Staurosporine Exposure Research Articles

Abstract 2472: Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy

Abstract We have developed a live tumor fragment (LTF) platform for predicting clinical response to cancer drugs. The success of this approach depends on screening tissue fragments derived from biopsies and excisions before drug treatment to select those that have acceptable levels of viability and tumor content. To enable this screening, we are developing label-free methods for integrated assessment of LTF histology, viability, and metabolic status using intrinsic multiphoton-excited fluorescence lifetime microscopy (MP-FLIM). We cut live EMT6 tumors and mammary fat pad into rectangular fragments that were then sorted and cultured in glass-bottomed multi-well plates. To induce cancer cell death, we treated one group with a therapeutic agent, e.g., the multi-kinase inhibitor, staurosporine, or the alkylating agent, cisplatin. Before and after treatment, we imaged LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide dinucleotides (NAD(P)H) and flavin adenine dinucleotides (FAD) fluorescence intensity and lifetime using dual-excitation multiphoton microscopy. As a ground truth viability reference, we then stained and re-imaged the fragments using nuclear and cell viability probes such as Hoechst, SYTOX, TMRE and/or propidium iodide (PI). We analyzed the data by fitting fluorescence decay curves with dual or triple exponents to generate images of lifetime parameters, including the mean and individual component lifetimes and amplitudes. Finally, we co-registered the extrinsically labeled and autofluorescent lifetime images for 3D spatial analysis using commercial and custom software. We verified the methods using monolayer cell cultures and ATP luminescence and flow cytometry viability assays. Image spatial heterogeneity was quantified utilizing entropy metrics. Intrinsic contrast from multiphoton imaging revealed cellular and tissue structures. The median entropy was higher and its distribution negatively skewed for EMT6 (p<0.01), allowing us to distinguish tumor from its corresponding normal tissue of origin. In viable cells, nuclei were distinguishable from the cytoplasm via higher cytoplasmic NADH signal. Upon loss of cell viability, nuclear NADH signals increased, with characteristically short lifetimes, and the overall NADH intensity decreased, reducing the contrast between nuclei and their surrounding cytoplasm. We detected an increase in the whole fragment mean lifetime (τm p<0.002) and concomitant decrease in the short lifetime component (α1 p< 0.002) within 24 hours of staurosporine exposure. We have demonstrated that MP-FLIM can distinguish tumor from normal and live from dead in LTFs without labels. This approach will facilitate selection of fragments with acceptable viability and tumor content for subsequent drug treatment as part of a platform built for personalizing cancer therapy. Citation Format: Jonathan Ouellette, John Rafter, Kelsey Tweed, Leung Kau Tang, Christina Scribano, Pichet Adstamongkonkul, Mikaela Schultz, Justine Coburn, Maged Aldeeb, Neil Anthony, Christin Johnson, Kevin Eliceiri, Jonathan Oliner, Tomasz Zal. Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2472.

Anti-human Interleukin(IL)-4 Clone 8D4-8 Cross-Reacts With Myosin-9 Associated With Apoptotic Cells and Should Not Be Used for Flow Cytometry Applications Querying IL-4 Expression.

Interleukin(IL)-4 is produced by T cells and other leukocytes and is a critical mediator of monocyte and B cell responses. During routine flow cytometry panel validation for the investigation of intracellular cytokines, we observed unique IL-4 expression patterns associated with the widely available monoclonal antibody 8D4-8. Namely, IL-4 (8D4-8) expression was observed in the absence of cellular activation and enhanced following staurosporine exposure. Mass spectrometry analysis of immunoprecipitates from peripheral blood lymphocytes (PBL) revealed that 8D4-8 cross-reacts with the ubiquitous cytoskeletal protein myosin-9. We confirmed these results by western blotting immunoprecipitates, using immunofluorescence among staurosporine-treated Caco-2 cells, and by surface-labeling PBL for 8D4-8 and myosin-9 and analyzing by flow cytometry. Although previously reported from several independent groups, we found no evidence to support the hypothesis that IL-4 is produced by apoptotic cells. Rather, this appears to have been myosin-9. Our data indicate clone 8D4-8 should not be used in the flow cytometric study of IL-4. Furthermore, our work calls for a reevaluation of previous flow cytometric studies that have used this clone for IL-4 analysis and highlights the importance of validation in antibody-based assays.

Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control.

