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Bax
Kinds of Bax Terms modified by Bax Selected AbstractsSuppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposureENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2009Melisa Bunderson-Schelvan Abstract The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1,, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma-type cancers. Environ. Mol. Mutagen. 2009. © 2009 Wiley-Liss, Inc. [source] Maintenance of the relative proportion of oligodendrocytes to axons even in the absence of BAX and BAKEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2009Kumi Kawai Abstract Highly purified oligodendroglial lineage cells from mice lacking functional bax and bak genes were resistant to apoptosis after in-vitro differentiation, indicating an essential role of the intrinsic apoptotic pathway in apoptosis of oligodendrocytes in the absence of neurons (axons) and other glial cells. These mice therefore provide a valuable tool with which to evaluate the significance of the intrinsic apoptotic pathway in regulating the population sizes of oligodendrocytes and oligodendroglial progenitor cells. Quantitative analysis of the optic nerves and the dorsal columns of the spinal cord revealed that the absolute numbers of mature oligodendrocytes immunolabeled for aspartoacylase and adult glial progenitor cells expressing NG2 chondroitin sulfate proteoglycan were increased in both white matter tracts of adult bax/bak -deficient mice and, to a lesser extent, bax -deficient mice, except that there was no increase in NG2-positive progenitor cells in the dorsal columns of these strains of mutant mice. These increases in mature oligodendrocytes and progenitor cells in bax/bak -deficient mice were unexpectedly proportional to increases in numbers of axons in these white matter tracts, thus retaining the oligodendroglial lineage to axon ratios of at most 1.3-fold of the physiological numbers. This is in contrast to the prominent expansion in numbers of neural precursor cells in the subventricular zones of these adult mutant mice. Our study indicates that homeostatic control of cell number is different for progenitors of the oligodendroglial and neuronal lineages. Furthermore, regulatory mechanism(s) operating in addition to apoptotic elimination through the intrinsic pathway, appear to prevent the overproduction of highly mitotic oligodendroglial progenitor cells. [source] No evidence for association of the TP53 12139 and the BAX,248 polymorphisms with endemic pemphigus foliaceus (fogo selvagem)INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 2 2006K. F. Köhler Summary Pemphigus foliaceus (PF) is an autoimmune bullous epidermal disease, characterized by autoantibodies specific to the desmosomal protein desmoglein 1 (dsg1) and by acantholysis, the rupture of the cellular junctions among keratinocytes. Known also as fogo selvagem (wild fire) in Brazil, the disease has distinct epidemiological characteristics, being endemic in certain regions of South America. It is a multifactorial (complex) disease, with oligo- or polygenic disease susceptibility. In view of the previously reported evidences of a role for apoptosis dysregulation in pemphigus pathogenesis, we hypothesized that genetic variants of molecules participating in apoptosis may contribute to interindividual variation of susceptibility to PF. The TP53 12139(G,C) and the BAX,248(G,A) single nucleotide polymorphisms (SNP) were analysed in a genetic association study. The allelic, genotypic and allele carrier frequencies for these SNPs did not differ statistically between the patient and the control groups, for both the Euro- and the Afro-Brazilian population strata. The results of this study lead us to conclude that, although the TP53 and BAX alleles analysed differ functionally, this variation does not alter the functionality of the molecules in a way that would interfere with the development of the disease. [source] Pro-apoptotic activity of transiently expressed BCL-2 occurs independent of BAX and BAKJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2003T. Subramanian Abstract BCL-2 suppresses apoptosis induced by a wide variety of stimuli in multiple cell types. Most of the in vitro studies that have examined the activity of BCL-2 have employed stable cell lines that ectopically express BCL-2. We have reported that BCL-2 is expressed at high levels in the absence of the 5,- and 3,-UTRs of the Bcl-2 gene and transient high level of expression results in potent cell death (Uhlmann et al., [1998]: JBC 278:17926,17932). Expression of BCL-2 under the transcriptional control of the cognate 5,- and 3,-UTRs express lower levels of BCL-2 and does not cause cell death. Our present results suggest that in contrast to BCL-2, transient expression of BCL-xL does not induce cell death and coexpression of BCL-xL with the pro-apoptotic BCL-2 does not suppress cell death. The pro-apoptotic activity of BCL-2 appears to involve activation of the cytochrome c/caspase 9/caspase 3 pathway. Elevated levels of BCL-2 expression results in N-terminal cleavage of BCL-2 at a novel site different from a previously identified caspase cleavage site at Asp 34 by a non-caspase protease. Transient expression of a BCL-2 mutant lacking aa 51,85 within the loop region induces efficient cell death and N-terminal