Produce Cell Death Research Articles

Experimental Study of Renal Tubular Cells Apoptosis Subsequent to Infectious by Influenza Virus (H9N2) in SPF Chickens

Influenza virus produces cell death in animals and human. Since cell death can be caused by either necrosis or apoptosis. We investigated the types of cell death that occur in chickens infected with avian influenza virus, A/chicken/Iran/772/2000(H9N2). In experimental study 60 SPF chickens at 3 weeks old were divided to two groups. The first group was infected with 10 EID50 titer of the virus intranasaly and the 7.5 second group was treated with saline normal. Following 72 hrs, renal tissues were collected and fixed in 10% formalin solution. The prepared microscopic sections with the thickness of 5-6 micron were stained using TUNEL method. In comparison to the control group, there were significant mean difference of apoptotic cells in renal tubular cells of the infected group (p<0.005). We demonstrated that A/chicken/Iran/772/2000 (H9N2) is able to induce apoptosis in renal tubular cells.

Cerebral blood flow alteration in neuroprotection following cerebral ischaemia

The best neuroprotectant for acute ischaemic stroke would always be the rapid return of oxygen and glucose to physiological levels. This is currently provided by thrombolysis which restores blood flow to the ischaemic region. The attempt to confer neuroprotection by targeting the brain parenchyma has shown promise in experimental stroke models, but has unequivocally failed to translate to the clinic. Neuroprotective therapy primarily targets the biochemical cascade that produces cell death following cerebral ischaemia. However, these agents may also alter signal transduction that controls cerebral blood flow, for example glutamate, which may affect the outcome after ischaemia. In these cases, neuroprotection may potentially be due to the improved access to oxygen and glucose rather than biochemical prevention of cell death. Improvement in cerebral blood flow is an important but often overlooked effect of neuroprotective therapy, analogous to the protective effects of drug-induced hypothermia. This short review will discuss cerebral blood flow alteration and protection of the brain in the context of ischaemic preconditioning, oxygen sensing and thrombolysis. Future neuroprotection studies in cerebral ischaemia require stringent monitoring of cerebral blood flow, plus other physiological parameters. This will increase the chances that any protection observed may be able to translate to human therapy.

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAsp-PSI)

Potato aspartic proteases (StAPs) and their swaposin domain (StAsp-PSI) are proteins with cytotoxic activity which involves plasma membrane destabilization. The ability of these proteins to produce cell death varies with the cellular type. Therefore, StAPs and StAsp-PSI selective cytotoxicity could be attributed to the different membrane lipid compositions of target cells. In this work we investigate the possible mechanism by which StAPs and StAsp-PSI produce selective membrane destabilization. Results obtained from leakage assays show that StAsp-PSI is a potent inducer of the leakage of LUVs containing anionic phospholipids, especially those containing phosphatidylglycerol. Based in these results, we suggest that the cytotoxic activity of StAsp-PSI on pathogenic microorganisms could be mediated by the attraction between the exposed positive domains of StAsp-PSI and the negatively charged microorganism membrane. On the other hand, our circular dichroism spectroscopic measurements and analysis by size exclusion chromatography and followed by electrophoresis, indicate that hydrophobic environment is necessary to StAsp-PSI oligomerization and both StAsp-PSI disulfide bounds and membrane with negative charged phospholipids are required by StAsp-PSI to produce membrane destabilization and then induce cell death in tumors and microorganism cell targets. Additionally, we demonstrate that the presence of cholesterol into the LUV membranes strongly diminishes the capacity of StAsp-PSI to produce leakage. This result suggests that the lack of hemolytic and cytotoxic activities on human lymphocytes of StAsp-PSI/StAPs may be partly due by the presence of cholesterol in these cell membrane types.

