Active Caspase-3 Assay Research Articles

Galectin-3 protects distal convoluted tubules in rhabdomyolysis-induced kidney injury

Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.

Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line.

Colon cancer is a major cause of morbidity and mortality in the world. Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim, and it has various pharmacological effects such as antiviral, antibacterial, and anticancer. In our study, we aimed to investigate the effect of juglone on CCL-228-SW 480 colon carcinoma cell line in monolayer and spheroid culture medium. The CCL-228-SW 480 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. ID50 inhibition was determined on the dose for juglone and after it was found 20 μM was applied to the cells to examine the effect of juglone on CCL-228-SW 480 colon carcinoma cell line. After Juglone was applied the BrdU marking index, Transferase dUTP Nick ends Labeling (TUNEL) assay, active caspase-3 assay, apoptosis-inducing factor (AIF) assay were determined by immunohistochemistry in both the monolayer and spheroid cultures. The control group had a healthy pattern of S-phase fraction, and many of the CCL-228-SW 480 cells nuclei were observed to be positive for BrdU. Terminal Deoxynucleotidyl TUNEL-positive cells, active Caspase-3, and AIF were detected after treatment with juglone in both the monolayer and spheroid cultures. The dead cell count was higher in the CCL-228-SW 480 cell lines with juglone applied than in the controls. Juglone significantly inhibits the proliferation and induces the apoptosis of CCL-228-SW 480 cells in vitro.

Pentoxifylline-Induced Apoptosis in Chronic Lymphocytic Leukemia: New Insights into Molecular Mechanism.

Chronic lymphocytic leukemia (CLL) is an indolent B-lineage neoplasm, characterized by clonal expansion of CD5 positive B cells with constitutive activation of survival pathways including NF-kB. Pentoxifylline, a xanthine-derivative compound indicated for the treatment of microvascular disturbancies, has been suggested to have anti-proliferative and anti-metastatic activities in various types of cancer. In the present study we extend these data showing one of the potential molecular mechanisms through which Pentoxifylline may promote apoptosis in CLL clonal lymphocytes. Peripheral blood mononuclear cells were isolated from 15 CLL patients 0 RAI stage and 15 healthy volunteers and treated for 24 and 48 hours with Pentoxifylline. Apoptosis induction was evaluated through Annexin V and TUNEL assays. Mitochondrial membrane potential depolarization analysis, active Caspase-3 assay, reactive oxygen species generation and Western Blot were assessed to further investigate the alterations induced by Pentoxifylline. We observed a statistically significant occurrence of apoptosis, DNA fragmentation and active Caspase-3 in lymphocytes from CLL patients compared to healthy volunteers after 48 hours of Pentoxifylline treatment. To clarify the molecular mechanism of the drug, we also evaluated the expression levels of NF-kB/p65 and its related proteins. In treated CLL cells, NF-kB/p65 was significantly decreased in comparison to normal cells, whereas we observed a less marked reduction of Bcl-2 expression. The treatment also induced a decrease of Mcl-1 in CLL cells with a greater down-regulation of the anti-apoptotic alternatively spliced isoform. These findings showed that Pentoxifylline induced apoptosis in leukemic cells through a molecular mechanism that involves the NF-kB signaling.

Jatrophane diterpenes and cancer multidrug resistance – ABCB1 efflux modulation and selective cell death induction

