Sample records for acinar cell death

  1. Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

    PubMed Central

    Athwal, T; Huang, W; Mukherjee, R; Latawiec, D; Chvanov, M; Clarke, R; Smith, K; Campbell, F; Merriman, C; Criddle, D; Sutton, R; Neoptolemos, J; Vlatković, N

    2014-01-01

    Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore

  2. Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis.

    PubMed

    Gukovskaya, Anna S; Gukovsky, Ilya; Jung, Yoon; Mouria, Michelle; Pandol, Stephen J

    2002-06-21

    Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.

  3. Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yu, Ge; Wan, Rong; Hu, Yanling

    2014-01-31

    Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration ofmore » sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.« less

  4. Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.

    PubMed

    Sun, Zhen; Gou, Wenyu; Kim, Do-Sung; Dong, Xiao; Strange, Charlie; Tan, Yu; Adams, David B; Wang, Hongjun

    2017-11-01

    The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 10 5 or 1 × 10 6 GFP + ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP + ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP + ASCs migrated to pancreas and differentiated into amylase + cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase + cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  5. PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

    PubMed

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z; Gittes, George K

    2016-02-17

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

  6. PNA lectin for purifying mouse acinar cells from the inflamed pancreas

    PubMed Central

    Xiao, Xiangwei; Fischbach, Shane; Fusco, Joseph; Zimmerman, Ray; Song, Zewen; Nebres, Philip; Ricks, David Matthew; Prasadan, Krishna; Shiota, Chiyo; Husain, Sohail Z.; Gittes, George K.

    2016-01-01

    Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer. PMID:26884345

  7. Calcium signalling in the acinar environment of the exocrine pancreas: physiology and pathophysiology.

    PubMed

    Gryshchenko, Oleksiy; Gerasimenko, Julia V; Peng, Shuang; Gerasimenko, Oleg V; Petersen, Ole H

    2018-02-09

    Ca 2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Ca 2+ signals evoked by K + -induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca 2+ signals in acinar but not in stellate cells. Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca 2+ signals in response to membrane depolarization. The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol-related acute pancreatitis, but they became sensitive to stimulation with trypsin. Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca 2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. Physiological Ca 2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca 2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca 2+ signals in the peri-acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca 2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization-evoked Ca 2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca 2+ signals in response to membrane depolarization. The principal agent evoking Ca 2+ signals in the stellate cells is bradykinin, but in experimental alcohol

  8. Cholecystokinin-8-induced hypoplasia of the rat pancreas: influence of nitric oxide on cell proliferation and programmed cell death.

    PubMed

    Trulsson, Lena M; Gasslander, Thomas; Svanvik, Joar

    2004-10-01

    The background of cholecystokinin-8 (CCK-8)-induced hypoplasia in the pancreas is not known. In order to increase our understanding we studied the roles of nitric oxide and NF-kappaB in rats. CCK-8 was injected for 4 days, in a mode known to cause hypoplasia, and the nitric oxide formation was either decreased by means of N(omega)-nitro-L-arginine (L-NNA) or increased by S-nitroso-N-acetylpencillamine (SNAP). The activation of NF-kappaB was quantified by ELISA detection, apoptosis with caspase-3 and histone-associated DNA-fragmentation and mitotic activity in the acinar, centroacinar and ductal cells were visualized by the incorporation of [(3)H]-thymidine. Pancreatic histology and weight as well as protein- and DNA contents were also studied. Intermittent CCK injections reduced pancreatic weight, protein and DNA contents and increased apoptosis, acinar cell proliferation and nuclear factor kappaB (NF-kappaB) activation. It also caused vacuolisation of acinar cells. The inhibition of endogenous nitric oxide formation by L-NNA further increased apoptosis and NF-kappaB activation but blocked the increased proliferation and vacuolisation of acinar cells. The DNA content was not further reduced. SNAP given together with CCK-8 increased apoptosis and other pathways of cell death, raised proliferation of acinar cells and strongly reduced the DNA content in the pancreas. Histological examination showed no inflammation in any group. We conclude that during CCK-8-induced pancreatic hypoplasia, endogenously formed nitric oxide suppresses apoptosis but increases cell death along non-apoptotic pathways and stimulates regeneration of acinar cells. Exogenous nitric oxide enhances the acinar cell turnover by increasing both apoptotic and non-apoptotic cell death and cell renewal. In this situation NF-kappaB activation seems not to inhibit apoptosis nor promote cell proliferation.

  9. Acinar cell cystadenoma of the pancreas: a benign neoplasm or non-neoplastic ballooning of acinar and ductal epithelium?