cleavage of BCL-2 while a different deletion mutant lacking aa 30,91 induces reduced levels of cell death in the absence of BCL-2 cleavage suggesting that N-terminal processing of BCL-2 may be an amplification event in BCL-2-mediated cell death. Overexpression of BCL-2 in a Bax-null human colon cancer cell line (HCT116Bax,/,) induces efficient cell death. The pro-apoptotic activity of BCL-2 is also observed in a Bax-null cells in which BAK expression is inhibited by stable RNAi expression. Our results suggest that BCL-2 contains an intrinsic pro-apoptotic activity and can induce apoptosis independent of BAX and BAK under specific conditions. © 2003 Wiley-Liss, Inc. [source] Evaluation of a Polymerase Chain Reaction,Based System for Detecting Salmonella Species from Pork Carcass Sponge SamplesJOURNAL OF FOOD SCIENCE, Issue 3 2003Chih-Chuan Wu ABSTRACT: Pork carcass sponge samples (n = 230) collected from 10 Taiwanese slaughter plants were screened for Salmonella using 2 methods: BAX® for Screening/Salmonella, a polymerase chain reaction-based detection system and a culture method using SM-ID agar as the selective plating medium. The BAX method identified 14 samples as positive for Salmonella. The 8 samples identified as Salmonella-positive by the SM-ID method were also BAX-positive. Inoculation studies showed BAX detected Salmonella in samples having initial 1.4 × 101 cfu/mL Salmonella inoculum prior to the enrichment process in the presence of 3.0 × 106 cfu/mL of non- Salmonella florae. BAX provides a rapid screening alternative to the culture method for Salmonella detection. [source] Apoptosis-associated gene expression in HIV-infected patients in response to successful antiretroviral therapy,JOURNAL OF MEDICAL VIROLOGY, Issue 2 2007Emanuela Balestrieri Abstract The simultaneous expression of 19 apoptosis-related genes was analyzed by RNA-protection assay in peripheral blood mononuclear cells of HIV-infected patients before and during successful antiretroviral therapy (ART). After 12 months of therapy, the expression of the pro-apoptotic genes FAS, FAS-L, FAF-1, FADD, CASPASE-8, DR3, TRAIL, TNFR-1, TRADD, and BAX was significantly downregulated with respect to time 0, while that of BCL-2, BCL-XL, and MCL-1 was significantly upregulated. The data suggest that inhibition of cell death in HIV-positive patients under successful therapy is the result of a complex network of multifactor signaling, correlated with both death and survival of lymphocytes. J. Med. Virol. 79:111,117, 2007. © 2006 Wiley-Liss, Inc. [source] LOCALISATION OF BAX, BCL-2 AND FAS mRNA IN A MODEL OF FOCAL AND SEGMENTAL GLOMERULOSCLEROSISNEPHROLOGY, Issue 3 2000Wang W [source] Microsatellite instability and alteration of E2F-4 gene in adenosquamous and squamous cell carcinomas of the stomachPATHOLOGY INTERNATIONAL, Issue 9 2000Dong Kyun Woo Microsatellite instability (MSI) due to defective DNA mismatch repair (MMR) is a form of genomic instability underlying the tumorigenesis of various human neoplasms. To evaluate the roles of MSI in the pathogenesis of gastric carcinomas with squamous differentiation, 17 primary stomach cancer patients (15 adenosquamous and two squamous cell carcinomas) were examined for MSI frequency using five microsatellite markers and the criteria for MSI recommended by the National Cancer Institute Workshop. The molecular causes and consequences of MSI in these neoplasms were further researched through the immunohistochemistry of MMR proteins and the mutational analysis of cancer-associated genes targeted by MSI, respectively. Two of the 17 (12%) cases demonstrated MSI at the most examined loci and were classified as having high level MSI (MSI-H). These tumors also exhibited frame-shift mutations at mononucleotide repeats in the target genes, including TGF,RII, IGFIIR, BAX, and hMSH6. It is interesting to note that the mutations of the serine (AGC)13 repeats within the E2F-4 gene were found only in the squamous cell carcinoma portions of them, whereas such alterations were not detected in any of the adenocarcinomatous portions. This suggests that E2F-4 might be implicated in the transformation of adenocarcinoma into squamous cell carcinoma and further studies are needed to understand its role in squamous differentiation. [source] Coptis japonica root extract induces apoptosis through caspase3 activation in SNU-668 human gastric cancer cellsPHYTOTHERAPY RESEARCH, Issue 3 2005H. J. Park Abstract Apoptosis-modulating approaches offer an attractive opportunity for therapeutic use for many tumors. We investigated the effects of the roots of Coptis japonica var. dissecta (Ranunculaceae) on human gastric cancer cells, SNU-668. The cytotoxicity of Coptis japonica at 100 µg[sol ]ml (methanol extract) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 13.89 ± 1.91% of control value. Considering the features by 4,6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, it was confirmed that the death of SNU-668 cells was due to apoptosis. In the apoptosis-regulating genes, BCL2 expression was diminished out, whereas BAX and CASP3 expressions were increased, compared with control. Furthermore, the activity of caspase3 was significantly increased by Coptis japonica treatment. These results suggest that Coptis japonica could induce apoptotic anticancer effect through caspase3 activation on SNU-668 human