BackgroundModulation of P-glycoprotein (ABCB1) and evaluation of the collateral sensitivity effect are among the most promising approaches to overcome multidrug resistance (MDR) in cancer. In a previous study, two rare 12,17-cyclojatrophanes (1–2) and other novel jatrophanes (3–4), isolated from Euphorbia welwitschii, were screened for collateral sensitivity effect. Herein, the isolation of another jatrophane (5) is presented, being the broader goal of this work to investigate the role of euphowelwitschines A (1) and B (2), welwitschene (3), epoxywelwitschene (4) and esulatin M (5) as ABCB1 modulators and/or collateral sensitivity agents. MethodsCompounds 1–5 were evaluated for ABCB1 modulation ability through combination of transport and chemosensitivity assays, using a mouse T-lymphoma MDR1-transfected cell model. Moreover, the nature of interaction of compound 4 with ABCB1 was studied, using an ATPase assay. The MDR-selective antiproliferative activity of compound 5 was evaluated against gastric (EPG85-257) and pancreatic (EPP85-181) human cancer cells and their drug-selected counterparts (EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, EPP85-181RNOV). The drug induced cell death was investigated for compounds 4 and 5, using the annexin V/PI staining and the active caspase-3 assay. ResultsThe jatrophanes 1–5 were able to modulate the efflux activity of ABCB1, and at 2µM, 3–5 maintained the strong modulator profile. Structure activity results indicated that high conformational flexibility of the twelve-membered ring of compounds 3–5 favored ABCB1 modulation, in contrast to the tetracyclic scaffold of compounds 1 and 2. The effects of epoxywelwitschene (4) on the ATPase activity of ABCB1 showed it to interact with the transporter and to be able to reduce the transport of a second subtrate. Drug combination experiments also corroborated the anti-MDR potential of these diterpenes due to their synergistic interaction with doxorubicin (combination index <0.7). Esulatin M (5) showed a strong MDR-selective antiproliferative activity against EPG85-257RDB and EPP85-181RDB cells, with IC50 of 1.8 and 4.8 µM, respectively. Compounds 4 and 5 induced apoptosis via caspase-3 activation. A significant discrimination was observed between the resistant cell lines and parental cells. ConclusionsThis study strengthens the role of jatrophane diterpenes as lead candidates for the development of MDR reversal agents, higlighting the action of compounds 4 and 5.

Liraglutide prevents beta-amyloid-induced neurotoxicity in SH-SY5Y cells via a PI3K-dependent signaling pathway

Objectives: The aim of the study was to investigate the effects of the GLP-1 analog liraglutide on beta-amyloid (Aβ)-induced neurotoxicity in the human neuroblastoma cell line SH-SY5Y and study the underlying mechanisms.Methods: Cultured SH-SY5Y cells in vitro were randomly divided into normal control group, beta-amyloid (Aβ) group (20, 40, and 80 uM), and liraglutide pre-treatment group (10, 100, and 200 nM). Cell viability was determined by CCK-8 and lactate dehydrogenase (LDH). Based on its higher protection potentials, the effect of the liraglutide (100 nM) and wortmannin (200 nM) on beta-amyloid (Aβ) group (40 uM) damage in human SH-SY5Ycells was examined by DAPI fluorescence staining and flow cytometry. Caspase-3, Bcl-2, Bax, Cyt-C, Akt, and P-Akt expression were detected by western blotting.Results: We found that exposure of SH-SY5Y to Aβ (25–35)-induced cytotoxicity, increased lactate dehydrogenase (LDH) leakage, and cellular apoptosis. Interestingly, pre-treatment with liraglutide reversed these reactions. Liraglutide afforded protection against Aβ (25–35)-induced toxicity by inhibiting apoptosis, which was also confirmed by the activated caspase-3 assay. P-Akt and Bcl-2/Bax expression increased after pre-treatment with liraglutide in SH-SY5Y cells exposed to Aβ (25–35), whereas cytochrome-c release decreased. This effect could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase).Discussion: These findings suggest that liraglutide prevented Aβ (25–35)-induced neurotoxicity by inhibiting neuronal apoptosis and liraglutide may have a neuroprotective effect through activation of the PI3K/Akt signaling pathway. Thus, liraglutide may be a preventive or therapeutic agent for Alzheimer’s disease.

Puerarin protects against β-amyloid-induced microglia apoptosis via a PI3K-dependent signaling pathway.