Enhancing immunotoxin cell-killing activity via combination therapy with ABT-737

Immunotoxins are antibody–toxin fusion proteins directed to kill cancer cells displaying specific target antigens on their surface. Remarkably, immunotoxins directed to CD22 on hairy cell leukemia have produced complete remissions in approximately 60% of patients enrolled in phase I/II trials. For reasons that are not yet clear, 40% of patients responded less well. In addition, patients with other CD22-positive malignancies have not yet achieved complete remissions. In trying to understand ‘resistance’ to immunotoxin therapy, a number of challenging issues have been raised. These include insufficient dosing, the production of neutralizing anti-immunotoxin antibodies, poor access to malignant cells, and resistance to toxin killing. In designing immunotoxins, we employ truncated Pseudomonas exotoxin, which enzymatically inactivates protein synthesis and produces cell death in sensitive cells. To begin to address toxin resistance we have explored combination therapy with the BH3-only mimetic, ABT-737. Our results indicate that immunotoxin–ABT combinations often exhibit greater killing activity than either compound alone and in some instances overcome resistance. Expression of high levels of prosurvival Bcl-2 proteins may contribute to toxin resistance.

Estrogen Receptor beta produces cell death in TCAM2 human seminoma cell line binding through Sp1 on the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) promoter gene

Testicular germ cell tumors of adults and adolescents (TGCTs) are the most common tumor in male. We focused on seminoma, by using the TCAM‐2 cell line, containing typical features of human seminoma. As both PTEN and estradiol (E2) appear to have an important role in the testis development and function, we evaluated a potential action of E2 on PTEN promoter gene. Increasing E2 concentrations were able to induce PTEN gene expression and transactivation. By transient trasfections, ChIP and EMSA assays, we evidenced the 5′‐flanking region of human PTEN gene important for E2‐responsiveness, the ERβ binding to the Sp1 regulates PTEN activity. The functional significance of the link between ERβ/PTEN was tested on cell viability, an increase in cell death was observed. The effect was abolished by trasfecting the TCAM2 cells with negative dominant of ERβ or by using mithramycin, confirming that the E2‐induced effect involves both ERβ and Sp1. E2 decreases AKT, while FHKR and BAD increased. The PI3K/AKT pathway is shared to the autophagy, and E2 increased autophagy‐related markers such as PI3III, Beclin 1, AMBRA and UVRAG. Our study suggesting a tumor suppressor role for the ERβ in human seminoma, indicates that the E2 induces cell death in TCAM2 mainly through autophagy, supporting estrogen‐dependency of human seminoma and candidating the ERβ‐ligands for a therapeutic use in the treatment of this pathological condition.

Multinomial model-based formulations of TCP and NTCP for radiotherapy treatment planning

Hit and target models of tumor growth, typically assume that all surviving cells have a constant and homogeneous sensitivity during the radiotherapy period. In this study, we propose a new multinomial model based on a discrete-time Markov chain, able to take into account cell repair, cell damage heterogeneity and cell proliferation. The proposed model relies on the ‘Hit paradigm’ and ‘Target’ theory in radiobiology and assumes that a cancer cell contains m targets which must be all deactivated to produce cell death. The surviving cell population is then split up into m categories to introduce the variation of cancer cell radio-sensitivity according to their damage states. New expressions of the Tumor Control Probability (TCP) and Normal Tissue Complication Probability (NTCP) are provided. Moreover, we show that hit and target models may be regarded as particular cases of the multinomial model. Numerical results should permit to keep the efficiency of treatment with a lower total radiation dose then that given by the typical hit models, which allow to decrease side effects.

Tipifarnib-Induced Apoptosis in Acute Myeloid Leukemia and Multiple Myeloma Cells Depends on Ca2+Influx through Plasma Membrane Ca2+Channels

A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.

Cell Cycle-Associated Kinases as Targets for Therapy in Lung Cancer

Cell Cycle-Associated Kinases as Targets for Therapy in Lung Cancer

Effect of Low Mg2+ and Bicuculline on Cell Survival in Hippocampal Slice Cultures

ABSTRACTA reliable model system of epileptiform insult would facilitate investigation into the underlying biological mechanisms. Epileptiform insult was induced in hippocampal slice cultures by lowering extracellular Mg2+, (+)-bicuculline, or (−)-bicuculline methochloride, a stable salt form of bicuculline (both forms block GABAA receptors). Cell death was assessed by propidium iodide uptake. Low Mg2+ or (+)-bicuculline did not produce cell death regardless of dose or incubation period. Exposure to 100 μM (−)-bicuculline methochloride for 48 hr resulted in prominent CA1 cell death. These findings demonstrate that not all pro-epileptic drugs/ion changes used routinely for electrophysiological recording of seizure activity lead to cell death in hippocampal slice cultures and that treatment with bicuculline methochloride can be used as a reliable model for epileptiform insult.

