DNA Damage-inducible Gene Research Articles

Electrical stimulation of cerebellar fastigial nucleus promotes the expression of growth arrest and DNA damage inducible gene β and motor function recovery in cerebral ischemia/reperfusion rats

This study focused on the effects of electrical stimulation of cerebellar fastigial nucleus on the expression of growth arrest and DNA damage inducible gene β (Gadd45β) and on motor function recovery after focal cerebral ischemia/reperfusion (I/R) in rats. Sprague–Dawley (SD) rats were randomly divided into 4 groups: sham I/R (control group), I/R (I/R group), I/R with sham stimulation and I/R with electrical stimulation at 6h, 12h, 24h, 2d and 3d after I/R. Cerebral ischemia and reperfusion was established by nylon monofilament occlusion method. Fastigial nucleus (FN) electrical stimulation was applied at 2h after ischemia for 1h. The changes in the expression of Gadd45β were analyzed by immunohistochemistry, real-time polymerase chain reaction (PCR) and Western-blot respectively. Another group of rats were divided into the same 4 groups. Montoya staircase test score was used to test the motor function of affected forelimb. The levels of Gadd45β were significantly elevated after I/R injury. FN electrical stimulation treatment elevated the expression of Gadd45β further and improved motor function recovery. These results suggest that FN electrical stimulation can promote the expression of Gadd45β and motor function recovery after focal cerebral ischemia.

Abstract B52: Design and synthesis of GADD45β agonists for sorefenib-resistance hepatocellular carcinoma cells

Abstract Background: Growth arrest DNA damage-inducible gene 45β (GADD45β) have been reported in regulating cellular stress response, survival, senescence, and apoptosis in cancer cells. Our previous data showed that GADD45β was induced in sorafenib-treated hepatocellular carcinoma (HCC) cell line and xenograft models. Sorafenib induction was not induced after the treatment ERK kinase inhibitor (U1206) and Raf inhibitor (ZM336372), suggesting that Raf/MEK/ERK kinase signaling pathway did not involve in induction of GADD45β by sorafenib. Methods: We have designed and synthesized a series of SC-compounds. These compounds were treated with and analyzed in terms of GADD45β expression in HCC cell lines (Huh-7, Huh7R). Also, Effects of SC-compounds and sorafenib induced cell apoptosis were evaluated. Results: In seeking new GADD45β agonists, we have screened a series of compounds with GADD45β expression assay. SC-20, a small molecule with N-(4-(quinolin-4-yloxy)phenyl)benzamide skeleton, has shown significantly in induction of GADD45β expression. Interestingly, SC-20 has no inhibition in phosphorylation of ERK, indicating that GADD45β induction by SC-20 is independent of ERK signaling pathway. In addition, SC-20 is able to inhibit HCC cell growth with MTT assay and further induce cell apoptosis. Moreover, SC-20 is able to overcome sorafenib resistance cell line by activating GADD45β expression. Conclusions: SC-20 provides a new concept that increasing GADD45β along will be applicable in sorafenib-sensitive and resistant HCC therapy. Grant support: NSC-100-2325-B-002-042, DOH100-TD-B-111-001, NSC-100-2325-B-010-007

Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone‐induced cell cycle arrest and apoptosis in human liver cells

Androgen receptor (AR) signaling plays an important role in the development and progression of several liver diseases, including hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Dihydrotestosterone (DHT) is the active metabolite of the major circulating androgen, testosterone. In this study, we investigated the effect of DHT on human liver cells. We found that DHT not only induces cell cycle arrest but also initiates apoptosis in androgen-sensitive liver cells, such as SMMC-7721 and L02. Importantly, DHT/AR induces the activation of RNA-dependent protein kinase (PKR)/eukaryotic initiation factor-2 alpha (eIF2α) cascades in androgen-sensitive liver cells. PKR/eIF2α activation-induced growth arrest and DNA damage-inducible gene 153 (GADD153) and heat shock protein 27 (Hsp27) expression contribute to cell cycle arrest in response to DHT. It is notable that DHT administration results in androgen-sensitive liver cells apoptosis, at least in part, through PKR/eIF2α/GADD153 cascades. These results suggest that the androgen/AR pathway plays a pivotal role in liver cell growth and apoptosis regulating, whose deregulation might be involved in the pathogenesis of liver diseases.

