Limited Cell Death Research Articles

Abstract T P343: Resveratrol Rescues Neuron Survival in Pimary Postnatal Cultures

Objective: Our goal is to identify supplements that can improve neuronal survival of postnatal neuronal cultures. Here, we hypothesize that resveratrol could rescue neurons that are stressed during the brain dissociation and plating procedures. Background: The standardized B27 supplement with neurobasal medium has been used to facilitate neuronal growth and attenuates glial proliferation. We have documented that resveratrol (3,4’5 trihydroxystilbene), a natural polyphenol and a bioactive component of red wine, have numerous neuroprotective properties both in vivo and after oxygen-glucose deprivation protocols. We have proposed that this stilbene would not likely act as a direct antioxidant but act as a bioactive agent capable to induce an intracellular cytoprotective cascade. Methods: Postnatal cultures of cortical neurons were derived from 0- to 1-day-old C57BL/6 mice. Brains were dissociated and neurons plated and maintained in three different media: neurobasal medium without any supplement (NBM), neurobasal medium without any supplement but containing resveratrol 10μM (NBM+R10μM), and neurobasal medium with B27 supplement (NBM+B27). Neuronal survival was assessed with the live/dead cell calcein AM/EthD-1), lactate dehydrogenase (LDH) and (MTT) assays after plating in these three different media. Results: Significant increase in live cells (52.9+/-5.5% and 53.6+/-4.5%, p<0.0001) was recorded in cultures incubated in NBM+R10μM and NBM+B27 respectively, when compared to NBM by calcein-AM assay and increase in viability (50.8+/-8.5% and 54.3+/-6.5%) by MTT assay. Significant decrease in dead cells (73.9+/-6.3% and 74.7+/-5.3%, p<0.0001) was recorded in cultures incubated in NBM+R10μM and NBM+B27 respectively, when compare to NBM by EthD-1 assay and decrease in cytotoxicity (17.9+/-2.5% and 19.4+/-2.8) by LDH assay. Conclusions: Results indicated that growth of primary neurons in resveratrol containing medium was most effective suggesting that resveratrol itself is sufficient to enhance neuronal attachment and survival during plating. Efforts are currently being conducted to characterize the mechanism by which resveratrol can limit cell death and increase neuron survival.

Necrostatin-1 reduces neurovascular injury after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the most common form of hemorrhagic stroke, accounting for 15% of all strokes. ICH has the highest acute mortality and the worst long-term prognosis of all stroke subtypes. Unfortunately, the dearth of clinically effective treatment options makes ICH the least treatable form of stroke, emphasizing the need for novel therapeutic targets. Recent work by our laboratory identified a novel role for the necroptosis inhibitor, necrostatin-1, in limiting neurovascular injury in tissue culture models of hemorrhagic injury. In the present study, we tested the hypothesis that necrostatin-1 reduces neurovascular injury after collagenase-induced ICH in mice. Necrostatin-1 significantly reduced hematoma volume by 54% at 72 h after-ICH, as compared to either sham-injured mice or mice administered an inactive, structural analogue of necrostatin-1. Necrostatin-1 also limited cell death by 48%, reduced blood-brain barrier opening by 51%, attenuated edema development to sham levels, and improved neurobehavioral outcomes after ICH. These data suggest a potential clinical utility for necrostatin-1 and/or novel necroptosis inhibitors as an adjunct therapy to reduce neurological injury and improve patient outcomes after ICH.