Puerarin extracted from Radix puerariae is well-known for its anti-oxidative and neuroprotective activities. In this study, we investigated the protective effect of puerarin on amyloid-β protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that pretreatment with puerarin afforded protection against Aβ-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. This result was also confirmed by the activated caspase-3 assay. Phospho-Akt and Bcl-2 expression increased after pretreatment with puerarin in BV-2 and primary microglial cells exposed to Aβ, whereas Bax expression and cytochrome c release decreased. In addition, puerarin treatment prevented the loss of mitochondrial membrane potential and reactive oxygen species production. Interestingly, these effects of puerarin against Aβ insult were abolished by LY294002, an inhibitor of PI3K phosphorylation. Taken together, these findings suggest that puerarin prevents Aβ-induced microglial apoptosis via the activation of PI3K/Akt signaling pathway, and might be a potential preventive or therapeutic agent for Alzheimer's disease.

The DNA methyl transferase inhibitor, 5′-aza-2-deoxycitidine, enhances the apoptotic effect of Mevastatin in human leukemia HL-60 cells

Statins induce antiproliferative effects and apoptotic response in various cancer cell types. Moreover, they also sensitize tumor cell lines from different origins to many agents. We aimed to investigate possible effects of Mevastatin (Mev) alone and sequential treatment of 5'-aza-2-deoxycitidine (DAC) and Mev on HL-60 cell line using XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay, lactate dehydrogenase release assay, flourescence microscopy, DNA fragmentation analysis, determination of DNA synthesis rate, and active caspase-3 assay. Messenger RNA (mRNA) expression of apoptotic and antiapoptotic genes were also evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) for BAX, BCL2, and XIAP genes and quantitative Real-time PCR for CASP3, CASP8, and CASP9 genes. We showed that treatment with Mev alone and DAC followed by Mev resulted in apoptotic response in a time- and dose-dependent manner. We also found that pretreatment with DAC sensitized HL-60 cells to Mev and caused more apoptotic cell death than Mev-alone treatment via caspase-3 activation and DNA fragmentation. Moreover, sequential addition of Mev after DAC diminished DNA synthesis rate more effectively than Mev-alone treatment. Furthermore, DAC pretreatment significantly increased CASP3 and CASP9 mRNA expression even with lower doses of Mev. BAX, BCL2, and XIAP gene mRNA levels were also found to be changed in the presence of DAC and Mev. Determination of the exact molecular effects of statins and DAC would allow us to identify new molecular targets to develop more effective treatment regimens for cancer.

Detection and quantification of farnesol-induced apoptosis in difficult primary cell cultures by TaqMan protein assay

Apoptosis can be detected reliably by assaying for cleaved caspase-3, for which active caspase-3 antibodies are used in several methods, such as immunocytochemistry, enzyme-linked immunosorbent assay, and western blot. In this study, we used TaqMan protein assay (TPA), a novel method for protein detection and quantification that detects proteins by amplification of substitute DNA templates. TPA uses antibodies and proximity ligation for quantitative real-time PCR. Meningiomas are primarily benign intracranial tumors. Primary cell cultures of meningiomas are often unsuitable for sensitive protein detection methods. We optimized a TPA to detect active caspase-3 and evaluated its ability to detect farnesol-induced apoptosis in primary meningioma cells. The specificity and sensitivity of the inactive and active caspase-3 assay were determined using recombinant caspase-3. Apoptosis was induced in meningiomas in the presence of 0.2 μM farnesol as shown by immunocytochemistry of single-stranded DNA. Also, viability decreased by over 90 % after treatment with 1.2 μM farnesol for 24 h. The TPA detected a significant increase in active caspase-3 after treatment with 2 and 4 μM farnesol for 2 h, which could not be detected using standard methods such as western blot and immunofluorescence. In addition, TPA determined that meningiomas show disparate sensitivities to low concentrations of farnesol. Caspase-3 expression fell significantly in cells that were treated with 0.25 μM farnesol for 2 h. Further, by TPA, active caspase-3 peaked after 2 h and declined with longer incubation times. This study demonstrates that cleaved caspase-3 is detected and quantified reliably in meningiomas by TPA.