Abstract 4356: Cytotoxicity of the anti-CD22 immunotoxin HA22 against pediatric acute lymphoblastic leukemia (ALL)

Abstract Cure rates for children with acute lymphoblastic leukemia (ALL) have steadily improved, but a proportion of patients still relapse despite intensive and prolonged chemotherapy. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. We have developed recombinant anti-CD22 immunotoxins composed of the Fv portion of the anti-CD22 mAb RFB4 linked to a 38kDa truncated protein derived from Pseudomonas exotoxin A (PE38). These produce cell death through a unique and highly specific mechanism of action, entailing binding to cell surface CD22, rapid internalization, intracellular processing, and inhibition of protein synthesis through ADP-ribosylation of elongation factor-2. In phase I and II trials of patients with hairy cell leukemia the first generation agent, BL22, produced complete responses (CR) of 61% and 47% respectively. However, activity was modest in children with ALL (Blood 2007;110:262a). A modified agent, HA22, was derived by complementarity-determining region mutagenesis resulting in a significantly higher binding affinity for the CD22 antigen. We cultured pre-B ALL cells from newly diagnosed (n = 13) and relapsed children (n= 22), as well as human ALL cells derived from xenografts in immunodeficient mice (n = 4) and the cell line KOPN-8 on telomerase-immortalized human bone marrow mesenchymal cells. On day 2 of culture, HA22 was added at concentrations ranging from 0 to 500ng/ml and the cells were incubated for an additional 72 hours. Dexamethasone 10umol/L was used as a positive control. The number of viable CD19+ cells at the end of the cultures was determined by flow cytometry. Viability was assessed using gates set for light-scatter properties and Annexin-PE/7-AAD staining on Days 2 and 5. The percentage of CD19+ viable cells relative to the control was determined and the 50% inhibitory concentration (IC50) calculated. Mesenchymal-supported cell cultures maintained ALL cell viability (mean 77%, median 52%, range 6% - 280% viable cells at 96 hours). ALL samples varied in their sensitivity to HA22. IC50s ranged from 1-80 ng/mL in 29 of 40 samples. In 11 samples 50% killing was not achieved. Resistance to cytotoxicity from dexamethasone was also observed, which correlated with resistance to HA22 for samples from newly diagnosed patients (r = 0.55, p = 0.053). Peripheral blood blasts appeared to be more sensitive to HA22 in comparison to bone marrow derived blasts (p = 0.008). Blasts with higher CD22 site densities showed increased cytotoxicity to HA22 (r = −0.34, p = 0.16). These results indicate that most cases of childhood B-lineage ALL are sensitive to HA22 at concentrations well below clinically achievable blood levels. Clinical testing of HA22 in children with drug-resistant ALL has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4356.

Abstract 3571: The Thioredoxin Reductase Inhibitor Chaetocin has Potent Antineoplastic Effects in Solid Tumors