The DeISGylase USP18 limits TRAIL-induced apoptosis through the regulation of TRAIL levels

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising molecule for anti-cancer therapies. Unfortunately, cancer cells frequently acquire resistance to rhTRAIL. Various co-treatments have been proposed to overcome apoptosis resistance to TRAIL. Here we show that downregulation of the deISGylase USP18 sensitizes cancer cells to rhTRAIL, whereas, elevate levels of USP18 inhibit TRAIL-induced apoptosis, in a deISGylase-independent manner. USP18 influences TRAIL signaling through the control of the IFN autocrine loop. In fact, cells with downregulated USP18 expression augment the expression of cellular TRAIL. Downregulation of cellular TRAIL abrogates the synergism between TRAIL and USP18 siRNA and also limits cell death induced by rhTRAIL. By comparing the apoptotic responsiveness to TRAIL in a panel of cancer cell lines, we have discovered a correlation between TRAIL levels and the apoptotic susceptibility to rhTRAIL, In cells expressing high levels of TRAIL-R2 susceptibility to rhTRAIL correlates with TRAIL expression. In conclusion, we propose that cellular TRAIL is an additional factor that can influence the apoptotic response to rhTRAIL.

Apigenin Sensitizes Colon Cancer Cells to Antitumor Activity of ABT-263

Apigenin is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. Apigenin treatment resulted in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between apigenin and ABT-263 in colon cancer cells. We observed a synergistic effect between apigenin and ABT-263 on apoptosis of colon cancer cells. ABT-263 alone induced limited cell death while upregulating expression of Mcl-1, a potential mechanism for the acquired resistance to ABT-263. The presence of apigenin antagonized ABT-263-induced Mcl-1 upregulation and dramatically enhanced ABT-263-induced cell death. Meanwhile, apigenin suppressed AKT and ERK activation. Inactivation of either AKT or ERK by lentivirus-transduced shRNA or treatment with specific small-molecule inhibitors of these pathways enhanced ABT-263-induced cell death, mirroring the effect of apigenin. Moreover, the combination response was associated with upregulation of Bim and activation of Bax. Downregulation of Bax eliminated the synergistic effect of apigenin and ABT-263 on cell death. Xenograft studies in SCID mice showed that the combined treatment with apigenin and ABT-263 inhibited tumor growth by up to 70% without obvious adverse effects, while either agent only inhibited around 30%. Our results demonstrate a novel strategy to enhance ABT-263-induced antitumor activity in human colon cancer cells by apigenin via inhibition of the Mcl-1, AKT, and ERK prosurvival regulators.

A Mortalin/HSPA9-Mediated Switch in Tumor-Suppressive Signaling of Raf/MEK/Extracellular Signal-Regulated Kinase

Dysregulated Raf/MEK/extracellular signal-regulated kinase (ERK) signaling, a common hallmark of tumorigenesis, can trigger innate tumor-suppressive mechanisms, which must be inactivated for carcinogenesis to occur. This innate tumor-suppressive signaling may provide a potential therapeutic target. Here we report that mortalin (HSPA9/GRP75/PBP74) is a novel negative regulator of Raf/MEK/ERK and may provide a target for the reactivation of tumor-suppressive signaling of the pathway in cancer. We found that mortalin is present in the MEK1/MEK2 proteome and is upregulated in human melanoma biopsy specimens. In different MEK/ERK-activated cancer cell lines, mortalin depletion induced cell death and growth arrest, which was accompanied by increased p21(CIP1) transcription and MEK/ERK activity. Remarkably, MEK/ERK activity was necessary for mortalin depletion to induce p21(CIP1) expression in B-Raf(V600E)-transformed cancer cells regardless of their p53 status. In contrast, in cell types exhibiting normal MEK/ERK status, mortalin overexpression suppressed B-Raf(V600E)- or ΔRaf-1:ER-induced MEK/ERK activation, p21(CIP1) expression, and cell cycle arrest. Other HSP70 family chaperones could not effectively replace mortalin for p21(CIP1) regulation, suggesting a unique role for mortalin. These findings reveal a novel mechanism underlying p21(CIP1) regulation in MEK/ERK-activated cancer and identify mortalin as a molecular switch that mediates the tumor-suppressive versus oncogenic result of dysregulated Raf/MEK/ERK signaling. Our study also demonstrates that p21(CIP1) has dual effects under mortalin-depleted conditions, i.e., mediating cell cycle arrest while limiting cell death.

