Induced Cell Death (induced + cell_death)

Distribution by Scientific Domains

Selected Abstracts

Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell death

Cedric Raoul
Abstract Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [source]

BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtin

Rebecca Leon
Abstract Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the N-terminus of the huntingtin protein. It is characterized by a selective loss of medium spiny neurons in the striatum. It has been suggested that impaired proteasome function and endoplasmic reticulum (ER) stress play important roles in mutant huntingtin (mHtt)-induced cell death. However, the molecular link involved is poorly understood. In the present study, we identified the essential role of the extra long form of Bim (Bcl-2 interacting mediator of cell death), BimEL, in mHtt-induced cell death. BimEL protein expression level was significantly increased in cell lines expressing the N-terminus of mHtt and in a mouse model of HD. Although quantitative RT-PCR analysis indicated that BimEL mRNA was increased in cells expressing mHtt, we provided evidence showing that, at the post-translational level, phosphorylation of BimEL played a more important role in regulating BimEL expression. Up-regulation of BimEL facilitated the translocation of Bax to the mitochondrial membrane, which further led to cytochrome c release and cell death. On the other hand, knocking down BimEL expression prevented mHtt-induced cell death. Taken together, these findings suggest that BimEL is a key element in regulating mHtt-induced cell death. A model depicting the role of BimEL in linking mHtt-induced ER stress and proteasome dysfunction to cell death is proposed. [source]

Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-,,induced apoptosis in cultured mouse hepatocytes

HEPATOLOGY, Issue 1 2002
Hidenari Nagai
The effect of reduced glutathione (GSH) depletion by acetaminophen (APAP), diethylmaleate (DEM), or phorone on the mode of cell death and susceptibility to tumor necrosis factor (TNF)-induced cell death was studied in cultured mouse hepatocytes. Dose-dependent necrosis was the exclusive mode of cell death with APAP alone, but the addition of TNF-, induced a switch to about half apoptosis without changing total loss of viability. This effect was seen at 1 and 5 mmol/L but was inhibited at 10 and 20 mmol/L APAP. The switch to apoptosis was associated with increased caspase activities, release of cytochrome c, and DNA laddering and was inhibited by caspase inhibitors. DEM and phorone also induced dose-dependent necrosis. Treatment with TNF-, under these conditions lead to incremental cell death in the form of apoptosis at 0.25 and 0.5 mmol/L DEM and 0.1 and 0.2 mmol/L phorone. At 1.0 and 2.0 mmol/L DEM and 0.5 mmol/L phorone, 90% to 100% necrosis was observed with resistance to TNF-, effects. The apoptosis with TNF-, plus DEM was confirmed by DNA laddering and inhibition by caspase inhibitors. However, in the presence of caspase inhibitors, the increment in cell death induced by TNF-, persisted as an increase in necrosis. A combination of antioxidants, vitamin E, and butylated hydroxytoluene (BHT) markedly inhibited necrosis induced by APAP or DEM alone, but the sensitization to TNF-,,induced apoptosis was unaffected. GSH monoethylester (GSH-EE) protected against necrosis and apoptosis. In conclusion, depletion of GSH by APAP, DEM, or phorone causes oxidative stress-induced necrosis and sensitizes to an oxidative stress independent TNF-,,induced apoptosis. [source]

Inhibitors of the PI3-kinase/Akt pathway induce mitotic catastrophe in non-small cell lung cancer cells