NK cells from HCV-infected patients effectively induce apoptosis of activated primary human hepatic stellate cells in a TRAIL-, FasL- and NKG2D-dependent manner

In mouse models it has been shown that natural killer (NK) cells can attenuate liver fibrosis via killing of activated hepatic stellate cells (HSCs) in a NKG2D- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent manner. However, only little data exist regarding interactions of human NK cells with HSCs and their potential role in hepatitis C virus (HCV)-associated fibrogenesis. Therefore, purified NK cells from untreated HCV RNA(+) patients (n=33), interferon-α (IFN-α)-treated patients (n=17) and healthy controls (n=18) were coincubated with activated primary HSCs, and were tested for degranulation (CD107a expression) and secretion of IFN-γ and TNF-α, respectively. Induction of HSC apoptosis was analyzed using an active caspase-3 assay. We found that following coincubation with HSCs a significant increase in CD107a expression could be observed in both NK cells from HCV(+) patients and healthy controls, whereas only negligible secretion of IFN-γ and TNF-α could be detected. More importantly, NK cells from untreated HCV RNA(+) patients were significantly more effective in induction of HSC apoptosis (17.8±9.2%) than NK cells from healthy controls (6.2±2.1%; P<0.0001). Additionally, we observed an inverse correlation of liver fibrosis stage and the ability of NK cells to induce HSC apoptosis. Induction of HSC apoptosis was contact dependent and could partly be blocked by antibodies specific for TRAIL, NKG2D and FasL, respectively. It is noteworthy that NK cells from IFN-α-treated HCV(+) patients displayed the highest capability to kill HSCs (27.6±10.5%). Accordingly, pre-stimulation of NK cells with recombinant IFN-α significantly increased the ability of NK cells to induce cell death in primary HSCs and was dependent on upregulated expression of TRAIL. Here we demonstrate that NK cells from HCV-infected patients are highly efficient in inducing apoptosis of activated HSCs. Thus, NK cells may have an important anti-fibrotic role in chronic hepatitis C.

Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity in PC12 cells via a PI3K-dependent signaling pathway

Epidemiological data have indicated that estrogen replacement therapy (ERT) can decrease the risk of developing Alzheimer's disease (AD). Phytoestrogens have been proposed as potential alternatives to ERT. The aim of the present study was to assess the neuroprotective effects of puerarin, a phytoestrogen isolated from Pueraria lobata, against the toxicity of beta-amyloid (Aβ) in relation to the mitochondria-mediated cell death process, and to elucidate the role the activation of Akt and modulation of the pro- and antiapoptotic proteins in puerarin-induced neuroprotection. The present study shows that puerarin afforded protection against Aβ-induced toxicity through inhibiting apoptosis in PC12 cells. This result was also confirmed by the activated caspase-3 assay. P-Akt, Bcl-2 and p-Bad expression increased after pretreatment with puerarin in PC12 cells exposed to Aβ 25–35, whereas Bax expression and cytochrome c release decreased. Interestingly, these effects of puerarin against Aβ 25–35 insult were abolished by wortmannin, an inhibitor of PI3K phosphorylation. These findings suggest that puerarin prevent Aβ-induced neurotoxicity through inhibiting neuronal apoptosis, and might be a potential preventive or therapeutic agent for AD.

Effects of a high daily dose of soy isoflavones on DNA damage, apoptosis, and estrogenic outcomes in healthy postmenopausal women