Tyrosine kinase inhibition increases functional parkin‐ B eclin‐1 interaction and enhances amyloid clearance and cognitive performance

Tyrosine kinase inhibitors (TKIs) are effective therapies for leukaemia. Alzheimer is a neurodegenerative disease characterized by accumulation of β-amyloid (plaques) and hyper-phosphorylated Tau (tangles). Here we show that AD animals have high levels of insoluble parkin and decreased parkin-Beclin-1 interaction, while peripheral administration of TKIs, including Nilotinib and Bosutinib, increases soluble parkin leading to amyloid clearance and cognitive improvement. Blocking Beclin-1 expression with shRNA or parkin deletion prevents tyrosine kinase (TK) inhibition-induced amyloid clearance, suggesting that functional parkin-Beclin-1 interaction mediates amyloid degradation. Isolation of autophagic vacuoles (AVs) in AD mouse brain shows accumulation of parkin and amyloid, consistent with previous results in AD brains, while Bosutinib and Nilotinib increase parkin-Beclin-1 interaction and result in protein deposition in the lysosome. These data suggest that decreased parkin solubility impedes parkin-Beclin-1 interaction and amyloid clearance. We identified two FDA-approved anti-cancer drugs as potential treatment for AD.Two FDA-approved tyrosine kinase inhibitor drugs, Bosutinib and Nilotinib, are shown to ameliorate Alzheimer's disease pathology in mouse models by increasing soluble parkin and leading to amyloid clearance and cognitive improvement.

Avoidance and tolerance of freezing in ectothermic vertebrates

Ectothermic vertebrates have colonized regions that are seasonally or perpetually cold, and some species, particularly terrestrial hibernators, must cope with temperatures that fall substantially below 0°C. Survival of such excursions depends on either freeze avoidance through supercooling or freeze tolerance. Supercooling, a metastable state in which body fluids remain liquid below the equilibrium freezing/melting point, is promoted by physiological responses that protect against chilling injury and by anatomical and behavioral traits that limit risk of inoculative freezing by environmental ice and ice-nucleating agents. Freeze tolerance evolved from responses to fundamental stresses to permit survival of the freezing of a substantial amount of body water under thermal and temporal conditions of ecological relevance. Survival of freezing is promoted by a complex suite of molecular, biochemical and physiological responses that limit cell death from excessive shrinkage, damage to macromolecules and membranes, metabolic perturbation and oxidative stress. Although freeze avoidance and freeze tolerance generally are mutually exclusive strategies, a few species can switch between them, the mode used in a particular instance of chilling depending on prevailing physiological and environmental conditions.

The Multiple Aspects of Stroke and Stem Cell Therapy

Cumulative evidence shows that transplantation of stem cells (SC) derivatives can reduce the functional deficits induced by cerebral ischemia or hemorrhage in animals. Most SC sources have been applied to stroke models, with varying degrees of differentiation into neural derivatives and in varying number, timing and route of administration, with similar benefits on functional outcome. Pioneering clinical trials developed in parallel, and currently outnumber other applications of SC in neurological disorders. These trials reflect a paradigm shift from cell replacement therapy to disease-modeling effects, with increased used of non neural SC. This shift stems in experimental demonstration of paracrine effects of SC that attenuate inflammation, limit cell death through neurotrophic effects, and enhance endogenous recovery processes. Due to its pathogenic characteristics, stroke can uniquely benefit from this variety of actions.

Defined, Serum-Free Conditions for in Vitro culture of Primary Human T-ALL Blasts