Autophagic behavior in skeletal muscle cells

Autophagy represents a physiological mechanism responsible for cell homeostasis and its deregulation is involved in several conditions related to muscle mass loss such as aging, anorexia, inflammatory diseases, cancer, disuse and immobilization (1). In our previous work, double membrane vesicles, suggestive of autophagy, appeared after chemotherapeutic treatments in C2C12 myotubes (2). Here, to better understand the autophagic behavior, skeletal muscle cells have been exposed to cisplatin, etoposide and staurosporine and their effects have been investigated by means of morphological, cytofluorimetric and functional analyses. Ultrastructural observations evidenced the presence of autophagic vacuoles containing abnormal mitochondria, nuclear materials and membranes. Flow cytometry evaluation of lysosomal compartment stability revealed an autophagic pattern increase, particularly after cisplatin and staurosporine exposure, suggesting the presence of a cell death mechanism. Since autophagic deregulation is involved in different muscle diseases, further studies are in progress to evaluate possible strategies, such as protein ipo/iper supplementation, to prevent the abnormal autophagic activation induced by chemical triggers, which lead to muscle atrophy (3).

Staurosporine‐induced collapse of cochlear hair bundles

Early postnatal mouse cochlear cultures were treated with a small panel of kinase inhibitors to elucidate the mechanisms underlying the maintenance of hair-bundle structure in the developing inner ear. At low concentrations (1–10 nM), staurosporine causes the collapse and loss of hair bundles without provoking hair-cell death, as judged by lack of terminal transferase dUTP nick end labeling (TUNEL) labeling or reactivity to anti-activated caspase-3. Staurosporine exposure results in the fusion of the hair bundle’s stereocilia, a resorption of the parallel actin bundles of the stereocilia into the cytoplasm of the hair cell, a detachment of the apical, non-stereociliary membrane of the hair cell from the underlying cuticular plate, and a severing of the hair-bundle’s rootlets from the actin cores of the stereocilia. It does not block membrane retrieval at the apical pole of the hair cells, nor does it elicit the externalization of phosphatidylserine. Staurosporine treatment causes a reduction in levels of the phosphorylated forms of ezrin, radixin, and moesin in cochlear cultures during the period of hair-bundle loss, indicating the integrity of the hair bundle may be actively maintained by the phosphorylation status of these proteins. J. Comp. Neurol. 522:3281–3294, 2014. © 2014 Wiley Periodicals, Inc.

Oxidizing Effects of Exogenous Stressors in Huntington’s Disease Knock-in Striatal Cells—Protective Effect of Cystamine and Creatine

Huntington's disease (HD) is a polyglutamine-expansion disease associated to degeneration of striatal and cortical neurons. Previously, we showed that oxidative stress occurs in HD knock-in striatal cells, but little is known regarding cell antioxidant response against exogenous stimuli. Therefore, in the present study we analyzed cellular antioxidant profile following hydrogen peroxide (H2O2) and staurosporine (STS) exposure and tested the protective effect of cystamine and creatine in striatal cells expressing mutant huntingtin with 111 glutamines (STHdh (Q111/Q111); mutant cells) versus wild-type cells (STHdh (Q7/Q7)). Mutant cells displayed increased mitochondrial reactive oxygen species (ROS) and decreased NADPH oxidase and xanthine oxidase (XO) activities, reflecting lower superoxide cytosolic generation, along with increased superoxide dismutases (SODs) and components of glutathione redox cycle. Exposure to H2O2 and STS enhanced ROS in mutant cells and largely increased XO activity; STS further boosted the generation of mitochondrial ROS and caspase-3 activity. Both stimuli slightly increased SOD1 activity, without affecting SOD2 activity, and decreased glutathione reductase with a consequent rise in oxidized glutathione or glutathione disulfide in mutant cells, whereas H2O2 only increased glutathione peroxidase activity. Additionally, creatine and cystamine increased mutant cells viability and prevented ROS formation in HD cells subjected to H2O2 and STS. These results indicate that elevation of the antioxidant systems accompanies mitochondrial-driven ROS generation in mutant striatal cells and that exposure to noxious stimuli induces a higher susceptibility to oxidative stress by increasing XO activity and lowering the antioxidant response. Furthermore, creatine and cystamine are efficient in preventing H2O2- and STS-evoked ROS formation in HD striatal cells.