Abstract Background: We had previously reported that the natural product chaetocin has potent and selective in vitro, ex vivo and in vivo anti-myeloma activity attributable to the imposition of cellular oxidative stress (ROS) in part mediated via competitive inhibition of the redox enzyme thioredoxin reductase. Having also observed chaetocin-induced cytotoxicity in solid tumor cell lines, we now extend prior work to characterize the effects of chaetocin in solid tumor cell lines and in human umbilical vein endothelial cells (HUVECs). Methods: The effects of chaetocin in solid tumor cell lines were assessed using colony forming assays, trypan blue exclusion assays, apoptosis and autophagy assays, electron microscopy, transcriptional profiling, and the National Cancer Institute 60 cell line screen. Results: Chaetocin demonstrated potent anti-cancer activity in all assessed solid tumor cell lines with IC50 values between 2-10 nM (24 h exposures, colony forming assays). While apoptosis was induced in a cell line-dependant fashion, it was not required for chaetocin-induced cytotoxicity, as ZVAD-fmk prevented apoptosis but not cell death. Markers of autophagy were not altered by chaetocin treatment. Interestingly, results form the NCI 60 cell line screen showed that hematological cell lines were generally more resistant to chaetocin than solid tumor lines despite our prior report indicating the activity of chaetocin in myeloma. Transcriptional profiling results were consistent with those anticipated from an agent producing cell death via imposition of cellular ROS, with heme oxidase-1 prominently induced along with other transcripts in pathways related to inflammatory response and cell death. Results from OxyBlot protein oxidation kit analyses (Millipore, Billerica, MA) confirmed a generalized increase in the carbonyl modification of proteins, a hallmark of cellular oxidative damage, in response to chaetocin treatment. Experiments using Rho0 mitochondrial inactive cells indicated that cellular ROS is induced by chaetocin independent of respiratory functional mitochondria. Chaetocin was also shown to block the interleukin-, fibroblast growth factor- or EGM2 media-induced proliferation of HUVEC cells at low-nanomolar concentrations. Conclusions: Chaetocin has wide-ranging antineoplastic activity across not only hematological, but also solid tumor, cell lines and displays evidence of antiangiogentic activity in HUVEC proliferation assays. Overall, chaetocin appears to be an attractive agent for further development as a candidate anti-cancer therapeutic in a variety of neoplasms. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3571.

Downregulation of Epidermal Growth Factor Receptor Expression Contributes toα-TEA's Proapoptotic Effects in Human Ovarian Cancer Cell Lines

RRR-α-tocopherol derivative α-TEA (RRR-α-tocopherol ether-linked acetic acid analog) has been shown to be a potent antitumor agent both in vivo and in vitro. In this study, we investigated the effects of α-TEA on the expression of epidermal growth factor receptor (EGFR) family members, ErbB1, 2 and 3, and the role of ErbB 2 and 3 in α-TEA-induced apoptosis and suppression of Akt, FLIP and survivin in the cisplatin-sensitive (A2780S) and -resistant (A2780/CP70R) human ovarian cancer cell lines. Data show that α-TEA's ability to induced apoptosis was associated with reduced expression of ErbB1 (cisplatin-resistant cells), 2 and 3 (both cell types) and reduced levels of the phosphorylated (active) form of Akt; as well as, reduced levels of FLIP and survivin proteins in both cell types. Ectopic overexpression and siRNA knockdown studies showed that ErbB2, ErbB3, Akt, FLIP and survivin are involved in α-TEA-induce apoptosis and that α-TEA downregulates FLIP and survivin via suppression of pAkt, which is mediated by ErbB2 and ErB3. Thus, α-TEA is a potent pro-apoptotic agent for both cisplatin-sensitive and -resistant ovarian cancer cell lines in cell culture and it produces cell death, at least in part, by downregulation of members of the EGFR family.

A multinomial model of tumor growth treated by radiotherapy

A main challenge in radiotherapy is to personalize the treatment by adapting the dose fractionation scheme to the patient. One way is to model the treatment effect on the tumor growth. In this study, we propose a new multinomial model based on a discrete-time Markov chain, able to take into account both of cell repair and cell damage heterogeneity. The proposed model relies on the ‘Hit’ theory in Radiobiology and assumes that a cancer cell contains m targets which must be all deactivated to produce cell death. The malignant cell population is then split up into m categories to incorporate the variation of cancer cell radio-sensitivity according to their states. This work gives also a new formulation of the tumor control probability (TCP) suited to the perspective of dynamic fractionation schedules in radiotherapy.