Abstract Abstract 3537 Acute lymphoblastic leukemia (ALL) is the most common malignancy diagnosed in children, accounting for 30% of cancer diagnoses in patients under the age of 15 years. Of these cases, roughly 15% derive from the T-cell lineage (T-ALL). Steady improvements in treatment protocols have recently led to survival rates near 80%; however, the remaining 20% have uniformly poor outcomes. Thus far, the therapeutic strategies implemented have focused on intensifying multiagent chemotherapy. As undesirable toxicity arises with additional intensification of standard chemotherapy agents, more targeted molecular agents for T-ALL are urgently needed to further improve outcomes of this disease. Preclinical studies have largely relied on a relatively small number of established T-ALL cell lines that have been maintained in culture for long periods of time, as well as primary T-ALLs xenografted in immunodeficient mice. Recently, research efforts have focused on the development of robust in vitro culture models for efficient testing and validation of novel therapeutics on primary human T-ALL blasts. A relatively simple and efficient in vitro culturing system would in part eliminate the concerns of using cell lines that might not reflect the biology of a typical disease in patients, and mitigate the costly and time-consuming endeavor of xenografting T-ALL blasts. Moreover, personalized medicine initiatives would be dramatically improved by the ability to assess drug sensitivity profiles on patients' own tumor cells in direct in vitro culture assays. Several groups have devised methods for in vitro culture of patient T-ALL cells, the most successful of which have utilized stromal feeders. These methods have relied also upon serum-containing medium and thus have yielded variable results due in part to lot-to-lot variation in serum quality. Using a defined, serum-free media based on the WIT formulation initially described for in vitro propagation of normal and transformed human mammary epithelial cells, we have now optimized culture conditions required for primary human T-ALL samples. We demonstrate this serum-free culture system to consistently outperform serum-containing medium, supporting expansion of primary T-ALL samples 2- to 200-fold within 3 weeks of initial explantation from either xenografted mice or patient biopsy material and with only limited cell death. When carried for longer periods of time (up to 6 weeks), cultures maintained at higher cell densities attained maximal expansions of 40- to 100-fold while those maintained at lower cell densities expanded up to 10,000-fold. Importantly, T-cell receptor γ heteroduplex analysis confirmed expansion of the original clone throughout the culture period and flow cytometric analysis confirmed cultures to be composed of immature human T-lineage cells. Of note, cultures tended to regress after 30–40 days in vitro, suggesting that long-term renewing cells are not supported under these conditions. The use of defined, serum-free medium in this culture system has facilitated systematic characterization of growth factor requirements. We show that supplemental IL-7 and insulin/IGF1 are critical for short-term culture expansion, whereas SCF and Flt3L are dispensable. This culture system will also enable us to assess other “stem” supporting factors and genes (by lentiviral transduction) for their contribution to long-term culture maintenance. Finally, we demonstrate the facility of this culture system for testing of novel therapeutic compounds. By allowing direct study of primary samples in vitro, this culture system will advance basic mechanistic studies in T-ALL biology as well as preclinical studies aimed at developing individually tailored therapy protocols. Disclosures: Ince: Stemgent (2008–10): Consultancy, TAI receives royalties for WIT-P, -I, and -T media Other.

MODULATION OF OXIDATIVE STRESS INDUCED APOPTOSIS AND AUTOPHAGY IN CARDIOMYOCYTES BY INTERMEDIN

The heart is subjected to oxidative stress in conditions of increased reactive oxygen species (ROS) production, such as doxorubicin (DOX) chemotherapy. A major cause of associated ventricular dysfunction is cardiomyocyte...

The APC/C gets Trim'd down for apoptosis

Huang et al. describe how the anaphase-promoting complex (APC/C) limits cell death and how this inhibition is relieved following DNA damage. Bcl-2 family proteins such as Bax stimulate apoptosis by promoting the release of cytochrome c from mitochondria. A protein called modulator of apoptosis-1 (