Data-Driven Modeling of Src Control on the Mitochondrial Pathway of Apoptosis: Implication for Anticancer Therapy Optimization

Src tyrosine kinases are deregulated in numerous cancers and may favor tumorigenesis and tumor progression. We previously described that Src activation in NIH-3T3 mouse fibroblasts promoted cell resistance to apoptosis. Indeed, Src was found to accelerate the degradation of the pro-apoptotic BH3-only protein Bik and compromised Bax activation as well as subsequent mitochondrial outer membrane permeabilization. The present study undertook a systems biomedicine approach to design optimal anticancer therapeutic strategies using Src-transformed and parental fibroblasts as a biological model. First, a mathematical model of Bik kinetics was designed and fitted to biological data. It guided further experimental investigation that showed that Bik total amount remained constant during staurosporine exposure, and suggested that Bik protein might undergo activation to induce apoptosis. Then, a mathematical model of the mitochondrial pathway of apoptosis was designed and fitted to experimental results. It showed that Src inhibitors could circumvent resistance to apoptosis in Src-transformed cells but gave no specific advantage to parental cells. In addition, it predicted that inhibitors of Bcl-2 antiapoptotic proteins such as ABT-737 should not be used in this biological system in which apoptosis resistance relied on the deficiency of an apoptosis accelerator but not on the overexpression of an apoptosis inhibitor, which was experimentally verified. Finally, we designed theoretically optimal therapeutic strategies using the data-calibrated model. All of them relied on the observed Bax overexpression in Src-transformed cells compared to parental fibroblasts. Indeed, they all involved Bax downregulation such that Bax levels would still be high enough to induce apoptosis in Src-transformed cells but not in parental ones. Efficacy of this counterintuitive therapeutic strategy was further experimentally validated. Thus, the use of Bax inhibitors might be an unexpected way to specifically target cancer cells with deregulated Src tyrosine kinase activity.

MLH1 function is context dependent in colorectal cancers

Background and aimsLoss of mismatch repair (MMR) function in sporadic colorectal cancer occurs most commonly because of inactivation of MLH1, and it causes an increase in mutation rate. However, it...

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Caspase-dependent and -independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53wt (wild-type p53) HeLa cells compared with p53mt (mutated p53) C-33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase-9/caspase-3 mitochondrial-dependent apoptotic pathway and via a delayed caspase-independent pathway. In contrast with p53wt cells, p53mt C-33A cells exhibit firstly caspase-8 activation leading to caspase-3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP-1 [poly(ADP-ribose) polymerase-1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z-VAD-fmk points toward a major involvement of a caspase-dependent pathway in staurosporine-induced apoptosis in p53wt HeLa cells, which is not the case in p53mt C-33A cells. Meanwhile, the use of 3-aminobenzamide, a PARP-1 inhibitor known to prevent AIF (apoptosis-inducing factor) release, significantly decreases staurosporine-induced death in these p53mt carcinoma cells, suggesting a preferential implication of caspase-independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin-alpha, isolated as a suppressor of p53-mediated transactivation, or by PRIMA-1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio-nuclear AIF translocation in staurosporine-induced apoptosis of cervical carcinoma cells. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase-8 or caspase-9 signalling cascades and via caspase-independent cell death, as well as through p53 activity.

Young WI-38 human fibroblasts apoptosis after to staurosporine exposure: a preliminary assessment

AbstractHuman fibroblasts cell culture is an important technique for the study of cell biology under various conditions, as exposure to apoptotic stimuli such as UV radiation or hydrogen peroxide administration or chemotherapeutic agent derivatives from staurosporine (STP). STP, isolated from Streptomyces staurospores, is a general inhibitor of protein kinases, and is commonly used to induce apoptosis in a variety of cell in cultures as tumor cell lines, lymphocytes, neurons and osteoblasts. The mechanism by which STP induce cell death is still unkown. Some studies refer that STP induced cell death courses with cytochrome c release, caspase-dependent pathway activation, especially caspase-3, and is inhibited by Bcl-2 overexpression. However cell death still occurs after caspase inhibition by z-VAD-fmk suggesting that caspase independent pathway is also activated.