    PubMed

    Singhi, Aatur D; Norwood, Stephanie; Liu, Ta-Chiang; Sharma, Rajni; Wolfgang, Christopher L; Schulick, Richard D; Zeh, Herbert J; Hruban, Ralph H

    2013-09-01

    Acinar cell cystadenoma (ACA) of the pancreas was initially described as a non-neoplastic cyst of the pancreas and, at that time, referred to as "acinar cystic transformation." In subsequent studies, these lesions were given the designation of "-oma," despite the relative lack of evidence supporting a neoplastic process. To characterize these lesions further, we examined the clinical, pathologic, and immunohistochemical features of 8 ACAs. The majority of patients were female (7 of 8, 88%) and ranged in age from 18 to 57 years (mean, 43 y). Grossly, the cysts involved the head (n=5), body (n=1), or the entire pancreas (n=2). ACAs were either multilocular (n=4) or unilocular (n=4) and ranged in size from 1.8 to 15 cm (mean, 6.8 cm). Histologically, multilocular ACAs were lined by patches of acinar and ductal epithelium. Immunolabeling, including double-labeling for cytokeratin 19 and chymotrypsin, highlighted the patchy pattern of the ductal and acinar cells lining the cysts. In some areas, the cysts with patches of acinar and ductal differentiation formed larger locules with incomplete septa as they appeared to fuse with other cysts. In contrast, the unilocular cases were lined by 1 to 2 cell layers of acinar cells with little intervening ductal epithelium. Nuclear atypia, mitotic figures, necrosis, infiltrative growth, and associated invasive carcinoma were absent in all cases. In addition, we assessed the clonal versus polyclonal nature of ACAs, occurring in women, using X-chromosome inactivation analysis of the human androgen receptor (AR) gene. Five of 7 cases were informative and demonstrated a random X-chromosome inactivation pattern. Clinical follow-up information was available for all patients, and follow-up ranged from 10 months to 7.8 years (mean, 3.6 y), with no evidence of recurrence or malignant transformation. We hypothesize that early lesions are marked by acinar dilatation that expands into and incorporates smaller ductules and later larger ducts

  10. Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury.

    PubMed

    Grady, T; Mah'Moud, M; Otani, T; Rhee, S; Lerch, M M; Gorelick, F S

    1998-11-01

    The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10(-7) M) hyperstimulation and bombesin (10(-5) M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

  11. In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes

    PubMed Central

    Li, Weida; Nakanishi, Mio; Zumsteg, Adrian; Shear, Matthew; Wright, Christopher; Melton, Douglas A; Zhou, Qiao

    2014-01-01

    Direct lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin+ β-cells, glucagon+ α-cells, and somatostatin+ δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to β-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and β-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001 PMID:24714494

  12. Age-related alterations in IL-1beta, TNF-alpha, and IL-6 concentrations in parotid acinar cells from BALB/c and non-obese diabetic mice.

    PubMed

    Yamakawa, M; Weinstein, R; Tsuji, T; McBride, J; Wong, D T; Login, G R

    2000-08-01

    IL-1beta, TNF-alpha, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3varepsilon, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1beta and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1beta and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033-1041,2000)

  13. Morphology and function of lacrimal gland acinar cells in primary culture.

    PubMed

    Hann, L E; Tatro, J B; Sullivan, D A

    1989-01-01

    The objectives of the current investigation were fourfold: (1) to establish an effective procedure for the isolation of acinar cells from the rat lacrimal gland; (2) to evaluate the functional capacity of freshly isolated cells; (3) to determine defined culture conditions which permit maintenance of viable, differentiated cells, as well as secretory component (SC) production, during long-term culture; and (4) to characterize the morphological features of cultured cells. Acinar cells were isolated by serial incubation of gland fragments in chelating and enzymatic solutions, followed by centrifugation through a Ficoll gradient. The yield of viable cells/gland appeared to be age-dependent: cell recovery was inversely proportional to the age of the animals. Immunofluorescence analysis of freshly isolated cells showed the presence of SC, the IgA antibody receptor, within isolated cells. In addition, experiments with a labeled analog (Nle4-D-Phe7-alpha MSH) of alpha-melanocyte-stimulating hormone (alpha-MSH) demonstrated specific binding sites on freshly isolated cells; alpha-MSH is a known modulator of acinar protein secretion. Maximum binding of the alpha-MSH analog occurred within 30 min, was dependent upon cell density and was reduced by coincubation with unlabeled alpha-MSH. To determine the culture requirements of acinar cells, cells were cultured on a variety of substrates (plastic or modified plastic [Primaria], coated with or without extracellular matrix [Matrigel]) in the presence or absence of various supplements and/or fetal calf serum (FCS) for 0.7 to 3.5 weeks. Cell attachment, function and long-term viability required an extracellular matrix. Moreover, in long term cultures (25 days), acinar cell attachment was enhanced by the inclusion of supplements to media containing 10% FCS. Replacement of serum with fibroblast growth factor, high-density lipoprotein and an increased concentration of epidermal growth factor resulted in a distinct "cobblestone

  14. Alcohols enhance caerulein-induced zymogen activation in pancreatic acinar cells

    PubMed Central

    LU, ZHAO; KARNE, SURESH; KOLODECIK, THOMAS; GORELICK, FRED S.