Chikungunya virus–induced autophagy delays caspase-dependent cell death

Autophagy is an important survival pathway and can participate in the host response to infection. Studying Chikungunya virus (CHIKV), the causative agent of a major epidemic in India, Southeast Asia, and southern Europe, we reveal a novel mechanism by which autophagy limits cell death and mortality after infection. We use biochemical studies and single cell multispectral assays to demonstrate that direct infection triggers both apoptosis and autophagy. CHIKV-induced autophagy is mediated by the independent induction of endoplasmic reticulum and oxidative stress pathways. These cellular responses delay apoptotic cell death by inducing the IRE1α-XBP-1 pathway in conjunction with ROS-mediated mTOR inhibition. Silencing of autophagy genes resulted in enhanced intrinsic and extrinsic apoptosis, favoring viral propagation in cultured cells. Providing in vivo evidence for the relevance of our findings, Atg16L(HM) mice, which display reduced levels of autophagy, exhibited increased lethality and showed a higher sensitivity to CHIKV-induced apoptosis. Based on kinetic studies and the observation that features of apoptosis and autophagy were mutually exclusive, we conclude that autophagy inhibits caspase-dependent cell death but is ultimately overwhelmed by viral replication. Our study suggests that inducers of autophagy may limit the pathogenesis of acute Chikungunya disease.

The WRN and MUS81 proteins limit cell death and genome instability following oncogene activation

Oncogene-induced replication stress is recognized as the primary cause of accumulation of DNA damage and genome instability in precancerous cells. Although the molecular mechanisms responding to such type of replication perturbation are not fully characterized, it has been speculated that their dysfunction may enhance genome instability and accelerate tumor progression. Here, we show that the WRN protein, a member of the human RecQ helicases, is necessary to sustain replication fork progression in response to oncogene-induced replication stress. Loss of WRN affects cell cycle progression and results in enhanced accumulation of double-strand breaks and instability at common fragile sites in cells experiencing oncogene-induced replication stress. Moreover, we demonstrate that double-strand breaks, observed upon oncogene over-expression, depend on the MUS81 endonuclease, which represents a parallel pathway collaborating with WRN to prevent cell death. Overall, our findings give insights into the mechanisms protecting replication forks in cells experiencing oncogene-induced replication stress, and identify factors that, when mutated or dysfunctional, may enhance genome instability in precancerous cells. In addition, because concomitant depletion of WRN and MUS81 causes synthetic sickness in cells growing under oncogene-induced replication stress, our results support the possibility of targeting cancer cells with an impaired replication fork recovery pathway by a specific inactivation of the other parallel pathway.

Enzymatic catalysed synthesis and gelation of ionic peptides for 3D cell culture applications.

ABSTRACTThe enzymatic catalyzed synthesis and gelation of an ionic peptide and its use to create hydrogels for 3D cell culture is discussed. Time resolved small angle scattering in conjunction with imaging technique allowed the structural changes occurring through this enzymatic reaction to be assessed. In turn, the structural information about the fibrillar network and its local density proved key in facilitating the understanding of the relationships between self-assembly behavior, local nanostructure and final physical properties of the materials. The understanding of the gelation process of these materials allowed the design of a simple and efficient methodology to prepare gels for cell culture. Tetrapeptide/enzyme solution containing cells could be injected into cell culture plate with subsequent gelation of the materials leading to encapsulation of the cells into a 3D network. This system was evaluated for the 3D cell culture of human dermal fibroblasts (HDF). Microscopy showed that cells were uniformly distributed within the gel matrix. Cell counting and live/dead staining showed proliferation of HDF with limited cell death over 10 days.

Integrative Genomic Analyses of Sporadic Clear Cell Renal Cell Carcinoma Define Disease Subtypes and Potential New Therapeutic Targets

Sporadic clear cell renal cell carcinoma (ccRCC), the most common type of adult kidney cancer, is often associated with genomic copy number aberrations on chromosomes 3p and 5q. Aberrations on chromosome 3p are associated with inactivation of the tumor suppressor gene von-Hippel Lindau (VHL), which activates the hypoxia-inducible factors HIF1α and HIF2α. In contrast, ccRCC genes on chromosome 5q remain to be defined. In this study, we conducted an integrated analysis of high-density copy number and gene expression data for 54 sporadic ccRCC tumors that identified the secreted glycoprotein STC2 (stanniocalcin 2) and the proteoglycan VCAN (versican) as potential 5q oncogenes in ccRCCs. In functional assays, STC2 and VCAN each promoted tumorigenesis by inhibiting cell death. Using the same approach, we also investigated the two VHL-deficient subtypes of ccRCC, which express both HIF1α and HIF2α (H1H2) or only HIF2α (H2). This analysis revealed a distinct pattern of genomic aberrations in each group, with the H1H2 group displaying, on average, a more aberrant genome than the H2 group. Together our findings provide a significant advance in understanding ccRCCs by offering a molecular definition of two subtypes with distinct characteristics as well as two potential chromosome 5q oncogenes, the overexpression of which is sufficient to promote tumorigenesis by limiting cell death.