gastric cancer cells. Copyright © 2005 John Wiley & Sons, Ltd. [source] Formalin fixation and immunoreactivity in prostate cancer and benign prostatic tissuesAPMIS, Issue 5 2010SARA JONMARKER JARAJ Jaraj SJ, Egevad L. Formalin fixation and immunoreactivity in prostate cancer and benign prostatic tissue. APMIS 2010; 118: 383,8. For better fixation, formalin injection of radical prostatectomy (RP) specimens has been suggested. We aimed to assess its effect on immunoreactivity using immunohistochemistry (IHC). A tissue microarray of cancer and benign tissues from 42 RP specimens was constructed. Twenty-one of the prostates had been injected with formalin prior to formalin immersion. IHC staining was performed using 15 antibodies, including nuclear and cytoplasmic markers known to be positive in prostate tissue: pan cytokeratin, P504S, high molecular weight (HMW) keratin, PSA, vimentin, actin HHF35, thioredoxin-1, peroxiredoxin-2, PDX-1, BAX, p27, androgen receptor (AR) and heat shock proteins (HSP) 27, 60 and 70. Differences in staining intensity in cancer and benign tissues were compared separately except for HMW keratin. Only 7 of 29 analyses showed significant differences between groups, including 5 of 15 antibodies. The expression of AR and HSP 27 was stronger in formalin-injected tissue, while the opposite was true for HSP 60, HSP 70 and peroxiredoxin-2. For most antibodies, formalin injection does not significantly affect immunoreactivity in prostate tissue. The staining variability caused by inter- and intratumoral heterogeneity may be greater than that caused by the fixation method. [source] BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Gregory J. Cost Abstract Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX - and BAK -deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production. Biotechnol. Bioeng. 2010; 105: 330,340. © 2009 Wiley Periodicals, Inc. [source] Identification of survival-related genes of the phosphatidylinositol 3,-kinase signaling pathway in glioblastoma multiformeCANCER, Issue 7 2008Yolanda Ruano BcSc Abstract BACKGROUND Knowledge of the molecular mechanisms involved in the biology of glioblastoma multiforme (GBM) is essential for the identification of candidate prognostic markers, new putative therapeutic targets, and early detection strategies predictive of survival. METHODS The authors performed expression-profiling analyses in a series of primary GBMs by using complementary DNA microarrays. Validation of putative targets was performed in large series of GBMs by immunohistochemistry on tissue microarrays, real-time quantitative reverse transcription-polymerase chain reaction analysis, and Western blot analysis. RESULTS The expression signature consisted of 159 up-regulated genes and 186 down-regulated genes. Most of these genes were involved in cell adhesion, signal transduction, cell cycle, apoptosis, and angiogenesis. Among the genes from the molecular signature, annexin 1 (ANXA1) and ubiquitin-specific protease 7 (USP7) were evaluated in wider series of GBMs. ANXA1 analysis carried out in different types of gliomas revealed exclusive overexpression in astrocytomas. Furthermore, survival analysis by using functional clusters of genes related with cancer and glioma biology revealed 7 genes involved in the PI3K-signaling pathway that presented a significant association with clinical outcome. Among these genes, positive expression of BCL2-associated X protein (BAX) was associated significantly with better survival in a larger series of tumors. In addition, activation of the PI3K/Akt pathway was demonstrated in this set of GBMs. CONCLUSIONS The authors concluded that there is a significant role for PI3K pathway survival-related genes in patients with GBM, and putative prognostic markers associated with glioma tumorigenesis were identified. The detailed study of these candidate genes and the molecular pathways regulating PI3K activation reveal that they are promising targets for the clinical management of patients with glioma. Cancer 2008. © 2008 American Cancer Society. [source] Increased MCL,1 Expression Is Associated with Poor Prognosis in Ovarian CarcinomasCANCER SCIENCE, Issue 5 2002Kazushi Shigemasa To investigate the potential role of the BCL,2 gene family (BAX, BCL,2, MCL,1, and BCL-XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi-quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL,1 in ovarian tumors was also examined. The expression levels of BAX and MCL,1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P<0.05). In contrast, the BCL,2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P<0.05). Expression of BCL-XL mRNA was no different between normal ovaries and ovarian tumors. Log-rank testing showed that low BAX mRNA expression and high MCL,1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P=0.05; MCL,1, P=0.02). Immunohistochemical analysis showed that diffuse-positive expression of MCL,1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P=0.03). In ovarian cancer cases, diffuse-positive expression of MCL,1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P<0.01; grade, P=0.01; survival, P=0.01). These results suggest that increased MCL,1 expression may play an important role in replacing the functions of increased BAX and decreased BCL,2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer. [source] Coenzyme