A phase I double-blind clinical trial was conducted to evaluate the effects of a high oral dose of soy isoflavones administered daily for 84 days to healthy postmenopausal women. Principal outcome measures included DNA damage, apoptosis, and changes indicative of estrogenic stimulation. After eligibility and equol-producer status were determined, stratified randomization was used to assign women to the isoflavone (active) or placebo group. Of the 30 women who completed the study, 18 were in the active group. DNA damage was assessed via COMET and apurinic/apyrimidinic site assays in lymphocytes. Apoptosis was evaluated via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and activated caspase-3 assays in lymphocytes. Estrogenic/antiestrogenic effects were assessed using a self-report questionnaire and by assaying for estrogen, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin in blood. In treated postmenopausal women, there was no indication that high doses of soy isoflavones caused DNA strand breakage, increased apurinic/apyrimidinic sites, or increased apoptosis in peripheral lymphocytes. There were no significant changes in mean values for estrogenic effects or other laboratory measurements. Very few adverse events occurred, and the only drug-related adverse events were mild or grade 1 in severity. Unconjugated soy isoflavones appear to be safe and well tolerated in healthy postmenopausal women at doses of 900 mg/day.

Effects of a high daily dose of soy isoflavones on DNA damage, apoptosis and estrogenic outcomes in healthy, post‐menopausal women ‐ a Phase I clinical trial

Objective—A Phase I double blinded clinical trial was conducted to evaluate the effects of a high oral dose of soy isoflavones administered daily for 84 days to healthy, postmenopausal women. Principal outcome measures included DNA damage, apoptosis and changes indicative of estrogenic stimulation. Design—After eligibility and equol-producer status were determined, stratified randomization was used to assign subjects to the isoflavone (active) or placebo group. Of the 30 subjects who completed the study, 18 were in the active group. DNA damage was assessed via COMET and apurinic/ apyrimidinic site assays in lymphocytes. Apoptosis was evaluated via TUNEL and activated Caspase-3 assays in lymphocytes. Estrogenic/antiestrogenic effects were assessed using a self-report questionnaire and by assaying for estrogen, follicle-stimulating hormone, luteinizing hormone, and sex hormone binding globulin in blood. Results—In treated postmenopausal women there was no indication that high doses of soy isoflavones caused DNA strand breakage, increased AP sites, or increased apoptosis in peripheral lymphocytes. There were no significant changes in mean values for estrogenic effects or other laboratory measurements. Very few adverse events occurred and the only drug-related adverse events were mild or grade 1 in severity. Conclusions—Unconjugated soy isoflavones appear to be safe and well tolerated in healthy postmenopausal women at doses of 900 mg per day.

Choline deficiency increases lymphocyte apoptosis and DNA damage in humans

Whereas deficiency of the essential nutrient choline is associated with DNA damage and apoptosis in cell and rodent models, it has not been shown in humans. The objective was to ascertain whether lymphocytes from choline-deficient humans had greater DNA damage and apoptosis than did those from choline-sufficient humans. Fifty-one men and women aged 18-70 y were fed a diet containing the recommended adequate intake of choline (control) for 10 d. They then were fed a choline-deficient diet for up to 42 d before repletion with 138-550 mg choline/d. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated. DNA damage and apoptosis were then assessed by activation of caspase-3, terminal deoxynucleotide transferase-mediated dUTP nick end-labeling, and single-cell gel electrophoresis (COMET) assays. All subjects fed the choline-deficient diet had lymphocyte DNA damage, as assessed by COMET assay, twice that found when they were fed the control diet. The subjects who developed organ dysfunction (liver or muscle) when fed the choline-deficient diet had significantly more apoptotic lymphocytes, as assessed by the activated caspase-3 assay, than when fed the control diet. A choline-deficient diet increased DNA damage in humans. Subjects in whom these diets induced liver or muscle dysfunction also had higher rates of apoptosis in their peripheral lymphocytes than did subjects who did not develop organ dysfunction. Assessment of DNA damage and apoptosis in lymphocytes appears to be a clinically useful measure in humans (such as those receiving parenteral nutrition) in whom choline deficiency is suspected.