Analysis of Localized Calcium Alteration During Neural Cell Death

Fluctuations in intracellular calcium ion (Ca2+i) levels are believed to participate in a myriad of physiological and pathological intracellular events. In an attempt to investigate localized alterations in Ca2+i dynamics in a cell-based neurodegeneration model, we used Fura-2/AM dye to monitor Ca2+i ion levels in the human SH-SY5Y neuroblastoma cells induced to undergo apoptosis with 500 nM staurosporine (STS) over a 24 h period. Using rapid illumination frequency at 5 Hz per 340/380 nm excitation wavelength pair, streaming image acquisition and analysis of 12 very small regions of interest (ROI) of ∼86.5 μm2 in either peri-nuclear (PN) or distal (DST) cytoplasmic locations, we captured micro-regional signals (“Ca2+i ripples”) at selected eight time points that were analyzed for dynamic changes in peak Ca2+i amplitudes and frequencies across both STS exposure times and CCh treatment with standard methods consisting of nonlinear curve-fitting and fast Fourier transform, respectively, and with a novel method of recurrence quantification analysis. Their results, although at times mixed or conflicting, showed that there may indeed be alterations in local Ca2+i signaling/dynamic which are biologically valid and sometimes differ from the global Ca2+i signaling/dynamic of whole cytoplasms. I propose, then, that, at least for intrinsic apoptosis, an early activation of caspases leads to cleavages of increasingly more IP3 receptors and sarco-/endoplasmic reticulum Ca2+ ATPase of ER which may enhance Ca2+i release by the former and inhibit uptake by the latter. Ultimately, this results in the accumulation of the ion that starts locally and heterogeneously until spreading by waves and becoming more apparent at the whole cytoplasmic scale.

Exploring mechanisms for regulating growth and survival of ectopic endometriotic cells using comprehensive DNA microarray analysis

OBJECTIVE: We previously reported that TNFα induced IL-8 expression in endometriotic stromal cells (ESCs). Bacterial endotoxin lipopolysaccaride (LPS), as an inducer of inflammation, enhanced TNFα and IL-8 production as well as cell proliferation in ESCs. On the other hand, altered apoptosis in endometriotic tissues is an aspect that illuminates the pathophysiology of endometriosis. To further define the nature of inflammatory reaction and abnormal survival of endometriotic cells, we sought to reveal the molecular mechanisms with regard to (a) the LPS induced-cell proliferation and (b) the resistance to apoptosis using cDNA microarray. DESIGN: Prospective study. MATERIALS AND METHODS: ESCs were obtained from the ovarian chocolate cysts. Eutopic endometrial stromal cells (EMSCs) were isolated from the endometrial tissue of premenopausal women who underwent hysterectomy for uterine myoma. LPS and staurosporine (SS) were exposed the cultured cells as the inflammatory reaction- and the apoptosis-inducing agent, respectively. Signaling pathway- or apoptosis related-gene expression profiles were analyzed using the pathway-focused cDNA microarray. Based on these microarray data, siRNAs directed against the target genes were transfected. The level of various gene and protein expression was evaluated by realtime RT-PCR, ELISA and western blotting. Cell proliferation was assessed by BrdU incorporation and WST-8 assay. RESULTS: ESCs exhibited the TNFα-induced cell proliferation and the ability to survive against SS exposure compared to EMSCs. In pathway finder array, knockdown of TNFα gene in ESCs repressed LPS-induced cIAP (inhibitor of apoptosis protein) -2 and IL-8 expression, the NFκB pathway responsive genes. After LPS addition, cIAP-2 and IL-8 expression in ESCs was higher than that in EMSCs. TNFα silencing also attenuated the LPS-induced ESCs proliferation. Adding NFκB inhibitor abolished these effects induced by TNFα silencing. On the other hand, in apoptosis gene array, cIAP-1, XIAP, and especially survivin, IAP family members, were highly expressed in ESCs. Survivin expression in ESCs after SS treatment was enhanced, whereas it was repressed in EMSCs, suggesting aberrant survivin expression in ESCs may be involved in the resistance to drug-induced apoptosis. EMSCs showed the potent activation of SS-induced caspase-3 or -7, whereas it was not detected in ESCs. CONCLUSIONS: TNFα and IAP family gene expression may be attributed to the mechanisms regulating mitosis and resistance to apoptosis in endometriotic tissues.