    2010-01-01

    Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10−10 to 10−7 M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of ≥2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation. PMID:11842000

  15. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

    PubMed

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-11-01

    The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP de-glycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger LAMPs' bulk de-glycosylation, but induces their degradation via CatB-mediated cleavage of LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, stimulates the basal and inhibits CCK-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis, and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction.

  16. Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis.

    PubMed

    Bhatia, Vandanajay; Rastellini, Cristiana; Han, Song; Aronson, Judith F; Greeley, George H; Falzon, Miriam

    2014-09-01

    Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrP(Δacinar)) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP(Δacinar) exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP(Δacinar) cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP(Δacinar) mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7-34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs. Copyright © 2014 the American Physiological Society.

  17. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress

    PubMed Central

    Antonucci, Laura; Fagman, Johan B.; Kim, Ju Youn; Todoric, Jelena; Gukovsky, Ilya; Mackey, Mason; Ellisman, Mark H.; Karin, Michael

    2015-01-01

    Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7Δpan mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7Δpan mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7Δpan mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures. PMID:26512112

  18. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

    PubMed Central

    Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

    2015-01-01

    Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

  19. Molecular Ghrelin System in the Pancreatic Acinar Cells: The Role of the Polypeptide, Caerulein and Sensory Nerves.

    PubMed

    Bonior, Joanna; Ceranowicz, Piotr; Gajdosz, Ryszard; Kuśnierz-Cabala, Beata; Pierzchalski, Piotr; Warzecha, Zygmunt; Dembiński, Artur; Pędziwiatr, Michał; Kot, Michalina; Leja-Szpak, Anna; Nawrot-Porąbka, Katarzyna; Link-Lenczowski, Paweł; Olszanecki, Rafał; Bartuś, Krzysztof; Jaworek, Jolanta

    2017-05-02

    Ghrelin (GHRL) is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Experimental studies showed that GHRL protects the stomach and pancreas against acute damage, but the effect of GHRL on pancreatic acinar cells was still undetermined. To investigate the effect of GHRL and caerulein on the functional ghrelin system in pancreatic acinar cells taking into account the role of sensory nerves (SN). Experiments were carried out on isolated pancreatic acinar cells and AR42J cells. Before acinar cells isolation, GHRL was administered intraperitoneally at a dose of 50 µg/kg to rats with intact SN or with capsaicin deactivation of SN (CDSN). After isolation, pancreatic acinar cells were incubated in caerulein-free or caerulein containing solution. AR42J cells were incubated under basal conditions and stimulated with caerulein, GHRL or a combination of the above. Incubation of isolated acinar cells with caerulein inhibited GHS-R and GHRL expression at the level of mRNA and protein in those cells. Either in rats with intact SN or with CDSN, administration of GHRL before isolation of acinar cells increased expression of GHRL and GHS-R in those cells and reversed the caerulein-induced reduction in expression of those parameters. Similar upregulation of GHS-R and GHRL was observed after administration of GHRL in AR42J cells. GHRL stimulates its own expression and expression of its receptor in isolated pancreatic acinar cells and AR42J cells on the positive feedback pathway. This mechanism seems to participate in the pancreatoprotective effect of GHRL in the course of acute pancreatitis.

  20. Sulforaphane Protects Pancreatic Acinar Cell Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3 Inflammatory Pathway

    PubMed Central

    Dong, Zhaojun; Shang, Haixiao; Chen, Yong Q.; Pan, Li-Long

    2016-01-01

    Acute pancreatitis (AP) is characterized by early activation of intra-acinar proteases followed by acinar cell death and inflammation. Cellular oxidative stress is a key mechanism underlying these pathological events. Sulforaphane (SFN) is a natural organosulfur antioxidant with undescribed effects on AP. Here we investigated modulatory effects of SFN on cellular oxidation and inflammation in AP. AP was induced by cerulean hyperstimulation in BALB/c mice. Treatment group received a single dose of 5 mg/kg SFN for 3 consecutive days before AP. We found that SFN administration attenuated pancreatic injury as evidenced by serum amylase, pancreatic edema, and myeloperoxidase, as well as by histological examination. SFN administration reverted AP-associated dysregulation of oxidative stress markers including pancreatic malondialdehyde and redox enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). In acinar cells, SFN treatment upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes including quinoneoxidoreductase-1, heme oxidase-1, SOD1, and GPx1. In addition, SFN selectively suppressed cerulein-induced activation of the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome, in parallel with reduced nuclear factor- (NF-) κB activation and modulated NF-κB-responsive cytokine expression. Together, our data suggested that SFN modulates Nrf2-mediated oxidative stress and NLRP3/NF-κB inflammatory pathways in acinar cells, thereby protecting against AP. PMID:27847555

  1. Sulforaphane Protects Pancreatic Acinar Cell Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3 Inflammatory Pathway.