NAMPT pathway is involved in the FOXO3a-mediated regulation of GADD45A expression

Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD+. Despite the pivotal role of NAD+ in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD+ level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD+ levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD+-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.

Early stress responses in Atlantic salmon (Salmo salar) exposed to environmentally relevant concentrations of uranium

Uranium (U) is a naturally occurring heavy metal widely used in many military and civil applications. Uranium contamination and the associated potential adverse effects of U on the aquatic environment have been debated during recent years. In order to understand the effect and mode of action (MoA) of U in vivo, juvenile Atlantic salmon (Salmo salar) were exposed to 0.25mg/L, 0.5mg/L and 1.0mg/L waterborne depleted uranyl acetate, respectively, in a static system for 48h. The U concentrations in the gill and liver were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and the resulting biological effects were determined by a combination of analysis of gene expression and micronuclei formation. The hepatic transcriptional level of 12 biomarker genes from four stress–response categories, including oxidative stress (γ-glutamyl cysteine synthetase (GCS), glutathione reductase (GR), glutathione peroxidase (GPx)), DNA damage and repair (P53, cyclin-dependent kinase inhibitor 1 (P21), growth arrest and DNA damage-inducible gene gamma (Gadd45G), proliferating cell nuclear antigen (PCNA), Rad51), apoptosis (Bcl2-associated X protein (BAX), Bcl-x, Caspase 6A,) and protein degradation (Ubiquitin) were evaluated by quantitative real-time polymerase chain reaction (q-rtPCR). The results clearly showed accumulation of U in the gill and liver with increasing concentrations of U in the exposure water. The effects of U on differential hepatic gene expression also occurred in a concentration-dependent manner, although deviations from ideal concentration–response relationships were observed at the highest U concentration (1.0mg/L). All the genes tested were found to be up-regulated by U while no significant micronuclei formation was identified. The results suggest that U may cause oxidative stress in fish liver at concentrations greater than 0.25mg/L, giving rise to clear induction of several toxicologically relevant biomarker genes, although no significant adverse effects were observed after the relatively short exposure period.

The Apoptotic Effect of the Methanol Extract of <i>Polygonum cuspidatum</i> through Up-Regulation Death Receptor 5 and CHOP in HSC-2 Human Oral Cancer Cells

Polygonum cuspidatum is used as a traditional medicinal herb for the therapy of various diseases including several types of cancers. In the present study, we focused on addressing the anti-cancer activity and molecular mechanism of methanol extract of Polygonum cuspidatum (MEPC) in HSC-2 human oral cancer cells. The effect of MEPC on oral cancer cells was estimated by 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis. MEPC inhibited the cell viability and induced apoptosis through the induction of death receptor (DR) 5. MEPC also increased the expression of C/EBP homologous protein/growth arrest and the DNA damage-inducible gene 153 (CHOP), a transcription factor induced by ER stress. Thus, we concluded that the induction of CHOP leading to DR5 up-regulation is required for the anti-cancer activity of MEPC in HSC-2 cells and MEPC may be a promising drug candidate for oral cancer.

Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning

Event Abstract Back to Event Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning Kazuhiro Wada1*, Masahiko Kobayashi1 and Wan-Chun Liu2 1 Hokkaido Universituy, Department of Biological Sciences, Japan 2 The Rockefeller University, United States A complex animal behavior is not only genetically developed but also is affected with influence of environmental factors. Regulation of gene expression in the brain is controlled by species-specific genome information modified under epigenetic influences. Epigenetic molecular mechanisms, such as DNA methylation and histone modification, involve in regulation of gene expression in neuronal cells of the brain. Songbirds learn species-specific song pattern from a tutor during the critical period of vocal learning. For vocal learning and production, songbirds possess specific neural pathways called song system including vocal motor pathway (VMP) and anterior forebrain pathway (AFP). The VMP is critical for vocal production and the AFP is necessary for vocal learning. These pathways consist of a set of brain areas called song nuclei. In these song nuclei, many genes are known to be induced by singing. Several researches showed that some of these singing-induced genes are differentially regulated during the critical period of vocal learning, especially in the forebrain nucleus RA. We found an epigenetic change of one of these immediate-early genes, activity-regulated cytoskeleton-associated (Arc) that interacts with endocytosis-related proteins and facilitates the removal of AMPA receptors from the plasma membran. The DNA methylation in the upstream genome region of Arc was differentially regulated through the critical period in zebra finch, a closed-ended learner. This data suggests that epigenetic mechanisms involve in regulation of induction of singing-induced genes in the critical period. To further clarify the relationship between gene expression and epigenetics, we examined expression patterns of epigenetics-related genes, histone H3 variants (H3.3) and Growth arrest and DNA-damage inducible gene 45 (Gadd45) families. Both H3.3B and Gadd45b genes were up-regulated in song nuclei of both VMP and AFP by singing. The induction of gene expressions was differentially regulated during the critical period of song learning in zebra finch. To clarify whether these regulations depend on age or sensorimotor learning itself, we examined gene expression of these two genes after seasonally singing in the canary, an open-ended learner, which has ability to re-learn new songs even after sexually matured as adult. As the result, gene induction of H3.3B and Gadd45b was higher in RA of the canaries singing variable and plastic songs than the birds singing crystallized songs. These data suggest that histone H3.3B and Gadd45b may control duration of the critical period by epigenetic regulation for several gene expressions that are related with vocal learning. Keywords: critical period, DNA Methylation, epigenetics, Immediate early Gene, sensory motor learning, Song learning, songbird, vocalization Conference: Tenth International Congress of Neuroethology, College Park. Maryland USA, United States, 5 Aug - 10 Aug, 2012. Presentation Type: Poster Presentation (see alternatives below as well) Topic: Genes and Behavior Citation: Wada K, Kobayashi M and Liu W (2012). Epigenetic gene expression dynamics induced by singing in a critical period of vocal learning. Conference Abstract: Tenth International Congress of Neuroethology. doi: 10.3389/conf.fnbeh.2012.27.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2012; Published Online: 07 Jul 2012. * Correspondence: Dr. Kazuhiro Wada, Hokkaido Universituy, Department of Biological Sciences, Sapporo, Hokkaido, 060-0810, Japan, wada@sci.hokudai.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Google Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Google Scholar Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu PubMed Kazuhiro Wada Masahiko Kobayashi Wan-Chun Liu Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Knockdown of metallopanstimulin‐1 inhibits NF‐κB signaling at different levels: The role of apoptosis induction of gastric cancer cells

The ribosomal protein S27 (metallopanstimulin-1, MPS-1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS-1 was highly expressed in human gastric cancer. Knockdown of MPS-1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS-1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS-1 knockdown-induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS-1 inhibited NF-κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF-κB nuclear translocation, and down-regulating its DNA binding activity. Furthermore, data-mining the Gene-Regulatory-Network revealed that growth arrest DNA damage inducible gene 45β (Gadd45β), a direct NF-κB target gene, played a critical role in MPS-1 knockdown-induced apoptosis. Over-expression of Gadd45β inhibited MPS-1 knockdown-induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS-1/NF-κB/Gadd45β signal pathway, played an important role in MPS-1 knockdown-induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS-1/NF-κB in apoptosis and the possible use of MPS-1 targeting strategy in the treatment of gastric cancer.

Role of activating transcription factor 3 in low glucose- and thapsigargin-induced apoptosis in cultured mouse islets