Volatile Anesthetic Preconditioning Attenuates Myocardial Apoptosis in Rabbits after Regional Ischemia and Reperfusion via Akt Signaling and Modulation of Bcl-2 Family Proteins

We tested whether isoflurane preconditioning inhibits cardiomyocyte apoptosis and evaluated the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic preconditioning and determined whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in anesthetic preconditioning. Six-month-old New Zealand rabbits subjected to 40 min of myocardial ischemia followed by 180 min of reperfusion were assigned to the following groups: ischemia-reperfusion (I/R), isoflurane preconditioning and isoflurane plus PI3K inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002) (0.6 and 0.3 mg/kg i.v., respectively). Sham-operated, wortmannin+I/R, wortmannin+sham, LY294002+I/R, and LY294002+sham groups were also included. Infarct size was assessed by triphenyltetrazolium chloride staining. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and activated caspase-3 assays. Akt phosphorylation, Bax, Bcl-2, Bad, and phosphorylated Bad (phospho-Bad) expression was assessed by immunoblotting. Isoflurane preconditioning reduced infarct size compared with the I/R group: 22+/-4 versus 41+/-5% (p<0.05). The percentage of apoptotic cells decreased in the isoflurane group (3.8+/-1.2%) compared with the I/R group (12.4+/-1.6%; p<0.05). These results were also confirmed by the activated caspase-3 assay. Wortmannin and LY294002 inhibited the effects of isoflurane. Myocardial infarction increased to 44+/-3 and 45+/-2% and the percentage of apoptotic cells was 11.9+/-2.1 and 11.7+/-3.3%, respectively. Akt phosphorylation and Bcl-2 and phospho-Bad expression increased after isoflurane preconditioning, whereas Bax expression decreased. These effects were inhibited by wortmannin and LY294002. The data indicate that isoflurane preconditioning reduces infarct size and myocardial apoptosis after I/R. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play a role in isoflurane-induced myocardial protection.

TEMPORAL‐SPATIAL REGULATION OF APOPTOSIS IN EXPERIMENTAL BILIARY ATRESIA

Background: Biliary atresia results from a fibrosing cholangitis that leads to bile duct obstruction. Recent studies using a mouse model of rhesus rotavirus (RRV)-induced biliary atresia demonstrate increased TNF-alpha levels in liver. Based on the established role of TNF-alpha in apoptosis, we hypothesized that RRV triggers bile duct apoptosis in experimental biliary atresia. Methods: Balb/c mice were inoculated with RRV or saline on day 1 of life, and the liver and EHBD were studied at days 2-18. Hepatic mononuclear cells were isolated for gene expression analysis by real-time PCR. TUNEL and active caspase-3 assays were performed on paraffin-embedded liver and EHBD sections, while mRNA expression of caspases was assessed using snap-frozen samples. Results: RRV infection resulted in obstruction of the EHBD after day 6. At this time, there was a marked increase in mRNA expression of FasL, perforin, and granzyme B in hepatic mononuclear cells compared to controls (p < 0.02). From days 6-18, intrahepatic portal tracts consistently showed higher numbers of TUNEL-positive cells than controls (p < 0.02). To investigate temporal dynamics of apoptosis in the EHBD, we counted TUNEL-positive cells at days 2-14. TUNEL labeling was localized to duct epithelial cells at day 4 and peaked in cells occluding the duct lumen at day 5 (mean ± SD: 20.9 ± 4.3 vs. 0.53 ± 0.29; p < 0.01). The active caspase-3 assay confirmed these results, showing maximal cell staining at day 5. mRNA expression of caspases-1 and -4 was higher in both liver and EHBD, with fold changes ranging from 2.0-7.4. Conclusions: RRV challenge induces: 1) the expression of pro-apoptotic molecules in hepatic mononuclear cells, 2) infiltration of portal tracts with TUNEL-labeled cells, and 3) a peak in TUNEL and active caspase-3 labeling in the EHBD at the time of obstruction. These results support a pathogenic role for apoptosis in experimental biliary atresia.

Lactate dehydrogenase-elevating virus induces apoptosis in cultured macrophages and in spinal cords of C58 mice coincident with onset of murine amyotrophic lateral sclerosis

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2–3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.

The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.