Therese H Hemström
Abstract Non-small cell lung cancer cells (NSCLC) are more resistant to anticancer treatment as compared with other types of cancer cells. Recently (Hemström et al., Exp Cell Res 2005;305:200,13) we showed that apoptosis of U1810 NSCLC cells induced by the staurosporine analog PKC 412 correlated with inhibition of Akt and ERK1/2, suggesting the involvement of these kinases in cell survival. Here we investigated the contribution of the PI3-kinase/Akt and MEK/ERK pathways to survival of NSCLC cells. The two signaling pathways were studied by using different combinations of the PI3-kinase inhibitors LY-294002 and wortmannin, the Akt activator Ro 31-8220, the MEK inhibitor PD 98059 and PKC 412. PI3-kinase inhibitors induced apoptosis-like death in U1810 cells. H157 cells in general were relatively resistant to PI3 kinase/Akt inhibitors yet these compounds sensitized cells to the DNA-damaging drug VP-16, while Ro 31-8220 could not. PD 98059 only had a sensitizing effect on H157 cells when combined with PI3-kinase inhibition and VP-16. Morphological data indicated that LY-294002 and PKC 412 induced cell death at anaphase and metaphase, respectively, suggesting death by mitotic catastrophe. Analyzes of cells blocked in G2/M-phase by nocodazol revealed that LY-294002 increased, while PKC 412 decreased histone H3 phosphorylation, suggesting that LY-294002 allowed, while PKC 412 inhibited cells to leave M-phase. Flow cytometric analysis of cell cycle distribution demonstrated that LY-294002 allowed cells to leave G2/M phase, while PKC 412 inhibited cytokinesis, resulting in formation of multinucleated cells. These results indicate that sensitization of NSCLC cells by PI3-kinase inhibition involves interplay between cell cycle regulation, mitotic catastrophe and apoptosis. © 2006 Wiley-Liss, Inc. [source]

Antioxidative and Anti-Inflammatory Protection of Oleanolic Acid and Ursolic Acid in PC12 Cells

Shih-Jei Tsai
ABSTRACT:, PC12 cells were used to examine the in vitro antioxidative and anti-inflammatory effects of oleanolic acid (OA) and ursolic acid (UA). PC12 cells were pretreated with OA or UA at 20 and 40 ,M and followed by exposure of hydrogen peroxide (H2O2) or 1-methyl-4-phenylpyridinium ion (MPP+) to induce cell injury. Results showed that H2O2 - or MPP+ -treatment significantly decreased cell viability and increased lactate dehydrogenase (LDH) release (P < 0.05). The pretreatment from OA or UA significantly and concentration-dependently reduced subsequent H2O2 - or MPP+ -induced cell death and LDH release (P < 0.05). Either H2O2 - or MPP+ -treatment significantly increased malonyldialdehyde (MDA) formation, decreased glutathione (GSH) content, and diminished glutathione peroxidase (GPX), catalase, and superoxide dismutase (SOD) activities (P < 0.05). The pretreatment from OA or UA significantly retained GSH, and reversed H2O2 - and MPP+ -induced impairment in catalase and SOD activities (P < 0.05), and decreased MDA formation (P < 0.05). Either H2O2 - or MPP+ -treatment significantly elevated interleukin-6 (IL-6) and tumor necrosis factor (TNF)-, levels (P < 0.05). The pretreatments from OA or UA significantly attenuated subsequent H2O2 - or MPP+ -induced release of IL-6 and TNF-, (P < 0.05). Based on the observed antioxidative and anti-inflammatory activities from OA and UA, these 2 compounds were potent agents against neurodegenerative disorder. [source]

Vascular endothelial growth factor protects hepatoma cells against oxidative stress-induced cell death

Shinji Osada
Abstract Background:, The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease. Methods:, Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis. Results:, The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 µmol/L hydrogen peroxide (H2O2) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H2O2, rapid phosphorylation of both ERK1/2 and c-Jun NH2 -terminal kinase (JNK) was observed. In the first 6 h, H2O2 induced cell death for 58.4 ± 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 ± 4.2%. The VEGF significantly decreased H2O2 -induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 µmol/L H2O2, expression of VEGF protein was detected. Furthermore, H2O2 -induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK. Conclusion:, Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF. [source]

Fibroblast growth factor 9 prevents MPP+ -induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Jui-Yen Huang
Abstract Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP+ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP+ intoxication and melatonin protection. We found that MPP+ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP+ -induced cell death of DA neurons. Melatonin co-treatment attenuated MPP+ -induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro. Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP+, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP+ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection. [source]

22R -Hydroxycholesterol protects neuronal cells from ,-amyloid-induced cytotoxicity by binding to ,-amyloid peptide