    PubMed

    Dong, Zhaojun; Shang, Haixiao; Chen, Yong Q; Pan, Li-Long; Bhatia, Madhav; Sun, Jia

    2016-01-01

    Acute pancreatitis (AP) is characterized by early activation of intra-acinar proteases followed by acinar cell death and inflammation. Cellular oxidative stress is a key mechanism underlying these pathological events. Sulforaphane (SFN) is a natural organosulfur antioxidant with undescribed effects on AP. Here we investigated modulatory effects of SFN on cellular oxidation and inflammation in AP. AP was induced by cerulean hyperstimulation in BALB/c mice. Treatment group received a single dose of 5 mg/kg SFN for 3 consecutive days before AP. We found that SFN administration attenuated pancreatic injury as evidenced by serum amylase, pancreatic edema, and myeloperoxidase, as well as by histological examination. SFN administration reverted AP-associated dysregulation of oxidative stress markers including pancreatic malondialdehyde and redox enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). In acinar cells, SFN treatment upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes including quinoneoxidoreductase-1, heme oxidase-1, SOD1, and GPx1. In addition, SFN selectively suppressed cerulein-induced activation of the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome, in parallel with reduced nuclear factor- (NF-) κ B activation and modulated NF- κ B-responsive cytokine expression. Together, our data suggested that SFN modulates Nrf2-mediated oxidative stress and NLRP3/NF- κ B inflammatory pathways in acinar cells, thereby protecting against AP.

  2. Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

    PubMed

    Yoon, Mi Na; Kim, Min Jae; Koong, Hwa Soo; Kim, Dong Kwan; Kim, Se Hoon; Park, Hyung Seo

    2017-09-01

    Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100 mM reversibly suppressed CCh-induced Ca 2+ oscillation in a dose-dependent manner. Pretreatment of ethanol has no effect on the store-operated calcium entry induced by 10 μM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. [Pancreatic acinar neoplasms : Comparative molecular characterization].

    PubMed

    Bergmann, F

    2016-11-01

    Pancreatic acinar cell carcinomas are biologically aggressive neoplasms for which treatment options are very limited. The molecular mechanisms of tumor initiation and progression are largely not understood and precursor lesions have not yet been identified. In this study, pancreatic acinar cell carcinomas were cytogenetically characterized as well as by molecular and immunohistochemical analyses. Corresponding investigations were carried out on pancreatic ductal adenocarcinomas and pancreatic neuroendocrine neoplasms augmented by functional analyses. We show that pancreatic acinar cell carcinomas display a microsatellite stable, chromosomal unstable genotype, characterized by recurrent chromosomal imbalances that clearly discriminate them from pancreatic ductal adenocarcinomas and neuroendocrine neoplasms. Based on findings obtained from comparative genomic hybridization, candidate genes could be identified, such as deleted in colorectal cancer (DCC) and c-MYC. Furthermore, several therapeutic targets were identified in acinar cell carcinomas and other pancreatic neoplasms, including epidermal growth factor receptor (EGFR), L1 cell adhesion molecule (L1CAM) and heat shock protein 90 (HSP90). Moreover, L1CAM was shown to play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma. Functional analyses in cell lines derived from pancreatic neuroendocrine neoplasms revealed promising anti-tumorigenic effects using EGFR and HSP90 inhibitors affecting the cell cycle and in the case of HSP90, regulating several other oncogenes. Finally, based on mutational analyses of mitochondrial DNA, molecular evidence is provided that acinar cell cystadenomas (or better cystic acinar transformation) represent non-clonal lesions, suggesting an inflammatory reactive non-neoplastic nature.

  4. Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.

    PubMed

    Muili, Kamaldeen A; Ahmad, Mahwish; Orabi, Abrahim I; Mahmood, Syeda M; Shah, Ahsan U; Molkentin, Jeffery D; Husain, Sohail Z

    2012-04-15

    Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

  5. Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury

    PubMed Central

    Muili, Kamaldeen A.; Ahmad, Mahwish; Orabi, Abrahim I.; Mahmood, Syeda M.; Shah, Ahsan U.; Molkentin, Jeffery D.

    2012-01-01

    Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca2+ is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca2+ signaling is the Ca2+-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ−/−) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis. PMID:22323127

  6. Induced PTF1a expression in pancreatic ductal adenocarcinoma cells activates acinar gene networks, reduces tumorigenic properties, and sensitizes cells to gemcitabine treatment.

    PubMed

    Jakubison, Brad L; Schweickert, Patrick G; Moser, Sarah E; Yang, Yi; Gao, Hongyu; Scully, Kathleen; Itkin-Ansari, Pamela; Liu, Yunlong; Konieczny, Stephen F