13 Human plaque vascular smooth muscle cells show differential expression of genes associated with oxidative DNA damage

RationaleOxidative DNA damage is a prominent feature of human atherosclerotic plaques. DNA damage results in vascular smooth muscle cell (VSMC) premature senescence, cell cycle arrest and apoptosis, features observed in...

The inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse.

Acute myocardial infarction (AMI) initiates an intense inflammatory response that promotes cardiac dysfunction, cell death, and ventricular remodeling. The molecular events underlying this inflammatory response, however, are incompletely understood. In experimental models of sterile inflammation, ATP released from dying cells triggers, through activation of the purinergic P2X7 receptor, the formation of the inflammasome, a multiprotein complex necessary for caspase-1 activation and amplification of the inflammatory response. Here we describe the presence of the inflammasome in the heart in an experimental mouse model of AMI as evidenced by increased caspase-1 activity and cytoplasmic aggregates of the three components of the inflammasome--apoptosis speck-like protein containing a caspase-recruitment domain (ASC), cryopyrin, and caspase-1, localized to the granulation tissue and cardiomyocytes bordering the infarct. Cultured adult murine cardiomyocytes also showed the inducible formation of the inflammasome associated with increased cell death. P2X7 and cryopyrin inhibition (using silencing RNA or a pharmacologic inhibitor) prevented the formation of the inflammasome and limited infarct size and cardiac enlargement after AMI. The formation of the inflammasome in the mouse heart during AMI causes additional loss of functional myocardium, leading to heart failure. Modulation of the inflammasome may therefore represent a unique therapeutic strategy to limit cell death and prevent heart failure after AMI.

Cyclooxygenase-2 in the kidney: good, BAD, or both?

Hypertonic stress in the kidney inner medulla is common, yet inner medullary cells adapt to limit cell death. Küper et al. have identified a cell-survival response by which increased cyclooxygenase-2 (COX-2) stimulates a prostaglandin E(2) (PGE(2))/protein kinase A (PKA)-mediated inactivation of the pro-apoptotic protein BAD. However, the PGE(2)/PKA pathway is not the only means to inactivate BAD and limit cell death. This Commentary shows a broader picture of this pathway to examine the kidney's BAD options.

Ectodermal Influx and Cell Hypertrophy Provide Early Growth for All Murine Mammary Rudiments, and Are Differentially Regulated among Them by Gli3

Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3Xt-J/Xt-J mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3Xt-J/Xt-J MR1 is 20% smaller, and Gli3Xt-J/Xt-J MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3Xt-J/Xt-J MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3Xt-J/Xt-J MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals.

Histochemical studies on the accumulation of H2O2 and hypersensitive cell death in the nonhost resistance of pepper against Blumeria graminis f. sp. tritici

Defence reactions between Blumeria graminis f. sp. tritici (Bgt) and pepper cultivar ‘A11’ (nonhost) and susceptible wheat cultivar ‘Shan 225’ were studied histochemically. Conidia of Bgt developed normally on the wheat leaves with limited detectable whole-cell H2O2 accumulation and cell death. In contrast, the germination of conidia on the pepper leaves was delayed. High frequency of H2O2 accumulation and hypersensitive response (HR) were detected in the nonhost interactions. H2O2 accumulation was accompanied by HR and papilla formation. These results indicate that H2O2 and HR play important roles in preventing colonization and disease development of nonhost-pathogen Bgt in pepper plants.