Zhi-Xing Yao
Abstract 22R -hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. ,-Amyloid (A,) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R -hydroxycholesterol was found to protect, in a dose-dependent manner, against A,-induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R -hydroxycholesterol was found to be stereospecific because its enantiomer 22S -hydroxycholesterol failed to protect the neurons from A,-induced cell death. Moreover, the effect of 22R -hydroxycholesterol was specific for A,-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R -hydroxycholesterol was seen when using A,1,42 but not the A,25,35 peptide. To investigate the mechanism of action of 22R -hydroxycholesterol we examined the direct binding of this steroid to A, using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R -hydroxycholesterol to A,1,42 and A,17,40, but not A,25,35, was observed. These data suggest that 22R -hydroxycholesterol binds to A, and the formed 22R -hydroxycholesterol/A, complex is not toxic to rodent and human neurons. We propose that 22R -hydroxycholesterol offers a new means of neuroprotection against A, toxicity by inactivating the peptide. [source]

The galanin receptor 2/3 agonist Gal2-11 protects the SN56 cells against ,-amyloid25,35 toxicity

S. Pirondi
Abstract The neuropeptide galanin is a modulator of cholinergic function and may play a role in A, peptide-induced degeneration of cholinergic forebrain neurons. We have studied the effect of galanin and its galanin receptor subtype 2/3 agonist Gal2-11on toxicity induced by freshly-prepared ,-amyloid25,35 in the cholinergic cell line SN56. Both nuclear fragmentation and caspase-3 expression were analysed. ,-amyloid25,35 -exposure induced a significant increase in caspase-3 mRNA expression after 30, 60, 90 or 150 min of ,-amyloid25,35 exposure. These effects were abolished in the presence of Gal2-11 (10 nM). Similarly, ,-amyloid25,35 -induced nuclear fragmentation was prevented by the galanin agonist at all time points studied. These findings indicate that the galanin 2/3 agonist Gal2-11 protects SN56 cholinergic cells from ,-amyloid25,35 -induced cell death and that this action is mediated by an early reduction of caspase-3 expression. © 2009 Wiley-Liss, Inc. [source]

Increases in expression of 14-3-3 eta and 14-3-3 zeta transcripts during neuroprotection induced by ,9 -tetrahydrocannabinol in AF5 cells,

Jia Chen
Abstract The molecular mechanisms involved in N-methyl-D-aspartate (NMDA)-induced cell death and ,9-tetrahydrocannabinol (THC)-induced neuroprotection were investigated in vitro with an AF5 neural progenitor cell line model. By microarray analysis, Ywhah, CK1, Hsp60, Pdcd 4, and Pdcd 7 were identified as being strongly regulated by both NMDA toxicity and THC neuroprotection. The 14-3-3 eta (14-3-3,; gene symbol Ywhah) and 14-3-3 zeta (14-3-3,; gene symbol Ywhaz) transcripts were deceased by NMDA treatment and increased by THC treatment prior to NMDA, as measured by cDNA microarray analysis and quantitative real-time RT-PCR. Other 14-3-3 isoforms were unchanged. Whereas up-regulation of 14-3-3, expression was observed 30 min after treatment with THC plus NMDA, down-regulation by NMDA alone was not seen until 16 hr after treatment. By Western blotting, THC increased 14-3-3 protein only in cells that were also treated with NMDA. Overexpression of 14-3-3, or 14-3-3, by transient plasmid transfection increased 14-3-3 protein levels and decreased NMDA-induced cell death. These data suggest that increases in 14-3-3 proteins mediate THC-induced neuroprotection under conditions of NMDA-induced cellular stress. © 2007 Wiley-Liss, Inc. [source]

Photoprotection of bacterial-derived melanin against ultraviolet A,induced cell death and its potential application as an active sunscreen

J Geng
Abstract Background, The increase in the incidence of non-melanoma skin tumours, photoaging, and immunosuppression demand for more effective sunscreen on ultraviolet A (UVA) irradiation. Objectives, The aim of the study is to evaluate the photoprotective effects of a bacterial-derived melanin against UVA-induced damages in vitro and in vivo. Methods, Human fibroblasts were used to assess the role of the bacterial-derived melanin on cell viability against UVA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and nuclear morphology were employed to evaluate the photoprotection at the cellular level. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. Evaluations of the bacterial-derived melanin as a sunscreen were measured by transmission test and persistent pigment darkening on human skin. Results, Bacterial-derived melanin efficiently scavenged ROS in the fibroblasts after UVA irradiation. The cell viability of xeroderma pigmentosum (XP) fibroblast treated with varied doses of melanin increased dramatically in comparison with untreated control and the treated XP fibroblasts became more resistant to UVA-induced apoptosis than normal fibroblasts. Although the relative transmission didn't change too much with different concentration of bacterial-derived melanin, this melanin could keep UVA-irradiated skin from pigment darkening and act as an active sunscreen on skin. Conclusions, The bacterial-derived melanin provided significant protection to fibroblast cell and human skin against the UVA radiation. It has the potential to be developed as an active sunscreen for the patients with photosensitivity skin to sun exposure. [source]