Q10 prevents human lens epithelial cells from light-induced apoptotic cell death by reducing oxidative stress and stabilizing BAX,/,Bcl-2 ratioACTA OPHTHALMOLOGICA, Issue 3 2010Marcus Kernt Abstract. Background:, Cataract is one of the most prevalent eye disease and a major cause for legal blindness in the world. Beside others, cumulative light-exposure and apoptotic cell death are significantly associated with cataract development. In contrast, supplementation with antioxidants has been suggested to prevent premature cataractogenesis. This study investigates possible protective effects of Coenzyme Q10 (CoQ10) regarding light-induced stress and apoptotic cell death in human lens epithelial cells (LEC). Methods:, Human LEC were either pre-incubated with CoQ10 or not and then exposed to white light. After 10,40 min of irradiation viability, induction of intracellular reactive oxygen species (ROS), apoptosis and cell death was determined. Expression of apoptotic BAX and anti-apoptotic Bcl-2 protein and their mRNA were determined by RT-PCR and Western blot analysis. Results:, Light exposure decreased LEC viability and Bcl-2 expression and increased intracellular ROS, apoptotic cell death, and BAX expression in a time-of-irradiation-dependent manner. Phototoxic cell death and apoptosis, as well as decrease of Bcl-2 and increase in BAX expression was significantly reduced, when cells were pre-incubated with CoQ10. Conclusions:, In this study, CoQ10 significantly reduced light-induced LEC-damage and attenuated phototoxic effects on BAX and Bcl-2 expression. Therefore, CoQ10 supplementation might also be useful in preventing LEC death and consecutive cataract formation in vivo. [source] Developmental cell death during Xenopus metamorphosis involves BID cleavage and caspase 2 and 8 activationDEVELOPMENTAL DYNAMICS, Issue 8 2006D. Du Pasquier Abstract Elimination of tadpole organs during Xenopus metamorphosis is largely achieved through apoptosis, and recent evidence suggest involvement of the mitochondrial death route and bax-initiated caspase-3 and -9 deployment. However, events upstream of the activation of Bax are unknown. In other models, proteins of the BH3-only group such as BID are known to assure this function. We show that Xenopus bid transcript levels increase at metamorphosis in larval cells destined to disappear. This increase correlates with an abrupt rise in Caspase-2 and -8 mRNA levels and an enhanced activity of Caspase-2 and -8. In BIDGFP transgenic animal's tail regression is accelerated. The cleavage of BIDGFP fusion protein during natural or T3 -induced metamorphosis was specifically inhibited by caspase-8 inhibitors. Our results show that tail regression at metamorphosis implicates an apoptotic pathway inducible by T3 hormone in an organ autonomous manner and involving the cell death executioners BID and Caspases-2 and -8. Developmental Dynamics 235:2083,2094, 2006. © 2006 Wiley-Liss, Inc. [source] The role of cell death in sexually dimorphic muscle development: Male-specific muscles are retained in female bax/bak knockout miceDEVELOPMENTAL NEUROBIOLOGY, Issue 11 2008Dena A. Jacob Abstract The bulbocavernosus (BC) and levator ani (LA) muscles are present in males but absent or severely reduced in females, and the fate of these muscles controls the survival of motoneurons in the sexually dimorphic spinal nucleus of the bulbocavernosus. However, the mechanism underlying the sex difference in BC and LA development has been controversial. We examined the role of cell death in sexual differentiation of the bulbocavernosus BC/LA muscles in mice. Muscle development was mapped from embryonic day 16 (E16) to postnatal day 5 (P5). A sex difference (male > female) first arose on E17 (BC) or E18 (LA), and increased in magnitude postnatally. TUNEL labeling revealed dying cells in the BC and LA muscles of both sexes perinatally. However, females had a significantly higher density of TUNEL-positive cells than did males. A role for the proapoptotic factors, Bax and Bak, in BC/LA development was tested by examining mice lacking one or both of these proteins. In females lacking either Bax or Bak, the BC was absent and the LA rudimentary. Deletion of both bax and bak genes, however, rescued the BC, increased LA size ,20-fold relative to controls, and virtually eliminated TUNEL-positive cells in both muscles. We conclude that cell death plays an essential role in sexual differentiation of the BC/LA muscles. The presence of either Bax or Bak is sufficient for cell death in the BC/LA, whereas the absence of both prevents sexually dimorphic muscle cell death. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Sex differences in the level of Bcl-2 family proteins and caspase-3 activation in the sexually dimorphic nuclei of the preoptic area in postnatal ratsDEVELOPMENTAL NEUROBIOLOGY, Issue 13 2006Shinji Tsukahara Abstract In developing rats, sex differences in the number of apoptotic cells are found in the central division of the medial preoptic nucleus (MPNc), which is a significant component of the sexually dimorphic nucleus of the preoptic area, and in the anteroventral periventricular nucleus (AVPV). Specifically, male rats have more apoptotic cells in the developing AVPV, whereas females have more apoptotic cells in the developing MPNc. To determine the mechanisms for the sex differences in apoptosis in these nuclei, we compared the expression of the Bcl-2 family members and active caspase-3 in postnatal female and male rats. Western blot analyses for the Bcl-2 family proteins were performed using preoptic tissues isolated from the brain on postnatal day (PD) 1 (day of birth) or on PD8. In the AVPV-containing tissues of PD1 rats, there were significant sex differences in the level of Bcl-2 (female > male) and Bax (female < male) proteins, but not of Bcl-xL or Bad proteins. In the MPNc-containing tissues of PD8 rats, there were significant sex differences in the protein levels for Bcl-2 (female < male), Bax (female > male), and Bad (female < male), but not for Bcl-xL. Immunohistochemical analyses showed significant sex differences in the number of active caspase-3-immunoreactive cells in the AVPV on PD1 (female < male) and in the MPNc on PD8 (female > male). We further found that active caspase-3-immunoreactive cells of the AVPV and MPNc were immunoreactive for NeuN, a neuronal marker. These results suggest that there are sex differences in the induction of apoptosis via the mitochondrial pathway during development of the AVPV and MPNc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Dose and age-dependent axonal responses of embryonic trigeminal neurons to localized NGF via p75NTR receptorDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2005P. Hande Özdinler Abstract Nerve growth factor (NGF) and related neurotrophins are target-derived survival factors for sensory neurons. In addition, these peptides modulate neuronal differentiation, axon guidance, and synaptic plasticity. We tested axonal behavior of embryonic trigeminal neurons towards localized sources of NGF in collagen gel assays. Trigeminal axons preferentially grow towards lower doses of localized NGF and grow away from higher concentrations at earlier stages of development, but do not show this response later. Dorsal root ganglion axons also show similar responses to NGF, but NGF-dependent superior cervical ganglion axons do not. Such axonal responses to localized NGF sources were also observed in Bax,/, mice, suggesting that the axonal effects are largely independent of cell survival. Immunocytochemical studies indicated that axons, which grow towards or away from localized NGF are TrkA-positive, and TrkA,/, TG axons do not respond to any dose of NGF. We further show that axonal responses to NGF are absent in TG derived from mice that lack the p75 neurotrophin receptor (p75NTR). Collectively, our results suggest that localized sources of NGF can direct axon outgrowth from trigeminal ganglion in a dose- and age-dependent fashion, mediated by p75NTR signaling through TrkA expressing axons. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Different apoptotic responses of human and bovine pericytes to fluctuating glucose levels and protective role of thiamineDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2009Elena Beltramo Abstract Background Vascular cells in diabetes are subjected to daily fluctuations from high to low glucose. We aimed at investigating whether pulsed exposure to different glucose concentrations influences apoptosis in human retinal pericytes (HRP) versus bovine retinal pericytes (BRP), with consequences on the onset of diabetic retinopathy, and the possible protective role of thiamine. Methods BRP and HRP (wild-type and immortalized) were grown in physiological/high glucose for 7 days, and then returned to physiological glucose for another 24, 48 or 72 h. Cells were also kept intermittently at 48-h intervals in high/normal glucose for 8 days, with/without thiamine/benfotiamine. Apoptosis was determined through ELISA, TUNEL, Bcl-2, Bax and p53 expression/concentration. Results Continuous exposure to high glucose increased apoptosis in BRP, but not HRP. BRP apoptosis normalized within 24 h of physiological glucose re-entry, while HRP apoptosis increased within 24,48 h of re-entry. Intermittent exposure to high glucose increased apoptosis in HRP and BRP. Bcl-2/Bax results were consistent with DNA fragmentation, while p53 was unchanged. Thiamine and benfotiamine countered intermittent high glucose-induced apoptosis. Conclusions Human pericytes are less prone to apoptosis induced by persistently high glucose than bovine cells. However, while BRP recover after returning to physiological levels, HRP are more vulnerable to both downwardly fluctuating glucose levels and intermittent exposure. These findings reinforce the hypotheses that (1) glycaemic fluctuations play a role in the development of diabetic retinopathy and (2) species-specific models are needed. Thiamine and benfotiamine prevent human pericyte apoptosis, indicating this vitamin as an inexpensive approach to the prevention and/or treatment of diabetic complications. Copyright © 2009 John Wiley & Sons, Ltd. [source] Alteration of proteins expression in apoptotic FL cells induced by MCLRENVIRONMENTAL TOXICOLOGY, Issue 4 2008Ming-Luan Xing Abstract Microcystins (MCs) are a family of monocyclic heptapeptide hepatotoxins produced by freshwater species of cyanobacteria. Microcystin-LR (MCLR) is the most frequently studied and most toxic in over 80 MC congeners. Great deals of studies have demonstrated that MCLR can induce apoptosis in a wide variety of cell types. Although much evidence indicates