In vitro, survival and function of rat pancreatic β-cells are optimally preserved in the presence of 10mmol/l glucose (G10) and markedly altered by prolonged culture at either 2mmol/l glucose (G2) or 30mmol/l glucose (G30). The increase in islet cell apoptosis in G2 and G30 vs. G10 is preceded by parallel increases in the mRNA levels of the integrated stress response (ISR) gene activating transcription factor 3 (Atf3) and its putative target and proapoptotic gene growth arrest- and DNA damage-inducible gene 153 (Gadd153/Chop). In this study, we used islets from Atf3 knockout (Atf3−/−) mice to test the role of ATF3 in the stimulation of islet cell apoptosis under conditions associated with ISR activation. The glucose sensitivity of Atf3−/− and WT islets for the stimulation of insulin secretion and Xbp1 mRNA splicing during 18h culture was similar, demonstrating that glucose metabolism was unaffected by Atf3 deletion. However, the stimulation of islet cell apoptosis by the SERCA pump inhibitor thapsigargin was slightly but significantly reduced in Atf3−/− vs. WT islets despite similar level of expression of Gadd153 and Gadd34 mRNA. Also, the stimulation of islet cell apoptosis by 7days of culture in G2 was slightly but significantly reduced in Atf3−/− vs. WT islets, and this effect was accompanied by a significant reduction in Gadd153 mRNA expression. In conclusion, the increase in Atf3 gene expression induced by thapsigargin and low glucose concentrations slightly contributes to the stimulation of islet cell apoptosis under these culture conditions.

Silencing GADD153/CHOP gene expression protects against Alzheimer's disease-like pathology induced by 27-hydroxycholesterol in rabbit hippocampus.

Endoplasmic reticulum (ER) stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor, DNA damage inducible gene 153 (gadd153; also called CHOP). Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS), increase β-amyloid (Aβ) levels, disturb iron homeostasis and induce inflammation as well as cell death, which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) triggers AD-like pathology in organotypic slices. However, the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced Aβ production by mechanisms involving reduction in levels of β-amyloid precursor protein (APP) and β-secretase (BACE1), the enzyme that initiates cleavage of APP to yield Aβ peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-α activation, and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology, and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products.

Role of p53 in induction of growth arrest DNA damage-inducible gene 45 β in human hepatoma cells by hydroxyurea

Objective To identify the role of p53 in the induction of growth arrest DNA damageinducible gene 45 (βGADD45β) in hepatocellular carcinoma (HCC) cells by hydroxyurea.Methods Hep3B +p53 clone was established by the transfection of the full-length p53 sequence to Hep3B.Following hydroxyurea administration,quantitative real-time polymerase chain reaction (PCR) was employed to validate the expression changes of GADD45β.pGL3 basic luciferase plasmids including promoter fragments were synthesized in vitro and transfected into cells.The effects on promoter activity,cell growth and the cleavage of Caspase-3 were further focused on.Results Hep3B +p53 could express p53 protein stably.The transfection of p53 could enhance the induction of GADD45β in Hep3B by hydroxyurea.The promoter activity of fragments constructing nuclear factor-κB (NF-κB) and E2F-1 binding sites could be induced about 1.1 and 0.8 folds by transfection of p53.The colony formation and DNA syntheses were inhibited apparently in Hep3B +p53 with p53 by hydroxyurea (37.15％ and 85.29％ by 10 mmol/L Hydroxyurea,respectively).Moreover,the p53 transfection could trigger the cleavage of Caspase-3 more rapidly.Conclusion p53 may play a role in the induction of GADD45β in Hep3B by ribonucleotide reductase inhibitor. Key words: Carcinoma,hepatocellular; Hydroxyurea; p53; Promoter

Augmentation of endoplasmic reticulum stress in cerebral ischemia/reperfusion injury associated with comorbid type 2 diabetes

<title/>Objective: Diabetes is one of the major risk factors for ischemic stroke and is reported to aggravate the ischemic brain damage in different experimental models as well as clinical situations. However, the mechanisms underlying the exacerbated ischemia/reperfusion (I/R) brain injury associated with comorbid diabetes are still not clear. This study investigated the role of endoplasmic reticulum (ER) stress in pathophysiology of aggravated I/R brain injury associated with diabetes.Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 hours followed by 22 hours of reperfusion in high-fat diet-fed and low-dose streptozotocin-treated type 2 diabetic rats. Immunohistochemistry and western blotting analysis were performed to detect the changes in expression of various ER stress and apoptotic markers such as 78 kDa glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153), and caspase-12. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect the extent of DNA fragmentation and cell death.Results: The diabetic rats subjected to I/R manifested significantly larger brain infarct volume and severe deterioration in neurological deficits than their normal, non-diabetic counterparts. There was a marked upregulation of GRP78 observed in brains of diabetic rats after 22 hours of reperfusion. Furthermore, augmentation of CHOP/GADD153 expression and activation of caspase-12 (ER stress-induced apoptotic factors) were observed in parallel with enhanced TUNEL-positive cells or DNA fragmentation in diabetic rats compared to normal rats following cerebral I/R.Discussion: Taken together, the current experimental findings demonstrate that diabetes exacerbates brain I/R injury which may be mediated through enhanced ER stress and cell death involving CHOP/GADD153 and caspase-12 activation.