A mutation in the Arabidopsis mTERF-related plastid protein SOLDAT10 activates retrograde signaling and suppresses 1O2 -induced cell death

Rasa Meskauskiene
Summary The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen (1O2) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate 1O2 -mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of 1O2 -responsive genes after a dark-to-light shift they do not show a 1O2 -dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent 1O2 -mediated cell death response without suppressing 1O2 -dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as ,plastid signaling'. [source]

Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana

Reetta Ahlfors
Summary Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O3) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O3 -induced cell death. Treatment with O3 induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O3 individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O3 induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O3 -induced SA accumulation. The O3 -sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 (Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1), a mutant with decreased production of NO, was also O3 sensitive. This, together with experiments combining O3 and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O3 exposure, and that a functional NO production is needed for a proper O3 response. In summary, NO is an important signalling molecule in the response to O3. [source]

Overexpression of hepatocyte nuclear factor-3, induces apoptosis through the upregulation and accumulation of cytoplasmic p53 in prostate cancer cells,

THE PROSTATE, Issue 4 2010
Hyun Joo Lee
Abstract BACKGROUND Hepatocyte nuclear factor-3, (HNF-3,) has been known to act as a repressor in the pathogenesis of many cancers. Herein, we investigated the effect of HNF-3, overexpression in prostate cancer cells. METHODS HNF-3, was overexpressed in prostate cancer cells using an adenovirus recombinant expressing wild-type HNF-3,. The apoptosis of prostate cancer cells was determined by TUNEL, FACS, and caspase activity analyses. RESULTS Adenovirus-mediated overexpression of HNF-3, caused cell death in prostate cancer cells as assessed by changes in cellular and nuclear morphology, TUNEL analysis, and caspase activations. Furthermore, FACS analysis showed an increased sub-G1 phase of cell cycle as well as the G2/M phase with a corresponding decrease in S phases. HNF-3, overexpression caused the upregulation of p53 protein and its accumulation, together with HNF-3,, in the cytoplasm. It also causes Bax protein to localize to the mitochondria-enriched fraction. These findings suggest that multiple apoptotic pathways seem to be involved in the HNF-3,-induced cell death: pathways involving the accumulation of p53 protein in the cytoplasm and subsequent cytochrome c release, and other pathways involving death receptor signaling and caspase-8 activation. CONCLUSIONS The results of the current study suggest a novel function of HNF-3, as a killer of malignant prostate cancer cells, which reveals HNF-3, as a promising therapeutic molecule for prostate cancers. Prostate 70: 353,361, 2010. © 2009 Wiley-Liss, Inc. [source]

Alsin/Rac1 signaling controls survival and growth of spinal motoneurons

Arnaud Jacquier MSc
Objective Recessive mutations in alsin, a guanine-nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock-out mice do not develop overt signs of motoneuron degeneration. Methods To generate an alsin loss-of-function model in an ALS-relevant cell type, we developed a new small interfering RNA electroporation technique that allows efficient knock down of alsin in embryonic rat spinal motoneurons. Results After small interfering RNA,mediated alsin knockdown, cultured motoneurons displayed a reduced apparent size of EEA1-labeled early endosomes and an increased intracellular accumulation of transferrin and L1CAM. Alsin knockdown induced cell death in 32 to 48% of motoneurons and significantly inhibited axon growth in the surviving neurons. Both cellular phenotypes were mimicked by expression of a dominant-negative Rac1 mutant and were completely blocked by expression of a constitutively active Rac1 mutant. Expression of dominant-negative or constitutively active forms of Rab5 had no such effects. Interpretation Our data demonstrate that alsin controls the growth and survival of motoneurons in a Rac1-dependant manner. The strategy reported here illustrates how small interfering RNA electroporation can be used to generate cellular models of neurodegenerative disease involving a loss-of-function mechanism. Ann Neurol 2006;60:105,117 [source]