that mitochondria play a pivotal role in MCLR-induced apoptosis, the complicated apoptosis mechanisms induced by MCLR have not been completely characterized. It is possible that there are other apoptotic pathways existing in MCLR-induced apoptosis. The present study was undertaken to determine the expression of PP2A, CHOP, Bax, Bcl-2, and p53 proteins in MCLR-induced apoptosis in FL cells. The results showed that MCLR could induce apoptosis in FL cells and the process was accompanied with the upregulation of PP2A, Bax, and p53 proteins and the downregulation of Bcl-2 proteins. In addition, the CHOP protein was upregulated at most treatment groups and decreased at the highest concentration group. These results, especially the alteration of PP2A and CHOP proteins might provide new insights into MCLR-induced apoptosis. © 2008 Wiley Periodicals, Inc. Environ Toxicol 2008. [source] Is the Cell Death in Mesial Temporal Sclerosis Apoptotic?EPILEPSIA, Issue 6 2003Hilmi Uysal Summary: Purpose: Mesial temporal sclerosis (MTS) is characterized by neuronal loss in the hippocampus. Studies on experimental models and patients with intractable epilepsy suggest that apoptosis may be involved in neuronal death induced by recurrent seizures. Methods: We searched evidence for apoptotic cell death in temporal lobes resected from drug-resistant epilepsy patients with MTS by using the terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP (TUNEL) method and immunohistochemistry for Bcl-2, Bax, and caspase-cleaved actin fragment, fractin. The temporal lobe specimens were obtained from 15 patients (six women and nine men; mean age, 29 ± 8 years). Results: Unlike that in normal adult brain, we observed Bcl-2 immunoreactivity in some of the remaining neurons dispersed throughout the hippocampus proper as well as in most of the reactive astroglia. Bax immunopositivity was increased in almost all neurons. Fractin immunostaining, an indicator of caspase activity, was detected in ,10% of these neurons. Des pite increased Bax expression and activation of caspases, we could not find evidence for DNA fragmentation by TUNEL staining. We also could not detect typical apoptotic changes in nuclear morphology by Hoechst-33258 or hematoxylin counterstaining. Conclusions: These data suggest that either apoptosis is not involved in cell loss in MTS, or a very slow rate of cell demise may have precluded detecting TUNEL-positive neurons dying through apoptosis. Increased Bax expression and activation of caspases support the latter possibility. [source] NF-,B and apoptosis in colorectal tumourigenesisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2007M. M. Aranha Abstract Background, Nuclear factor-,B (NF-,B) may play an important role in colorectal tumourigenesis, controlling cell cycle and apoptosis gene expression. In addition, imbalances between cell proliferation and cell death are thought to underlie neoplastic development. The aims of this study were to investigate apoptosis and expression of several apoptosis-related proteins, and to determine correlations with colorectal tumour progression. Materials and methods, Apoptosis was evaluated by the TUNEL assay in 48 patient samples, including adenomas, adenocarcinomas and adjacent normal mucosas. Immunohistochemistry was performed for Bcl-2 and NF-,B. Expression levels of p53, Bax and I,B proteins were determined by immunoblotting. Cultured human colon cancer cells were used to evaluate NF-,B expression and nuclear translocation by immunocytochemistry and immunoblotting. Results, Apoptosis and NF-,B immunoreactivity were significantly higher in tumour tissue compared with normal mucosa (P < 0·01), increasing in association with histological tumour progression (P < 0·01). Bcl-2 was consistently higher in normal mucosa (P < 0·01) and inversely correlated with the percentage of apoptosis (P < 0·01). Phosphorylated p53 and Bax levels were similar in tumour tissue and normal mucosa; however, the NF-,B inhibitor, I,B, tended to decrease in tumours. In vitro, nuclear translocation of NF-,B was greater in proliferative than in resting phases of colon cancer cells. Conclusions, NF-,B expression and apoptosis are increased from adenoma to poorly differentiated adenocarcinoma tissues. Apoptosis is correlated with suppression of Bcl-2 expression, but appears to proceed through a p53- and Bax-independent pathway. Activation of NF-,B may play an important role in colorectal tumour progression. [source] Glutathione depletion and cardiomyocyte apoptosis in viral myocarditisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2004V. Kytö Abstract Background, The course of viral myocarditis is highly variable. Oxidative stress and Bcl-2 family genes may play a role in its pathogenesis by regulating the amount of cardiomyocyte apoptosis. Apoptosis is difficult to detect and quantify in vivo. Therefore, we set to look for indicators of this potentially preventable form of cell death during various phases of experimental murine coxsackievirus B3 myocarditis. Methods, BALB/c mice were infected with the cardiotropic coxsackievirus B3 variant. Glutathione (HPLC), cardiomyocyte apoptosis (TUNEL and caspase-3 cleavage), Bax and Bcl-XL mRNA expression (real time RT-PCR), histopathology and viral replication (plaque assay and real time RT-PCR) were measured from day 3 to day 20 after