kimberly R. Fake ◽  
Leona D. Samson
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Retinal Degeneration ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Death Process ◽  
Partial Protection ◽  
Cell Death Process ◽  
Base Excision ◽  
Damage Marker

DNA-alkylating agents are commonly used to kill cancer cells, but the base excision repair (BER) pathway they trigger can also produce toxic intermediates that cause tissue damage, such as retinal degeneration (RD). Apoptosis, a process of programmed cell death, is assumed to be the main mechanism of this alkylation-induced photoreceptor (PR) cell death in RD. Here, we studied the involvement of necroptosis (another programmed cell death process) and inflammation in alkylation-induced RD. Male mice exposed to a methylating agent exhibited a reduced number of PR cell rows, active gliosis, and cytokine induction and macrophage infiltration in the retina. Dying PRs exhibited a necrotic morphology, increased 8-hydroxyguanosine abundance (an oxidative damage marker), and overexpression of the necroptosis-associated genes Rip1 and Rip3. The activity of PARP1, which mediates BER, cell death, and inflammation, was increased in PR cells and associated with the release of proinflammatory chemokine HMGB1 from PR nuclei. Mice lacking the anti-inflammatory cytokine IL-10 exhibited more severe RD, whereas deficiency of RIP3 (also known as RIPK3) conferred partial protection. Female mice were partially protected from alkylation-induced RD, showing reduced necroptosis and inflammation compared to males. PRs in mice lacking the BER-initiating DNA glycosylase AAG did not exhibit alkylation-induced necroptosis or inflammation. Our findings show that AAG-initiated BER at alkylated DNA bases induces sex-dependent RD primarily by triggering necroptosis and activating an inflammatory response that amplifies the original damage and, furthermore, reveal new potential targets to prevent this side effect of chemotherapy.

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 Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

2002 ◽  
Vol 22 (1) ◽  
pp. 41-56 ◽  
Author(s):  
Marie-Claude Gingras ◽  
Claudia Champagne ◽  
Mélanie Roy ◽  
Josée N. Lavoie
Keyword(s):  
Cell Death ◽  
Adenovirus Type ◽  
Wild Type ◽  
Membrane Blebbing ◽  
Signaling Complex ◽  
Phosphorylated Proteins ◽  
Apoptotic Signal

ABSTRACT In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Src-mediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.

The absence of c-fos prevents light-induced apoptotic cell death of photoreceptors in retinal degeneration in vivo

1997 ◽  
Vol 3 (3) ◽  
pp. 346-349 ◽  
Author(s):  
Farhad Hafezi ◽  
Joachim P. Steinbach ◽  
Andreas Marti ◽  
Kurt Munz ◽  
Zhao-Qi Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Retinal Degeneration ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death

scholarly journals Mutant rab8 Impairs Docking and Fusion of Rhodopsin-bearing Post-Golgi Membranes and Causes Cell Death of TransgenicXenopus Rods

2001 ◽  
Vol 12 (8) ◽  
pp. 2341-2351 ◽  
Author(s):  
Orson L. Moritz ◽  
Beatrice M. Tam ◽  
Larry L. Hurd ◽  
Johan Peränen ◽  
Dusanka Deretic ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Membrane Trafficking ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Photoreceptor Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Golgi Membranes ◽  
Constitutively Active

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevismodel of retinal degenerative disease.

scholarly journals APR-246 Reveals a Therapeutic Potential Via Triggering Different Cell Death Mechanisms in Diffuse Large B Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3521-3521
Author(s):  
Yuheng Hong ◽  
Tianyuan Ren ◽  
Xiaoxuan Wang ◽  
Kai Fu ◽  
Xianhuo Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Missense Mutations ◽  
Wild Type ◽  
Tp53 Mutations ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy and is characterized by its pronounced genetic and clinical heterogeneity. Although the first-line therapy (R-CHOP) improves the curability of patients with DLBCL, nearly 40% remainder ultimately undergo relapsed or refractory disease. Emerging evidence has shown that TP53 mutations correlate with the recurrence and progression of DLBCL. APR-246, also known as PRIMA-1MET, can reactivate the trans-activation of TP53 mutants by facilitating their DNA binding to target genes, making it a promising therapeutic compound for mutated TP53 carcinomas. Although APR-246 in combination with other chemicals has been applied in clinical trials for non-Hodgkin lymphoma (NHL), the efficacy and the underlying molecular mechanisms of APR-246 on DLBCL remain unclear. The arms are to investigate the TP53 mutations and the correlation of mutated TP53 with the prognosis. Furthermore, we evaluated the effect of APR-246 on DLBCL in vitro and in vivo. Methods: Multiple datasets, including the Gene Expression Omnibus (GEO) and cBioPortal, were searched for available data for DLBCL. For evaluation of APR-246 effect on DLBCL in vitro, ten DLBCL cell lines harbouring different and representative molecular properties, especially distinct TP53 mutation status, were tested. To validate the therapeutic effect of APR-246 in vivo, NSG mice were injected subcutaneously with the DLBCL cells with TP53 mutations to establish a xenograft animal model, and then these mice received the APR-246 administration. The cell viability of DLBCL was measured post addition of APR-246. Pharmaceutical inhibition of different cell death pathways was applied to elucidate the mechanisms by which APR-246 functions. Results: Total 2204 patients with DLBCL were evaluated, of which nearly 15% contained TP53 mutations. The missense mutation of TP53 was up to 76% and mutations occurring in the DNA binding domain (DBD) was about 90%. Patients with TP53 mutations had poor OS (p=0.0118). Further, we found that patients with TP53 mutations in GCB and UNC subtypes exhibited inferior OS (p=0.043; p=0.049, respectively), but no in ABC subtype. Notably, TP53 single mutations located in the DBD (exon 5-8) led to an unfavorable survival (p=0.0263), while patients carrying TP53 multiple mutations and single mutations in other domains exhibited no difference in survival time than those with wild-type TP53. APR-246 induced the cell death in a dose dependent manner for all DLBCL cell lines. DLBCL cells with TP53 missense mutations in the DBD were the most sensitive to APR-246 than those with intact or other types of mutated TP53. APR-246 mediated the cell death via p53-dependent ferritinophagy for DLBCL cells with TP53 missense mutations in the DBD. In addition, APR-246 also induced the ferroptosis for other DLBCL cells harbouring wild type TP53 and other forms of TP53 mutations. Conclusions: Nearly 15% of patients with DLBCL harbouring TP53 mutations had inferior prognosis than those with wild-type TP53. Patients with TP53 single mutations occurred in the DBD (exon 5-8) showed poorer prognosis than those with TP53 multiple mutations and single mutations in other domains. APR-246 induced the cell death of DLBCL through different mechanisms, depend on different forms of TP53 mutations, which provided an evidence for clinical application of APR-246 in the treatment of DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals A unique mode of keratinocyte death requires intracellular acidification

2021 ◽  
Vol 118 (17) ◽  
pp. e2020722118
Author(s):  
Takeshi Matsui ◽  
Nanako Kadono-Maekubo ◽  
Yoshiro Suzuki ◽  
Yuki Furuichi ◽  
Keiichiro Shiraga ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Nuclear Dna ◽  
Receptor Potential ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Death Process ◽  
Intracellular Acidification

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.

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