Impaired activation-induced cell death promotes spontaneous arthritis in antigen (cartilage proteoglycan),specific T cell receptor,transgenic mice

Ferenc Boldizsar
Objective To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis. Methods We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAYQDK peptide of human cartilage PG. PG-specific TCR,transgenic (PG-TCR,Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG-TCR,Tg mice. Results Spontaneous arthritis developed as early as 5,6 months of age, and the incidence increased to 40,50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4+ T cells, enhanced interferon-, and interleukin-17 (IL-17) production, and elevated levels of serum autoantibodies. PG-TCR,Tg mice lacking IL-4 developed arthritis earlier and at a higher incidence than IL-4,sufficient mice. Antigen-specific activation,induced cell death was diminished in vitro in CD4+ T cells of PG-TCR,Tg mice with spontaneous arthritis, especially in those lacking IL-4. Conclusion The presence of CD4+ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL-4 appears to be a negative regulator of this disease, through attenuation of activation-induced cell death. [source]

Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fc, receptor I,directed immunotoxin

Joel A. G. Van Roon
Objective To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin-conjugated antibody CD64,ricin A (CD64-RiA) directed toward the high-affinity receptor for IgG (Fc,RI), exploiting the capacity of Fc,RI to efficiently endocytose antibody which it has bound. Methods Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64-RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light-scatter patterns and CD14 and Fc,RI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64-RiA,induced cell death of macrophages affected their capacity to stimulate antigen-induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64-RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated. Results Inflammatory macrophages from RA SF expressed elevated levels of Fc,RI and were selectively eliminated by CD64-RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of Fc,RI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen-induced lymphocyte proliferation and a reduction in tumor necrosis factor , (TNF,) release. Consistent with these effects on SF macrophages, CD64-RiA also inhibited TNF, production, interleukin-1, production, and cartilage-degrading activity of RA synovial tissue explants. Conclusion Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through Fc,RI-directed immunotoxins could be a novel approach to the treatment of RA. [source]

Brazilin and the extract from Caesalpinia sappan L. protect oxidative injury through the expression of heme oxygenase-1

BIOFACTORS, Issue 3 2007
Byung-Min Choi
Abstract In this study, we examined the protective effects of Caesalpinia sappan L. and its major component, brazilin, against tert-butylhydroperoxide (t-BHP)-induced cell death in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. We found that the extract of C. sappan L. and brazilin induced antioxidant response element (ARE)-luciferase activity and heme oxygenase-1 (HO-1) expression in a concentration-dependent manner. The inductive effect of brazilin was more potent than the extract of C. sappan L. and the expression of HO-1 reached a peak at 12 h after brazilin treatment. The extract and brazilin protected the cells against t-BHP-induced cell death. Their protective effects were abrogated by zinc protoporphyrin IX (ZnPP IX), a HO inhibitor. These results demonstrate that the extract of C. sappan L. and brazilin induce the expression of HO-1 and the enzyme diminishes t-BHP-induced cell death in HEI-OC1 cells. [source]

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice

CANCER, Issue 6 2005
Simeng Suy Ph.D.
Abstract BACKGROUND In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). METHODS The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. RESULTS Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6,7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2,3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5,10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 ± 272.83; tempo, 9.72 ± 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. CONCLUSIONS These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents. Cancer 2005. © 2005 American Cancer Society. [source]

Distinct effects of atypical 1,4-dihydropyridines on 1-methyl-4-phenylpyridinium-induced toxicity