infection. Results, Infection caused severe myocarditis and led to progressive decrease of plasma glutathione levels. Myocardial mRNA levels of pro-apoptotic Bax and antiapoptotic Bcl-XL were significantly increased from day 3 onwards. Bax mRNA and ratio of Bax to Bcl-XL correlated with cardiomyocyte apoptosis (r = 0·77, P = < 0·001 and r 0·51, P < 0·01, respectively). Cardiomyocyte apoptosis was highest on day 5, coinciding with a rapid decline in plasma glutathione (r = ,0·52, P = 0·003). Conclusions, Systemic oxidative stress as indicated by decreased plasma glutathione levels coincides with cardiomyocyte apoptosis in experimental coxsackievirus myocarditis. Decreased plasma glutathione levels and changes in cardiac Bax and Bcl-XL mRNA expression identify a phase of myocarditis in which the potentially preventable cardiomyocyte apoptosis is mostly observed. [source] Activation of p53 signalling in acetylsalicylic acid-induced apoptosis in OC2 human oral cancer cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003C.-C. Ho Abstract Background, Nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid (ASA, aspirin) are well known chemotherapeutic agents of cancers; however, the signalling molecules involved remain unclear. The aim of this study was to investigate the possible existence of a putative p53-dependent pathway underlying the ASA-induced apoptosis in OC2 cells, a human oral cancer cell line. Materials and methods, The methyl tetrazolium (MTT) assay was employed to quantify differences in cell viability. DNA ladder formation on agarose electrophoresis was used as apoptosis assay. The expression levels of several master regulatory molecules controlling various signal pathways were monitored using the immunoblotting techniques. Flow cytometry was used to confirm the effect of ASA on cell cycle. Patterns of changes in expression were scanned and analyzed using the NIH image 1·56 software (NIH, Bethesda, MD, USA). All the data were analyzed by anova. Results, Acetylsalicylic acid reduced cell viability and presence of internucleosomal DNA fragmentation. In the meanwhile, phosphorylation of p53 at serine 15, accumulation of p53 and increased the expression of its downstream target genes, p21 and Bax induced by ASA. The expression of cyclooxygenase-2 was suppressed. Disruption of p53-murine double minute-2 (MDM2) complex formation resulted in increasing the expression of MDM2 60-kDa cleavage fragment. Inhibited the activation of p42/p44 mitogen-activated protein kinase (MAPK) by PD98059, a specific inhibitor of extracellular regulatory kinase (ERK), significantly decreased cell viability and enhanced the expression of p53 induced by ASA. The result of the cell-cycle analysis showed that ASA and PD98059 induced the cell cycle arrested at the G0/G1 phase and resulted in apoptosis. Conclusion, Nonsteroidal anti-inflammatory drug-inhibited cyclooxygenase is not the only or even the most important mechanism of inhibition. Our study presents evidences that activation of p53 signalling involved in apoptosis induced by ASA. Furthermore, the apoptotic effect was enhanced by blocking the activation of p42/p44 MAPK in response to treatment with ASA, thus indicating a negative role for p42/p44 MAPK. [source] The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2002H.-G. Yu Summary Background Acetylsalicylic acid (ASA, aspirin), the most common nonsteroidal anti-inflammatory drug (NSAID), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the mechanism of its anticancer function remains unclear. The aim of this study was to determine the effects of acetylsalicylic acid on proliferation, apoptosis, and invasion in human cyclooxygenase-2 (COX-2) negative colorectal cancer cell lines. Materials and Methods After treatment with various concentrations of ASA, cell proliferation was measured in the human colon cancer cell line SW480. Apoptotic cells were identified by transmission electron microscopy, acridine orange staining, and flow cytometry. The invasive potential of SW480 cells was detected using an in vitro invasion assay. The production of carcinoembryonic antigen was measured by microparticle enzyme immunoassay. Expression of Bcl2, Bax, CD44v6, and nm23 were evaluated by immunocytochemistry. Results ASA significantly inhibited the proliferation of SW480 cells and stimulated apoptosis. Production of carcinoembryonic antigen and the invasive potential of SW480 cells were also inhibited by ASA. After treatment with ASA, down-regulation of Bcl2 and CD44v6 expression and up-regulation of nm23 expression were observed in SW480 cells. No obvious effect of ASA was found on Bax expression. Conclusion Our findings reveal that ASA inhibits the proliferation and promotes apoptosis in the human colon cancer cell line SW480. Down-regulation of Bcl2 expression might represent a potential mechanism by which ASA induces apoptosis in this COX-2 negative colon cancer cell line. Our results also suggest that ASA decreases the invasive potential of these colon cancer cells. Decreased CEA content and CD44v6 expression and elevated nm23 expression may contribute to the effect of ASA on invasive potential of SW480 colon cancer cells. [source] Proteasome inhibitor-induced apoptosis in human monocyte-derived dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2006Alessio Nencioni Dr. Abstract