ClpR Protein-like Regulator Specifically Recognizes RecA Protein-independent Promoter Motif and Broadly Regulates Expression of DNA Damage-inducible Genes in Mycobacteria

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.

Pathways of macrophage apoptosis within the interface membrane in aseptic loosening of prostheses

Aseptic loosening is a major cause of failure of total hip arthroplasty (THA). Macrophage apoptosis in interface membrane has been proved to play an important role in the pathogenesis of aseptic loosening. The purpose of current study was to identify the apoptotic mechanism of macrophages in the interface membrane of aseptic loosening. We collected periprosthetic interface membrane from 23 patients undergoing the revision operations for aseptic loosening of hip joint prostheses. To serve as the control group, samples of capsule were collected from 18 patients undergoing the primary hip arthroplasties for osteoarthritis (OA). The ultrastructure of interface membrane was examined by transmission electron microscopy (TEM), and in situ apoptotic macrophage identification was performed by TUNEL staining. Furthermore, using immunohistochemical methods we investigated the expression of some apoptosis-related markers such as inducible nitric oxide synthase (iNOS), peroxynitrite (ONOO −), cleaved caspase-3/4/8/9, cytochrome c, glucose regulated protein 78 (GRP78), and growth arrest and DNA damage-inducible gene 153 (GADD153) in macrophages. These markers were regarded as apoptotic inducers or specific indicators of different apoptotic pathways such as death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway. TEM showed that a great deal of wear debris was phagocytosed by macrophages, which displayed morphological changes characteristic of apoptosis. The results of TUNEL staining demonstrated that there were more apoptotic macrophages in interface membrane. The expression levels of iNOS, ONOO −, cleaved caspase-3/4/8/9, cytochrome c, GRP78 and GADD153 in macrophages in interface membrane were significantly higher than those in the control samples ( p < 0.05). Our results suggest that death receptor pathway, mitochondria/cytochrosome c caspase-dependent pathway and ER stress pathway are involved in the process of macrophage apoptosis. A therapeutic target to modulate the apoptotic pathways in macrophages may be a strategy to prevent and treat aseptic loosening.

Edaravone Offers Neuroprotection in a Diabetic Stroke Model via Inhibition of Endoplasmic Reticulum Stress

Recent investigations have postulated a link between oxidative stress and endoplasmic reticulum (ER) dysfunction in cerebral ischaemic/reperfusion (I/R) injury. Diabetes is common amongst elderly patients with stroke and has been postulated to aggravate brain I/R damage by triggering oxidative as well as ER stress. We investigated whether treatment with edaravone (1-10 mg/kg), a potent free radical scavenger protects against cerebral I/R injury in rats associated with comorbid type 2 diabetes. Diabetic rats exposed to 2-hr middle cerebral artery occlusion (MCAO) and 22 hr of reperfusion significantly had increased infarct, oedema volume and functional neurological deficits as compared to sham-operated rats. Also, the massive DNA fragmentation accompanied by significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) positive cells was noticed in the ipsilateral penumbral brain region of diabetic I/R rats. The effects of I/R injury were associated with significant up-regulation of 78 kDa-glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, markers of ER stress/apoptosis. Treatment with edaravone (3 and 10 mg/kg) significantly diminished the cerebral infarct, oedema volume and improved functional recovery of neurological deficits. In addition, edaravone treatment ameliorated the DNA fragmentation concomitantly with a significant decrease in induction of GRP78, CHOP/GADD153 immunoreactivity/expression and activation of caspase-12 in the ischaemic brain hemispheres. Overall, the present data indicate that edaravone offers good neuroprotection against diabetic stroke by interrupting the ER stress-mediated apoptotic pathways involving CHOP/GADD153 and caspase-12.

Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: A study of molecular, cellular, and anatomical parameters

Organophosphorus-ester induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral, distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken that results in mild ataxia in 7–14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 ml/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were euthanized at different time points such as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum, brainstem and spinal cord were quickly dissected and frozen for mRNA (northern) studies. Northern blots were probed with BCL2, GADD45, beta actin, and 28S RNA to investigate their expression pattern. Another set of hens was treated for a series of time points and perfused with phosphate buffered saline and fixative for histological studies. Various staining protocols such as Hematoxylin and Eosin (H&E); Sevier-Munger; Cresyl echt Violet for Nissl substance; and Gallocynin stain for Nissl granules were used to assess various patterns of cell death and degenerative changes. Complex cell death mechanisms may be involved in the neuronal and axonal degeneration. These data indicate altered and differential mRNA expressions of BCL2 (anti apoptotic gene) and GADD45 (DNA damage inducible gene) in various tissues. Increased cell death and other degenerative changes noted in the susceptible regions (spinal cord and cerebellum) than the resistant region (cerebrum), may indicate complex molecular pathways via altered BCL2 and GADD45 gene expression, causing the homeostatic imbalance between cell survival and cell death mechanisms. Semi quantitative analysis revealed that the order of severity of damage declines from the spino-cerebellar, ventral, and dorsal tract respectively, suggesting neuroanatomical specificity. Thus, early activation of cell death and cell survival processes may play significant role in the clinical progression and syndromic clinical feature presentation of OPIDN.

Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation

Endoplasmic reticulum (ER) stress has been postulated to play a crucial role in the pathophysiology of cerebral ischemic/reperfusion (I/R) injury and diabetes. Diabetes is a major risk factor and also common amongst the people who suffer from stroke. In this study, we have investigated the neuroprotective potential of sodium 4-phenylbutyrate (SPB; 30–300 mg/kg), a chemical chaperone by targeting ER stress in a rat model of transient focal cerebral ischemia associated with comorbid type 2 diabetes. Intraperitoneal treatment with SPB (100 and 300 mg/kg) significantly ameliorated brain I/R damage as evidenced by reduction in cerebral infarct and edema volume. It also significantly improved the functional recovery of various neurobehavioral impairments (neurological deficit score, grip strength and rota rod) evoked by I/R compared with vehicle-treatment. Further, SPB (100 mg/kg) significantly reduced the DNA fragmentation as shown by prominent reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells. This effect was observed concomitantly with significant attenuation in upregulation of 78 kDa glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, specific markers of ER stress/apoptosis. The neuroprotection observed with SPB was independent of its effect on cerebral blood flow and blood glucose. In conclusion, this study demonstrates the neuroprotective effect of SPB owing to amelioration of ER stress and DNA fragmentation. It also suggest that targeting ER stress might offer a promising therapeutic approach and benefits against ischemic stroke associated with comorbid type 2 diabetes.

Is there a common upstream link for autophagic and apoptotic cell death in human high-grade gliomas?