    2018-05-02

    Pancreatic acinar cells synthesize, package, and secrete digestive enzymes into the duodenum to aid in nutrient absorption and meet metabolic demands. When exposed to cellular stresses and insults, acinar cells undergo a dedifferentiation process termed acinar-ductal metaplasia (ADM). ADM lesions with oncogenic mutations eventually give rise to pancreatic ductal adenocarcinoma (PDAC). In healthy pancreata, the basic helix-loop-helix (bHLH) factors MIST1 and PTF1a coordinate an acinar-specific transcription network that maintains the highly developed differentiation status of the cells, protecting the pancreas from undergoing a transformative process. However, when MIST1 and PTF1a gene expression is silenced, cells are more prone to progress to PDAC. In this study, we tested whether induced MIST1 or PTF1a expression in PDAC cells could (i) re-establish the transcriptional program of differentiated acinar cells and (ii) simultaneously reduce tumor cell properties. As predicted, PTF1a induced gene expression of digestive enzymes and acinar-specific transcription factors, while MIST1 induced gene expression of vesicle trafficking molecules as well as activation of unfolded protein response components, all of which are essential to handle the high protein production load that is characteristic of acinar cells. Importantly, induction of PTF1a in PDAC also influenced cancer-associated properties, leading to a decrease in cell proliferation, cancer stem cell numbers, and repression of key ATP-binding cassette efflux transporters resulting in heightened sensitivity to gemcitabine. Thus, activation of pancreatic bHLH transcription factors rescues the acinar gene program and decreases tumorigenic properties in pancreatic cancer cells, offering unique opportunities to develop novel therapeutic intervention strategies for this deadly disease. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  7. Salivary gland acinar cells regenerate functional glandular structures in modified hydrogels

    NASA Astrophysics Data System (ADS)

    Pradhan, Swati

    Xerostomia, a condition resulting from irradiation of the head and neck, affects over 40,000 cancer patients each year in the United States. Direct radiation damage of the acinar cells that secrete fluid and protein results in salivary gland hypofunction. Present medical management for xerostomia for patients treated for upper respiratory cancer is largely ineffective. Patients who have survived their terminal diagnosis are often left with a diminished quality of life and are unable to enjoy the simple pleasures of eating and drinking. This project aims to ultimately reduce human suffering by developing a functional implantable artificial salivary gland. The goal was to create an extracellular matrix (ECM) modified hyaluronic acid (HA) based hydrogel culture system that allows for the growth and differentiation of salivary acinar cells into functional acini-like structures capable of secreting large amounts of protein and fluid unidirectionally and to ultimately engineer a functional artificial salivary gland that can be implanted into an animal model. A tissue collection protocol was established and salivary gland tissue was obtained from patients undergoing head and neck surgery. The tissue specimen was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Hematoxylin and eosin staining confirmed normal glandular tissue structures including intercalated ducts, striated ducts and acini. alpha-Amylase and periodic acid schiff stain, used for structures with a high proportion of carbohydrate macromolecules, preferentially stained acinar cells in the tissue. Intercalated and striated duct structures were identified using cytokeratins 19 and 7 staining. Myoepithelial cells positive for cytokeratin 14 were found wrapped around the serous and mucous acini. Tight junction components including ZO-1 and E-cadherin were present between both ductal and acinar cells. Ductal and acinar

  8. Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

    PubMed Central

    Hilgenberg, Jill D.; Rybalchenko, Volodymyr; Medina-Ortiz, Wanda E.; Gregg, Elaine V.; Koulen, Peter

    2011-01-01

    Purpose. Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca2+ homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca2+ concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca2+ release channels, transient receptor potential polycystin family (TRPP) channels. Methods. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca2+ channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. Results. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. Conclusions. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology. PMID:21508103

  9. Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells.

    PubMed Central

    Leite, M F; Dranoff, J A; Gao, L; Nathanson, M H

    1999-01-01

    The ryanodine receptor (RyR) is the principal Ca2+-release channel in excitable cells, whereas the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is primarily responsible for Ca2+ release in non-excitable cells, including epithelia. RyR also is expressed in a number of non-excitable cell types, but is thought to serve as an auxiliary or alternative Ca2+-release pathway in those cells. Here we use reverse transcription PCR to show that a polarized epithelium, the pancreatic acinar cell, expresses the type 2, but not the type 1 or 3, isoform of RyR. We furthermore use immunochemistry to demonstrate that the type 2 RyR is distributed throughout the basolateral and, to a lesser extent, the apical region of the acinar cell, but is excluded from the trigger zone, where cytosolic Ca2+ signals originate in this cell type. Since propagation of Ca2+ waves in acinar cells is sensitive to ryanodine, caffeine and Ca2+, these findings suggest that Ca2+ waves in this cell type result from the co-ordinated release of Ca2+, first from InsP3Rs in the trigger zone, then from RyRs elsewhere in the cell. RyR may play a fundamental role in Ca2+ signalling in polarized epithelia, including for Ca2+ signals initiated by InsP3. PMID:9882629

  10. Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

    PubMed Central

    Armstrong, Jane A.; Cash, Nicole J.; Ouyang, Yulin; Morton, Jack C.; Chvanov, Michael; Latawiec, Diane; Awais, Muhammad; Tepikin, Alexei V.; Sutton, Robert; Criddle, David N.

    2018-01-01

    Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1–10 μm), whereas higher levels (0.5–1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2. H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2. However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation. PMID:29626097

  11. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells*

    PubMed Central

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S.

