scholarly journals Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Deirdre Fahy ◽  
Marwa N. M. E. Sanad ◽  
Kerstin Duscha ◽  
Madison Lyons ◽  
Fuquan Liu ◽  
...  
Keyword(s):  
Cell Death ◽  
Simple Technique ◽  
Peroxisome Proliferation ◽  
Wild Type ◽  
Small Probe ◽  
Physiological Relevance ◽  
Potential Applications ◽  
Salt Susceptible ◽  
Synthesis Of Hormones

Abstract Plant peroxisomes maintain a plethora of key life processes including fatty acid β-oxidation, photorespiration, synthesis of hormones, and homeostasis of reactive oxygen species (ROS). Abundance of peroxisomes in cells is dynamic; however mechanisms controlling peroxisome proliferation remain poorly understood because measuring peroxisome abundance is technically challenging. Counting peroxisomes in individual cells of complex organs by electron or fluorescence microscopy is expensive and time consuming. Here we present a simple technique for quantifying peroxisome abundance using the small probe Nitro-BODIPY, which in vivo fluoresces selectively inside peroxisomes. The physiological relevance of our technique was demonstrated using salinity as a known inducer of peroxisome proliferation. While significant peroxisome proliferation was observed in wild-type Arabidopsis leaves following 5-hour exposure to NaCl, no proliferation was detected in the salt-susceptible mutants fry1-6, sos1-14, and sos1-15. We also found that N-BODIPY detects aggregation of peroxisomes during final stages of programmed cell death and can be used as a marker of this stage. Furthermore, accumulation of peroxisomes in an autophagy-deficient Arabidopsis mutant atg5 correlated with N-BODIPY labeling. In conclusion, the technique reported here enables quantification of peroxisomes in plant material at various physiological settings. Its potential applications encompass identification of genes controlling peroxisome homeostasis and capturing stress-tolerant genotypes.

scholarly journals Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

2010 ◽  
Vol 107 (5) ◽  
pp. 2319-2324 ◽  
Author(s):  
Adolfo Rivero-Müller ◽  
Yen-Yin Chou ◽  
Inhae Ji ◽  
Svetlana Lajic ◽  
Aylin C. Hanyaloglu ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Luteinizing Hormone Receptor ◽  
Wild Type ◽  
Gpcr Signaling ◽  
Physiological Relevance ◽  
Biosynthesis Activation ◽  
Mutant Receptors ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

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.

scholarly journals Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function

2020 ◽  
pp. jbc.RA120.016570
Author(s):  
Matthew L. Edin ◽  
Haruto Yamanashi ◽  
William E. Boeglin ◽  
Joan P. Graves ◽  
Laura M. DeGraff ◽  
...  
Keyword(s):  
Epoxide Hydrolase ◽  
Skin Barrier ◽  
Skin Barrier Function ◽  
Potential Contribution ◽  
Wild Type ◽  
Lipoxygenase Pathway ◽  
Physiological Relevance ◽  
Hydrolysis Of

The mammalian epoxide hydrolase EPHX3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo.  Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of trans-epidermal water loss detected a modest and statistically significant increase compared to the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal epoxide hydrolase (EPHX1/mEH) or soluble epoxide hydrolase (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p<0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (~85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide, and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.

scholarly journals Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage

2019 ◽  
Vol 116 (16) ◽  
pp. 8010-8017 ◽  
Author(s):  
Jean Defourny ◽  
Alain Aghaie ◽  
Isabelle Perfettini ◽  
Paul Avan ◽  
Sedigheh Delmaghani ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hair Cells ◽  
Cell Damage ◽  
Peroxisome Proliferation ◽  
Wild Type ◽  
Auditory Hair Cells ◽  
C Terminus ◽  
Cysteine Mutagenesis ◽  
Autophagic Degradation

Noise overexposure causes oxidative stress, leading to auditory hair cell damage. Adaptive peroxisome proliferation involving pejvakin, a peroxisome-associated protein from the gasdermin family, has been shown to protect against this harmful oxidative stress. However, the role of pejvakin in peroxisome dynamics and homeostasis remains unclear. Here we show that sound overstimulation induces an early and rapid selective autophagic degradation of peroxisomes (pexophagy) in auditory hair cells from wild-type, but not pejvakin-deficient (Pjvk−/−), mice. Noise overexposure triggers recruitment of the autophagosome-associated protein MAP1LC3B (LC3B; microtubule-associated protein 1 light chain 3β) to peroxisomes in wild-type, but not Pjvk−/−, mice. We also show that pejvakin–LC3B binding involves an LC3-interacting region within the predicted chaperone domain of pejvakin. In transfected cells and in vivo transduced auditory hair cells, cysteine mutagenesis experiments demonstrated the requirement for both C328 and C343, the two cysteine residues closest to the C terminus of pejvakin, for reactive oxygen species-induced pejvakin–LC3B interaction and pexophagy. The viral transduction of auditory hair cells from Pjvk−/− mice in vivo with both Pjvk and Lc3b cDNAs completely restored sound-induced pexophagy, fully prevented the development of oxidative stress, and resulted in normal levels of peroxisome proliferation, whereas Pjvk cDNA alone yielded only a partial correction of the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify defective pexophagy as a cause of noise-induced hearing loss. They suggest that pejvakin acts as a redox-activated pexophagy receptor/adaptor, thereby identifying a previously unknown function of gasdermin family proteins.

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&lt;0.01) and perforin −/− (P&lt;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&lt;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.

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.

Lower threshold to NFκB activity sensitizes murine β-cells to streptozotocin

2021 ◽  
Author(s):  
Clyde J. Wright ◽  
Sarah McKenna ◽  
Robyn De Dios ◽  
Brit H. Boehmer ◽  
Leanna Nguyen ◽  
...  
Keyword(s):  
Cell Death ◽  
Trypan Blue ◽  
Target Genes ◽  
Cell Injury ◽  
Wild Type ◽  
Β Cells ◽  
Β Cell ◽  
Trypan Blue Exclusion ◽  
Min6 Cell

The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we use streptozotocin (STZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8mM, 0-24h) ±TNFα (100ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ-/- and wild type (WT) mice were exposed to STZ and monitored for diabetes. The IκBβ-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes.

Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system

Development ◽  
1999 ◽  
Vol 126 (12) ◽  
pp. 2785-2797 ◽  
Author(s):  
S. Taraviras ◽  
C.V. Marcos-Gutierrez ◽  
P. Durbec ◽  
H. Jani ◽  
M. Grigoriou ◽  
...  
Keyword(s):  
Cell Death ◽  
Nervous System ◽  
Growth Factors ◽  
Tyrosine Kinase ◽  
Enteric Nervous System ◽  
Receptor Tyrosine Kinase ◽  
Enteric Neurons ◽  
Wild Type ◽  
Rat Embryos

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5–15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

scholarly journals Protection of cisplatin cytotoxicity by an inactive cyclin-dependent kinase

2010 ◽  
Vol 299 (1) ◽  
pp. F112-F120 ◽  
Author(s):  
Rawad Hodeify ◽  
Judit Megyesi ◽  
Adel Tarcsafalvi ◽  
Robert L. Safirstein ◽  
Peter M. Price
Keyword(s):  
Cell Death ◽  
Cyclin A ◽  
Binding Pocket ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Cytochrome C Release ◽  
Endonuclease G ◽  
Cdk2 Activity

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

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