The prognosis of patients with human high-grade gliomas (HGGs) remains dismal despite major advances in their management, due mainly to the high resistance of these infiltrative tumor cells to programmed cell death (PCD). Most therapeutic strategies for HGGs are aimed to maximize PCD type I, apoptosis or type II, autophagy. These are predominantly distinctive processes, but many studies suggest a cross-talk between the two. A better understanding of the link between PCD types I and II might allow development of more effective therapies for HGGs. In this study, we examined whether there is a common upstream signaling event responsible for both apoptotic and autophagic PCD using 3 chemotherapeutic agents in human HGG cells. Our study shows that each agent caused a significant decrease in cell viability in each of the HGG cell lines tested. The increase rate of apoptosis and autophagy varied among cell lines and chemotherapeutic agents used. Increased expression of cytidine-cytidine-adenosine-adenosine-thymidine (C)/enhancer binding protein (EBP) homologous transcription factor C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 (GADD153) was documented after use of either pro-autophagic or pro-apoptotic agents. The involvement of CHOP/GADD153 in both type I and type II PCD was confirmed by overexpression and gene-silencing studies. Gene silencing by small-interfering RNA-mediated CHOP/GADD153 resulted in increased cell viability, decreased upregulation of microtubule-associated protein light-chain 3' type II (LC3II) and cleaved caspase-3, and inhibition of apoptosis and autophagy. Exogenous expression of CHOP/GADD153 triggered apoptosis and autophagy in the absence of other stimuli. The clinical significance of these findings was supported by the evidence that celecoxib, a nonsteroidal anti-inflammatory drug known to induce GADD153-mediated apoptosis, strongly increases both type I and type II PCD in HGG cells when combined with another inducer of GADD153. These data suggest that CHOP/GADD153 should be investigated as a novel targetable signaling step to improve therapies for HGGs.

The novel compound OSI-461 induces apoptosis and growth arrest in human acute myeloid leukemia cells

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy. Treatment of patients suffering from high-risk AML as defined by clinical parameters, cytogenetics, and/or molecular analyses is often unsuccessful. OSI-461 is a pro-apoptotic compound that has been proposed as a novel therapeutic option for patients suffering from solid tumors like prostate or colorectal carcinoma. But little is known about its anti-proliferative potential in AML. Hence, we treated bone marrow derived CD34(+) selected blast cells from 20 AML patients and the five AML cell lines KG-1a, THP-1, HL-60, U-937, and MV4-11 with the physiologically achievable concentration of 1μM OSI-461 or equal amounts of DMSO as a control. Following incubation with OSI-461, we found a consistent induction of apoptosis and an accumulation of cells in the G2/M phase of the cell cycle. In addition, we demonstrate that the OSI-461 mediated anti-proliferative effects observed in AML are associated with the induction of the pro-apoptotic cytokine mda-7/IL-24 and activation of the growth arrest and DNA-damage inducible genes (GADD) 45α and 45γ. Furthermore, OSI-461 treated leukemia cells did not regain their proliferative potential for up to 8days after cessation of treatment following the initial 48 h treatment period with 1μM OSI-461. This indicates sufficient targeting of the leukemia-initiating cells in our in vitro experiments through OSI-461. The AML samples tested in this study included samples from patients who were resistant to conventional chemotherapy and/or had FLT3-ITD mutations demonstrating the high potential of OSI-461 in human AML.

The effects of the cadmium chloride on the DNA damage and the expression level of gadd gene in HepG2 cell line

To investigate the effects of the cadmium chloride on the DNA damage and the expression of the gadd153 and gadd45beta promoter and mRNA in HepG2 cells. DNA damage induced by cadmium chloride was detected by comet assay. The plasmids (pGADD153-Luc and pG45-Luc) containing DNA damage and repair inducible gene 153 and 45 (gadd153 and gadd45beta) promoter and luciferase and gadd45beta reporter gene were constructed. The activity of gadd153 and gadd45beta promoter were represented by the luciferase activity, the inducible luciferase activities was detected by bioluminescence. The expression of gadd153 and gadd45beta mRNA was detected by RT-PCR. The results of comet assay indicated that Olive Tail Moment induced by the cadmium chloride increased significantly at the dose of 100, 300 micromol/L, compared with the control (P < 0.05). The luciferase activity analysis showed that the expression levels of gadd153 promoter increased significantly in 1, 5, 10 micromol/L treatment group, compared with the control (P < 0.05). The expression levels of gadd45beta promoter in 5, 10 micromol/L treatment group were significantly higher than that in control group (P < 0.05). The expression levels of gadd153 mRNA induced by cadmium chloride at the doses of 1, 5, 10 micromol/L and the expression levels of gadd45beta mRNA induced at the doses of 5, 10 micromol/L were significantly higher than thoae in control group (P < 0.05). The cadmium chloride can induce the DNA damage and increase the expression levels of the gadd153 and gadd45beta promoters in HepG2 cells.