    2016-01-01

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca2+] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca2+-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca2+ entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca2+-dependent up-regulation of AQP5. These important findings reveal that the Ca2+-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. PMID:26903518

  12. Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

    2015-02-01

    The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

  13. Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

    PubMed

    Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

    2017-06-01

    Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

  14. Functional somatostatin receptors on a rat pancreatic acinar cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

    1988-07-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

  15. Pancreatic acinar cell carcinoma extending into the common bile and main pancreatic ducts.

    PubMed

    Yamaguchi, Rin; Okabe, Yoshinobu; Jimi, Atsuo; Shiota, Koji; Kodama, Takahito; Naito, Yoshiki; Yasunaga, Masafumi; Kinoshita, Hisafumi; Kojiro, Masamichi

    2006-10-01

    Acinar cell carcinoma (ACC) of the pancreas is relatively rare, accounting for only approximately 1% of all exocrine pancreatic tumors. A 69-year-old man was found to have a mass lesion measuring approximately 4 cm in diameter in the pancreatic head on ultrasound, abdominal dynamic CT, and percutaneous transhepatic cholangiography. Magnetic resonance cholangiopancreatography showed defect of the lower common bile duct (CBD) due to obstruction by the tumor cast. Histopathologically, the pancreatic head tumor invaded the main pancreatic duct (MPD) and CBD with extension into the CBD in a form of tumor cast. The tumor cells consisted of a solid proliferation with abundant eosinophilic cytoplasm and round nuclei in an acinar and trabecular fashion. A 55-year-old man with upper abdominal pain and nausea, had a cystic lesion approximately 3 cm in size in the pancreatic tail on CT. Histopathologically, the tumor was encapsulated by fibrous capsule and had extensive central necrosis with solid areas in the tumor periphery, and invaded with extension into the MPD in a form of tumor cast. The tumor cells resembled acinar cells in solid growths. Two resected cases of ACC with unusual tumor extension into the CBD and the MPD, respectively, are reported.

  16. Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.

    PubMed

    Fazio, Elena N; Young, Claire C; Toma, Jelena; Levy, Michael; Berger, Kurt R; Johnson, Charis L; Mehmood, Rashid; Swan, Patrick; Chu, Alphonse; Cregan, Sean P; Dilworth, F Jeffrey; Howlett, Christopher J; Pin, Christopher L

    2017-09-01

    Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3 -/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC. © 2017 Fazio et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. The nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis.

    PubMed

    Hale, Michael A; Swift, Galvin H; Hoang, Chinh Q; Deering, Tye G; Masui, Toshi; Lee, Youn-Kyoung; Xue, Jumin; MacDonald, Raymond J

    2014-08-01

    The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype. © 2014. Published by The Company of Biologists Ltd.

  18. Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

    PubMed

    Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

    1997-03-01

    Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

  19. ptf1a+ , ela3l- cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae.

    PubMed

    Schmitner, Nicole; Kohno, Kenji; Meyer, Dirk

    2017-03-01

    The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l- negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l -positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b In conclusion, we

  20. ptf1a+, ela3l− cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae

    PubMed Central

    Schmitner, Nicole; Kohno, Kenji

    2017-01-01

    ABSTRACT The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l-negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l-positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b. In

  1. Functional differences in the acinar cells of the murine major salivary glands.

    PubMed

    Kondo, Y; Nakamoto, T; Jaramillo, Y; Choi, S; Catalan, M A; Melvin, J E

    2015-05-01

    In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization. © International & American Associations for Dental Research 2015.

  2. Establishment of Functional Acinar-like Cultures from Human Salivary Glands

    PubMed Central

    Jang, S.I.; Ong, H.L.; Gallo, A.; Liu, X.; Illei, G.

    2015-01-01

    Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers—namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients. PMID:25416669

  3. Establishment of functional acinar-like cultures from human salivary glands.

    PubMed

    Jang, S I; Ong, H L; Gallo, A; Liu, X; Illei, G; Alevizos, I

    2015-02-01

    Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers-namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients. © International & American Associations for Dental Research 2014.

  4. Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

    PubMed

    Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

    2011-06-06

    Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

    PubMed Central

    Huang, Ze-bing; Wang, Hai-yan; Sun, Na-na; Wang, Jing-ke; Zhao, Meng-qin; Shen, Jian-xin; Gao, Ming; Hammer, Ronald P; Fan, Xue-gong; Wu, Jie

    2014-01-01

    Aim: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca2+ oscillations in mouse pancreatic acinar cells in vitro. Methods: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca2+ oscillations were monitored by whole-cell recording of Ca2+-activated Cl− currents and by using confocal Ca2+ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established. Results: Bath application of ACh (10 nmol/L) induced typical Ca2+ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca2+ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca2+ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca2+ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca2+ oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca2+ oscillations. Conclusion: Congo red significantly modulates intracellular Ca2+ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug. PMID:25345744

  6. Pancreatic panniculitis as a presentation symptom of acinar cell carcinoma.

    PubMed

    de Frutos Rosa, Diego; Espinosa Taranilla, Laura; González de Canales de Simón, Pilar; Vélez Velázquez, María Dolores; Guirado Koch, Cristina

    2018-05-01

    Pancreatic panniculitis is a rare skin manifestation associated with pancreatic conditions. This condition has similar characteristics to those of other panniculitis types and its course parallels the triggering condition and may occasionally precede it. We report the case of a female patient with asymptomatic pancreatic panniculitis; the etiologic study identified a pancreatic acinar cell carcinoma with liver metastases.