Mathematical model of a network of interaction between p53 and Bcl-2 during genotoxic-induced apoptosis

Ionizing radiation like UV light, gamma and X rays, can produce genotoxic damage in fibroblast cells. This injury can be reverted by activation of specific nuclear molecules. However, intense genotoxic damage induces the activation of the p53 dependent apoptotic pathway. Activated nuclear p53 has the role of a transcription factor that switches on the transcription of the Puma protein, which once released into the cytoplasm leads to the activation of a network of chemical processes that produces cell death. This network is built up with the chemical interaction between pro-apoptotic p53, Puma and Bax proteins and the anti-apoptotic Bcl2 and Bcl-x L proteins. In this work we present a mathematical model of this modular network under different regimes of Puma release into the cytoplasm. In this model we use a set of coupled nonlinear differential equations, which is solved by numerical methods, to determine the nature of the equilibrium points of the dynamical system. With this information we construct the phase portrait associated with Puma induced apoptosis and we found that once the genotoxic injury is produced, Bax levels continuously increase. In this work we propose that a possible mechanism for the control of apoptosis is the Bax level in the cytoplasm, i.e., Bax is continuously released into the cytoplasm, even in absence of Puma, according to the kinetic properties of the set of chemical interactions in which the Bcl2/Bax complex takes part. If the damage is reverted before the Bax level reaches a threshold value the apoptotic process is stopped, otherwise the process goes on until cell death.

APOMAB®, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy

BackgroundAntineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMAB® representing the DAB4 clone of a La/SSB-specific murine monoclonal autoantibody is a malignant cell-death ligand, which accumulates preferentially in tumors in an antigen-specific and dose-dependent manner after DNA-damaging chemotherapy. Here, we aim to image tumor uptake of APOMAB® (DAB4) and to define its biological correlates.Methodology/Principal FindingsBrisk tumor cell apoptosis is induced in the syngeneic EL4 lymphoma model after treatment of tumor-bearing mice with DNA-damaging cyclophosphamide/etoposide chemotherapy. Tumor and normal organ accumulation of Indium 111 (111In)-labeled La-specific DAB4 mAb as whole IgG or IgG fragments was quantified by whole-body static imaging and organ assay in tumor-bearing mice. Immunohistochemical measurements of tumor caspase-3 activation and PARP-1 cleavage, which are indicators of early and late apoptosis, respectively, were correlated with tumor accumulation of DAB4. Increased tumor accumulation of DAB4 was associated directly with both the extent of chemotherapy-induced tumor cell death and DAB4 binding per dead tumor cell. Tumor DAB4 accumulation correlated with cumulative caspase-3 activation and PARP-1 cleavage as tumor biomarkers of apoptosis and was directly related to the extended median survival time of tumor-bearing mice.Conclusions/SignificanceRadiolabeled La-specific monoclonal antibody, DAB4, detected dead tumor cells after chemotherapy, rather than chemosensitive normal tissues of gut and bone marrow. DAB4 identified late apoptotic tumor cells in vivo. Hence, radiolabeled DAB4 may usefully image responses to human carcinoma therapy because DAB4 would capture the protracted cell death of carcinoma. We believe that the ability of radiolabeled DAB4 to rapidly assess the apoptotic tumor response and, consequently, to potentially predict extended survival justifies its future clinical development as a radioimmunoscintigraphic agent.This article is part I of a two-part series providing proof-of-concept for the the diagnostic and therapeutic use of a La-specific monoclonal antibody, the DAB4 clone of which is represented by the registered trademark, APOMAB®.

Starving Neurons Show Sex Difference in Autophagy

Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or L-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.

Cell Death after Spinal Cord Injury Is Exacerbated by Rapid TNFα-Induced Trafficking of GluR2-Lacking AMPARs to the Plasma Membrane