Proteasome inhibitors possess potent antitumor activity against a broad spectrum of human malignancies. However, the effects of these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effects of proteasome inhibitors on dendritic cells (DC), antigen-presenting cells playing a key role in the initiation of immune responses. Exposure to the proteasome inhibitors bortezomib, MG132 or epoxomicin was found to promote apoptosis of human monocyte-derived DC and to reduce the yield of viable DC when given to monocytes early during differentiation to DC. DC apoptosis via proteasome inhibition was accompanied by mitochondria disruption and subsequent activation of the caspase cascade. Up-regulation and intracellular redistribution of Bcl-2-associated X,protein (Bax), a pro-apoptotic Bcl-2 family protein, were observed in DC treated with these compounds and represent a suitable mechanism leading to activation of the intrinsic apoptotic pathway. Finally, active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by proteasome inhibitors, since the translation inhibitor cycloheximide blocked all of the steps of the observed apoptotic response. In conclusion, induction of apoptosis in DC may represent a novel mechanism by which proteasome inhibitors affect the immune response at the antigen-presenting cell level. [source] The imbalance between Bim and Mcl-1 expression controls the survival of human myeloma cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2004Patricia Gomez-Bougie Abstract Multiple myeloma is a fatal B,cell malignancy characterized by the accumulation of plasma cells within the bone marrow. IL-6 is a major survival factor for myeloma cells. Bcl-2 protein family regulates pathways to apoptosis that are activated upon growth factor deprivation. Pro-apoptotic proteins that have only a single Bcl-2 homology domain, BH3-only, are potent inducers of apoptosis. In myeloma cells, Mcl-1 has been shown to be a major anti-apoptotic protein that appears to regulate cell survival through the JAK/STAT pathway. In this study, we examined the regulation of the BH3-only protein Bim and its interaction with Mcl-1. The three major Bim isoforms are expressed in myeloma cells and are negatively regulated by IL-6. Blockade of IL-6 signaling induces an up-regulation of Bim concomitant to Mcl-1 down-regulation. Of major interest, Bim is found strongly associated with Mcl-1 in viable myeloma cells while this interaction is disrupted under apoptosis induction. Of note, while Bim is also found strongly associated to Bcl-2, this interaction is not changed under apoptosis induction. Thus, in myeloma cells, Mcl-1 neutralizes Bim through complex formation and therefore prevents apoptosis. Under apoptosis induction, the disappearance of Mcl-1 allows Bim to exercise its pro-apoptotic function and to activate Bax. [source] BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtinEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Rebecca 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] The maintenance of specific aspects of neuronal function and behavior is dependent on programmed cell death of adult-generated neurons in the dentate gyrusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2009Woon Ryoung Kim Abstract A considerable number of new neurons are generated daily in the dentate gyrus (DG) of the adult hippocampus, but only a subset of these survive, as many adult-generated neurons undergo programmed cell death (PCD). However, the significance of PCD in the adult brain for the functionality of DG circuits is not known. Here, we examined the electrophysiological and behavioral characteristics of Bax -knockout (Bax -KO) mice in which PCD of post-mitotic neurons is prevented. The continuous increase in DG cell numbers in Bax -KO mice resulted in the readjustment of afferent and efferent synaptic connections, represented by age-dependent reductions in the dendritic arborization of DG neurons and in the synaptic contact ratio of mossy fibers with CA3 dendritic spines. These neuroanatomical changes were associated with reductions in synaptic transmission and reduced performance in a contextual fear memory task in 6-month-old Bax -KO mice. These results suggest that the elimination of excess DG neurons via Bax -dependent PCD in the adult brain is required for the normal organization and function of the hippocampus. [source] Ca2+/H+ antiporter-like activity of human recombinant Bax inhibitor-1 reconstituted into liposomesFEBS JOURNAL, Issue 8 2009Taeho Ahn We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes. When proteoliposomes were suspended in acidic solutions, encapsulated Ca2+ was released from the membranes, as previously suggested [Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee BJ, Cho SG, Kim S, Seo YR, Shin YJ et al. (2008) J Biol Chem283, 15946,15955]. Concomitantly, proton ions were internalized when assayed using the time-dependent change in the fluorescence of the pH-sensitive dye oxonol V entrapped in the proteoliposomes. The influx of proton ions was confirmed by observing tritium accumulation in the membranes. However, the external acidity of the membranes per se did not induce proton ion influx without internalized Ca2+. These results suggest that reconstituted Bax inhibitor-1 has a Ca2+/H+ antiporter-like activity. [source] | |||||||||||||||||||||||||||||||||||||