  7. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    PubMed

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  8. Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

    PubMed Central

    Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

    2012-01-01

    Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

  9. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis*

    PubMed Central

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F. Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M.

    2016-01-01

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

  10. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

    PubMed

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

    2016-07-08

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

    PubMed

    Willemer, S; Köhler, H; Naumann, R; Kern, H F; Adler, G

    1990-01-01

    Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

  12. Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease

    PubMed Central

    Teos, LY; Zheng, C-Y; Liu, X; Swaim, WD; Goldsmith, CM; Cotrim, AP; Baum, BJ; Ambudkar, IS

    2017-01-01

    Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

  13. Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

    PubMed

    Song, Eun Ah; Lim, Joo Weon; Kim, Hyeyoung

    2017-07-01

    Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Copyright © 2017. Published by Elsevier Ltd.

  14. Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells.

    PubMed

    Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

    2012-07-01

    Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  15. Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells

    PubMed Central

    1982-01-01

    We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin- vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic lobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels. Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins. PMID:6185502

  16. Establishment and characterization of a new human acinar cell carcinoma cell line, Faraz-ICR, from pancreas.

    PubMed

    Rezaei, Marzieh; Hosseini, Ahmad; Nikeghbalian, Saman; Ghaderi, Abbas

    Basic research in the field of acinar cell carcinoma (ACC) as a rare neoplasm of the pancreas is dependent on the availability of pragmatic model such as new pancreatic cancer cell lines. Thus, establishment and characterization of new pancreatic cancer cell lines from ACC origin are deemed important. Faraz-ICR cell line was derived from a 58-years old woman with pancreatic acinar cell carcinoma by the collagenase digestion protocol. We characterized the cell line by examining its morphology and cytostructural and functional profile. Faraz-ICR has a doubling time of 35 hours and grows in soft agar with a colony-forming efficiency of 25%. The cell had nearly normal pattern of chromosomes in karyotype analysis and Comparative Genomic Hybridization (CGH) array analysis. Evaluation of cells by flowcytometry showed that Faraz-ICR is negative for EpCAM and mesenchymal markers in different passages, and has epithelial nature. Immunofluorescence staining revealed that cells were strongly positive for vimentin, desmin, ezrin, S100, nestin and they were negative for pan-cytokeratins, chromogranin and alpha smooth muscle actin. We were able to establish a new pancreatic carcinoma cell line with partial aspects of Epithelial-mesenchymal transition and aggressiveness. This cell line might be suitable for studying various anticancer drugs and protein profile aiming to see any possible tumor associated marker for ACC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

  17. RNA synthesis in the pancreatic acinar cells of aging mice as revealed by electron microscopic radioautography.

    PubMed

    Nagata, Tetsuji

    2012-01-01

    For the purpose of studying the aging changes of macromolecular synthesis in the pancreatic acinar cells of experimental animals, we studied 10 groups of aging mice during development and aging from fetal day 19 to postnatal month 24. They were injected with 3H-uridine, a precursor for RNA synthesis, sacrificed and the pancreatic tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating RNA synthesis in pancreatic acinar cells in respective aging groups were analyzed qualitatively. The number of mitochondria per cell, the number of labeled mitochondria with silver grains and the number of silver grains in each cell in respective aging groups were analyzed quantitatively in relation to the aging of animals. The results revealed that the RNA synthetic activity as expressed by the incorporations of RNA precursor, i.e., the number of silver grains in cell nuclei, cell organelles, changed due to the aging of animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each pancreatic acinar cell. It was demonstrated that the number of mitochondria, the number of labeled mitochondria and the labeling indices showing RNA synthesis at various ages increased from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile stage at postnatal year 1 to 2, indicating the aging changes. Based upon our findings, available literature on macromolecular synthesis in mitochondria of various cells are reviewed.

  18. Assessing the secretory capacity of pancreatic acinar cells.

    PubMed

    Geron, Erez; Schejter, Eyal D; Shilo, Ben-Zion

    2014-08-28

    Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

  19. Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

    PubMed

    Brown, David A; Melvin, James E; Yule, David I

    2003-11-01

    The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

  20. The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma

    PubMed Central

    Krah, Nathan M; De La O, Jean-Paul; Swift, Galvin H; Hoang, Chinh Q; Willet, Spencer G; Chen Pan, Fong; Cash, Gabriela M; Bronner, Mary P; Wright, Christopher VE; MacDonald, Raymond J; Murtaugh, L Charles

    2015-01-01

    Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas. DOI: http://dx.doi.org/10.7554/eLife.07125.001 PMID:26151762

  1. A Single Injection of Interleukin-1 Induces Reversible Aqueous-tear Deficiency, Lacrimal Gland Inflammation, and Acinar and Ductal Cell Proliferation

    PubMed Central

    Zoukhri, Driss; Macari, Elizabeth; Kublin, Claire L.