Glutamate, the major excitatory neurotransmitter in the CNS, is implicated in both normal neurotransmission and excitotoxicity. Numerous in vitro findings indicate that the ionotropic glutamate receptor, AMPAR, can rapidly traffic from intracellular stores to the plasma membrane, altering neuronal excitability. These receptor trafficking events are thought to be involved in CNS plasticity as well as learning and memory. AMPAR trafficking has recently been shown to be regulated by glial release of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) in vitro. This has potential relevance to several CNS disorders, because many pathological states have a neuroinflammatory component involving TNFalpha. However, TNFalpha-induced trafficking of AMPARs has only been explored in primary or slice cultures and has not been demonstrated in preclinical models of CNS damage. Here, we use confocal and image analysis techniques to demonstrate that spinal cord injury (SCI) induces trafficking of AMPARs to the neuronal membrane. We then show that this effect is mimicked by nanoinjections of TNFalpha, which produces specific trafficking of GluR2-lacking receptors which enhance excitotoxicity. To determine if TNFalpha-induced trafficking affects neuronal cell death, we sequestered TNFalpha after SCI using a soluble TNFalpha receptor, and significantly reduced both AMPAR trafficking and neuronal excitotoxicity in the injury penumbra. The data provide the first evidence linking rapid TNFalpha-induced AMPAR trafficking to early excitotoxic secondary injury after CNS trauma in vivo, and demonstrate a novel way in which pathological states hijack mechanisms involved in normal synaptic plasticity to produce cell death.

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.

Conditioned medium from Ad-IFN-alpha-infected bladder cancer and normal urothelial cells is cytotoxic to cancer cells but not normal cells: further evidence for a strong bystander effect.

We have reported earlier that a bystander effect is seen in cancer cells that are resistant to high concentrations of the interferon-alpha protein (Intron A) when treated with adenoviral-mediated interferon-alpha (Ad-IFN-alpha). We now provide further evidence for this bystander effect using conditioned medium (CM) collected from Ad-IFN-alpha-infected cancer and normal urothelial cells. The CMs collected from UC-9 and KU7 bladder cancer cells as well as normal urothelial cells following transfection with Ad-IFN produce cell death when added to various cancer cell types in culture but not to normal urothelial cells. The CM could be filtered, frozen and thawed, and diluted to at least one part Ad-IFN CM to five parts fresh control medium and the diluted CM still shows a similar cytotoxicity as a 100% concentration of Ad-IFN CM. This cytotoxicity was observed by both flow cytometry and MTT assays as well as by phase microscopy, and a significant sub-G1 population was seen whether the CM was collected 48, 72 or 96 h after initial Ad-IFN treatment. In addition, the CM could be partially inactivated by exposure to 65 degrees C for 30 min and totally inactivated by placement at 92 degrees C for 3 min, whereas Intron A was not inactivated under the same conditions. Importantly, although significant caspase 8 and caspase 9 cleavage occurred in Ad-IFN-treated cells as a direct effect of Ad-IFN transfection, the Ad-IFN CM produced no activation of caspase 8 and caspase 9, indicating that a different mechanism of cell death was produced by the bystander factor(s) than the direct effect of Ad-IFN. This bystander effect in turn may play an important role in the efficacy of the current Ad-IFN clinical trial for superficial bladder cancer now underway.

Molecular Cloning of a cDNA Encoding Atypical Antimicrobial and Cytotoxic Brevinin-1Ja from the Skin of the Japanese Brown Frog, Rana japonica

Antimicrobial peptides (AMPs) are important components of the host innate defense system against pathogenic microbial invasion in many organisms. In the present study, we cloned by RT-PCR a cDNA from total RNA prepared from the skin of the Japanese brown frog Rana japonica. The cDNA directs the synthesis of a novel, non-C-terminally alpha-amidated peptide composed of 21 amino acid residues (FLGSLIGAAIPAIKQLLGLKK). The putative peptide showed limited sequence similarity to atypical acyclic brevinin-1OK family AMPs originally isolated from the skin of the Ryukyu brown frog (R. okinavana), which lacks the COOH-terminal cyclic domain commonly observed in typical brevinin-1 groups, but that contains a C-terminally alpha-amidated residue. Although it is unclear whether the peptide, designated brevinin-1Ja, is produced in R. japonica skin, a synthetic replicate of the peptide showed differential growth-inhibiting activity against the Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli (minimal inhibitory concentrations: 15 microM and 119 microM, respectively), and produced cell death of mammalian COS7 cells (LD50=28 microM).