Determination of Poly (ADP-ribose) polymerase (PARP) homologues in human ejaculated sperm and its correlation with sperm maturation

To investigate the presence of Poly (ADP-ribose) polymerase (PARP) and evaluate its function in ejaculated spermatozoa. Experimental study. Reproductive Research Center. Semen specimens from 18 healthy donors and 12 infertile males. Ejaculated spermatozoa were subjected to sperm fractionation with a double-layer density gradient, protein extraction, detection, immunoblotting, and mass spectrometry. Semen samples were exposed to PARP inducer staurosporine, or hydrogen peroxide with or without PARP inhibitor 3-aminobenzimide. Annexin V assay was examined for apoptosis by flow cytometry. We detected approximately 75 kDa, approximately 63 kDa, and approximately 60 kDa PARP homologues on the immunoblot of mature and immature sperm fraction isolated from human ejaculate, which were identified as PARP-1 ( approximately 75 kDa), PARP-9 ( approximately 63 kDa), and PARP-2 ( approximately 60 kDa), respectively. Western blot analysis showed a positive correlation between the amount of PARP protein and sperm maturity. PARP proteins (75 kDa, 63 kDa, and 60 kDa) were evaluated after inducing apoptosis by hydrogen peroxide and staurosporine exposure. PARP-2 may have a role in prevention of oxygen species/oxidative stress and chemical (staurosporine)-induced sperm cell apoptosis. The presence of the PARP homologue, that is, PARP-1 ( approximately 75 kDa), suggests its role in preventing damage of mature sperm. Additional studies are needed to delineate the role of PARP-9 in sperm physiology. The results from our study indicate an active role for PARPs in sperm cell physiology in preventing apoptosis.

Monoamine oxidase‐A modulates apoptotic cell death induced by staurosporine in human neuroblastoma cells

Monoamine oxidases (MAOs) are mitochondrial enzymes which control the levels of neurotransmitters in the brain and dietary amines in peripheral tissues via oxidative deamination. MAO has also been implicated in cell signalling. In this study, we describe the MAO-A isoform as functional in apoptosis induced by staurosporine (STS) in human dopaminergic neuroblastoma cells (SH-SY5Y). Increased levels of MAO-A activity were induced by STS, accompanied by increased MAO-A protein and activation of the initiator of the intrinsic pathway, caspase 9, and the executioner caspase 3. MAO-A mRNA levels were unaffected by STS, suggesting that changes in MAO-A protein are due to post-transcriptional events. Two unrelated MAO-A inhibitors reduced caspase activation. STS treatment resulted in sustained activation of the mitogen-activated protein kinase pathway enzymes extracellular regulated kinase, c-jun terminal kinase and p38, and depletion of the anti-apoptotic protein Bcl-2. These changes were significantly reversed by MAO inhibition. Production of reactive oxygen species was increased following STS exposure, which was blocked by both MAO inhibition and the antioxidant N-acetylcysteine. Therefore our data provide evidence that MAO-A, through its production of reactive oxygen species as a by-product of its catalytic activity on the mitochondrial surface, is recruited by the cell to enhance apoptotic signalling.

The role of α-, β-, and γ-ENaC subunits in distal lung epithelial fluid absorption induced by pulmonary edema fluid

Edema fluid (EF) increases epithelial Na(+) transport by rat fetal distal lung epithelia (FDLE) and induces net lung fluid absorption in fetal mouse lung explants [Rafii B, Gillie DJ, Sulowski C, Hannam V, Cheung T, Otulakowski G, Barker PM, O'Brodovich H. J Physiol (Lond) 544: 537-548, 2002]. We now show that EF increases fluid absorption across monolayers of rat FDLE in a dose-dependent manner. To study the role of subunits of the epithelial Na(+) channel (ENaC) in the phenomena, we cultured explants from the distal lungs of 16-day gestational age wild-type (WT) or alpha-, beta-, or gamma-ENaC knockout or heterozygote (HT) mice. WT explants cultured in media continuously expanded over time as a result of net fluid secretion. In contrast, when explants were exposed to EF for 24 h, net fluid absorption occurred. EF-exposed explants had normal histology, but marked changes were seen after Triton X-100 or staurosporine exposure. Transmission electron microscopy showed EF promoted lamellar body formation and abundant surfactant in the explants' lumens. EF-induced changes in explant size were similar in alpha-ENaC knockout, WT, and HT littermate fetal lung explants (P > 0.05). In contrast, EF's effect was attenuated in beta- and gamma-ENaC knockouts (P < 0.05) vs. WT and HT littermate fetal lung explants. EF exposure slightly decreased or had no effect on mRNA levels for alpha-ENaC in various mouse genotypes but decreased expression of beta- and gamma-ENaC subunit mRNAs (P < 0.01) across all genotype groups. We conclude that beta- and gamma-, but not alpha-, ENaC subunits are essential for EF to exert its maximal effect on net fluid absorption by distal lung epithelia.