Linda Klimaviciusa
Abstract Our previous data obtained from in vivo experiments demonstrated high neuroprotective effects of three novel atypical neuronal non-calcium antagonistic 1,4-dihydropyridine (DHP) derivatives cerebrocrast, glutapyrone and tauropyrone. The present studies were carried out in vitro to clarify, at least in part, their mechanism of action in primary culture of cerebellar granule cells by use of 1-methyl-4-phenylpyridinium (MPP+) as a neurotoxic agent which causes dramatic oxidative stress. Cerebrocrast (highly lipophilic, with a classical two-ring structure) dose-dependently (0.01,10.0,µM, EC50,=,13,nM) reduced MPP+ -induced cell death. At the same time, the calcium antagonist nimodipine (reference drug) protected cell death at much higher concentrations (EC50,=,12.4,µM). Cerebrocrast decreased also the generation of reactive oxygen species and loss of mitochondrial membrane potential. In contrast, low lipophilic amino acid-containing DHPs glutapyrone and tauropyrone (glutamate- and taurine-containing, correspondingly) were without significant effects indicating their distinct mode of action in comparison to cerebrocrast. We have demonstrated for the first time an ability of atypical non-calcium antagonistic DHP cerebrocrast (which has classical DHP structure elements and high lipophilicity) to protect MPP+ -induced deterioration of mitochondrial bioenergetics. One may suggest mitochondria as an essential intracellular target for the neuroprotective action of cerebrocrast and indicate its usefulness in the treatment of Parkinson's disease. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Preconditioning protects the retinal pigment epithelium cells from oxidative stress-induced cell death

Rajesh K. Sharma
Abstract. Purpose:, The cytotoxic effects of oxidative stress, which play an important role in ocular diseases, are well known. In this study, we investigated the effect of non-lethal doses of oxidative stress on various cell functions, namely cell viability, cell attachment and cell migration in a widely used retinal pigment epithelium (RPE) cell line (ARPE-19). Methods:, A single exposure to various concentrations of hydrogen peroxide (H2O2) was used to establish a dose response for H2O2 -induced cell death. Other cellular responses, such as changes in cell attachment and migration, were monitored after exposure to increasing doses. Finally, the effects of preconditioning cells with increasing non-lethal doses of H2O2, with and without a subsequent exposure to lethal doses of H2O2, were determined. Results:, The optimum dose for inducing cell death in ARPE-19 cells was between 900 and 1000 ,m H2O2. Preconditioning the cells with 1, 10 and 50 ,m of H2O2 provided a dose-dependent protection against cell death induced by a lethal dose (900,1000 ,m) of H2O2. Preconditioning with higher doses caused cells to become more susceptible to the cytotoxic effects of the lethal dose. Although H2O2 increased cell attachment in lower doses, it induced a dose-dependent inhibition of cell attachment to the substrate in higher doses. H2O2 did not affect cell migration in sub-lethal doses. Conclusion:, Preconditioning RPE cells with limited exposure to non-lethal oxidative stress confers significant protection against subsequent H2O2 -induced cell death. It also affects cell attachment in a dose-specific manner. This finding may help in understanding the pathogenesis of diseases in which oxidative stress plays an important role and in determining the suitability of certain treatment strategies, in particular RPE transplantation in the treatment of age-related macular degeneration. [source]

In vivo Remote Delivery of DNA Encoding for Hypoxia-inducible Factor 1 Alpha Reduces Myocardial Infarct Size

Gabor Czibik M.D.
Abstract We tested if remote gene delivery of hypoxia-inducible factor 1 alpha (HIF-1,) protected hearts against induced ischemia, hypothesizing that gene delivery into skeletal muscle may lead to secretion of proteins with actions elsewhere. Murine quadriceps muscles were transfected with DNA encoding for human HIF-1,, which resulted in a local, but lasting expression (mRNA and protein, where the latter had nuclear localization). Subjection of isolated hearts to global ischemia and reperfusion 1, 4, and 8 weeks after gene delivery resulted in infarct size reduction (p < 0.05). Supporting that this was due to paracrine effects, HL-1 cells treated with conditioned media from cells transfected with HIF-1, or serum from HIF-1,-treated mice were protected against H2O2 -induced cell death (p < 0.05, respectively). The latter protection was reduced when a heme oxygenase activity blocker was used. Taqman low-density array of 47 HIF-1,-regulated genes at the treatment site showed nine specific upregulations (p < 0.05). Of the corresponding proteins, PDGF-B and adrenomedullin were upregulated in the heart. HIF-1, treatment induced an increased vascularization of the heart and skeletal muscle. In conclusion, remote delivery of DNAfor HIF-1, was cardioprotective, represented by consistent infarct size reduction, which may be due to release of paracrine factors from the transfected muscle. [source]