Differential p53 Regulation of Neural Precursor Cell Death

Regulation of neural precursor cell (NPC) death is important for both normal nervous system development and brain tumor formation. We previously showed in primary cerebellar NPCs that genotoxin-induced NPC death requires new protein synthesis and PUMA, a p53 transcriptionally-regulated BH-3 only molecule. In contrast, staurosporine (STS), a broad spectrum protein kinase inhibitor, induced NPC death without requiring new protein synthesis or PUMA expression. Similar to primary cerebellar NPCs, genotoxin-induced death of the neural stem cell line C17.2 required new gene transcription, while STS induced NPC death did not. Genotoxic agents increased nuclear p53 immunoreactivity, while STS induced rapid cytoplasmic p53 accumulation. Total p53 protein levels were increased after both genotoxic injury and STS treatment. Immunocytochemical studies showed that the majority of cytoplasmic p53 after STS treatment did not colocalize with either mitochondrial markers or activated-Bax. Phosphorylation of p53 on ser 18 was highly increased after genotoxic injury, but not STS treatment. Deficiency of ataxia-telangiectaisa mutated (ATM), which phosphorylates p53 on ser 18 after DNA damage, decreased bleomycin-induced, but not STS-induced NPC death. Combined with our previous findings, these results suggest that genotoxic agents induce p53 transcription-dependent NPC death, while STS induces p53 transcription-independent NPC death. Whether differential post-translational modification of p53 leads to its different subcellular localization after genotoxin or STS exposure and how cytosolic p53 induces p53-dependent, transcription-independent NPC death requires further investigation. Supported by NIH grants NS35107 and NS41962.

Neuroprotective Efficacy of the Peroxisome Proliferator-Activated Receptor δ-Selective Agonists in Vitro and in Vivo

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARdelta subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARdelta agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARdelta agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARdelta agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPARdelta agonists could be useful tools for understanding the role of PPARdelta in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.

JNK- and p38 Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells

Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H(2)O(2), etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [(32)P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr(167) is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.

Stat3 Cleavage by Caspases: IMPACT ON FULL-LENGTH Stat3 EXPRESSION, FRAGMENT FORMATION, AND TRANSCRIPTIONAL ACTIVITY

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Dual effects of staurosporine on A431 and NRK cells: Microfilament disassembly and uncoordinated lamellipodial activity followed by cell death

The general protein kinase inhibitor staurosporine (STS) has dual effects on human epidermoid cancer cells (A431) and normal rat kidney fibroblasts (NRK). It almost immediately stimulated increased lamellipodial activity of both cell lines and after 2 h induced typical signs of apoptosis, including cytoplasmic condensation, nuclear fragmentation, caspase-3 activation and DNA degradation. In the early phase we observed disruption of actin-containing stress fibres and accumulation of monomeric actin in the perinuclear region and cell nucleus. Increased lamellipodial-like extensions were observed particularly in A431 cells as demonstrated by co-localisation of actin and Arp2/3 complex, whereas NRK cells shrunk and exhibited numerous thin long extensions. These extensions exhibited uncoordinated centrifugal motile activity that appeared to tear the cells apart. Both cofilin and ADF were translocated from perinuclear regions to the cell cortex and, as expected in the presence of a kinase inhibitor, all the cofilin was dephosphorylated. Myosin II was absent from the extensions, and a reduction of phosphorylated myosin light chains was observed within the cytoplasm indicating myosin inactivation. Microtubules and intermediate filaments retained their characteristic filamentous organisation after STS exposure even when the cells became rounded and disorganised. Simultaneous treatment of NRK cells with STS and the caspase inhibitor zVAD did not inhibit the morphological and cytoskeletal changes. However, the cells underwent cell death as verified by positive annexin-V-staining. Thus it seems likely that cell death induced by STS may not only be a consequence of the activation of caspase, instead the disruption of the many motile processes involving the actin cytoskeleton may by itself suffice to induce caspase-independent cell death.