    2011-01-01

    Emerging studies from our laboratory demonstrate that interleukin-1 (IL-1) family members play a major role in impairing lacrimal gland functions. Here we have extended our investigations to observe the effects of IL-1 on aqueous tear production, lacrimal gland secretion, lacrimal gland histology, and acinar and ductal cell proliferation. We demonstrate that a single injection of IL-1 into the lacrimal glands inhibited neurally- as well as agonist-induced protein secretion resulting in decreased tear output. Meanwhile, IL-1 injection induced a severe, but reversible (7–13 days), inflammatory response that led to destruction of lacrimal gland acinar epithelial cells. Finally, we demonstrate that as the inflammatory response subsided and lacrimal gland secretion and tear production returned to normal levels, there was increased proliferation of acinar and ductal epithelial cells. Our work uncovers novel effects of IL-1 on lacrimal gland functions and the potential regenerative capacity of the mouse lacrimal gland. PMID:17362931

  2. Nicotine promotes initiation and progression of KRAS-induced pancreatic cancer via Gata6-dependent dedifferentiation of acinar cells in mice.

    PubMed

    Hermann, Patrick C; Sancho, Patricia; Cañamero, Marta; Martinelli, Paola; Madriles, Francesc; Michl, Patrick; Gress, Thomas; de Pascual, Ricardo; Gandia, Luis; Guerra, Carmen; Barbacid, Mariano; Wagner, Martin; Vieira, Catarina R; Aicher, Alexandra; Real, Francisco X; Sainz, Bruno; Heeschen, Christopher

    2014-11-01

    Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting

  3. New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

    PubMed

    Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

    2018-04-01

    The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

  4. Snail1 is required for the maintenance of the pancreatic acinar phenotype

    PubMed Central

    Loubat-Casanovas, Jordina; Peña, Raúl; Gonzàlez, Núria; Alba-Castellón, Lorena; Rosell, Santi; Francí, Clara; Navarro, Pilar; de Herreros, Antonio García

    2016-01-01

    The Snail1 transcriptional factor is required for correct embryonic development, yet its expression in adult animals is very limited and its functional roles are not evident. We have now conditionally inactivated Snail1 in adult mice and analyzed the phenotype of these animals. Snail1 ablation rapidly altered pancreas structure: one month after Snail1 depletion, acinar cells were markedly depleted, and pancreas accumulated adipose tissue. Snail1 expression was not detected in the epithelium but was in pancreatic mesenchymal cells (PMCs). Snail1 ablation in cultured PMCs downregulated the expression of several β-catenin/Tcf-4 target genes, modified the secretome of these cells and decreased their ability to maintain acinar markers in cultured pancreas cells. Finally, Snail1 deficiency modified the phenotype of pancreatic tumors generated in transgenic mice expressing c-myc under the control of the elastase promoter. Specifically, Snail1 depletion did not significantly alter the size of the tumors but accelerated acinar-ductal metaplasia. These results demonstrate that Snail1 is expressed in PMCs and plays a pivotal role in maintaining acinar cells within the pancreas in normal and pathological conditions. PMID:26735179

  5. Sirtuin-1 regulates acinar-to-ductal metaplasia and supports cancer cell viability in pancreatic cancer.

    PubMed

    Wauters, Elke; Sanchez-Arévalo Lobo, Victor J; Pinho, Andreia V; Mawson, Amanda; Herranz, Daniel; Wu, Jianmin; Cowley, Mark J; Colvin, Emily K; Njicop, Erna Ngwayi; Sutherland, Rob L; Liu, Tao; Serrano, Manuel; Bouwens, Luc; Real, Francisco X; Biankin, Andrew V; Rooman, Ilse

    2013-04-01

    The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. ©2012 AACR.

  6. Gata6 is required for complete acinar differentiation and maintenance of the exocrine pancreas in adult mice.

    PubMed

    Martinelli, Paola; Cañamero, Marta; del Pozo, Natalia; Madriles, Francesc; Zapata, Agustín; Real, Francisco X

    2013-10-01

    Previous studies have suggested an important role of the transcription factor Gata6 in endocrine pancreas, while GATA6 haploinsufficient inactivating mutations cause pancreatic agenesis in humans. We aimed to analyse the effects of Gata6 inactivation on pancreas development and function. We deleted Gata6 in all epithelial cells in the murine pancreas at the onset of its development. Acinar proliferation, apoptosis, differentiation and exocrine functions were assessed using reverse transcriptase quantitative PCR (RT-qPCR), chromatin immunoprecipitation, immunohistochemistry and enzyme assays. Adipocyte transdifferentiation was assessed using electron microscopy and genetic lineage tracing. Gata6 is expressed in all epithelial cells in the adult mouse pancreas but it is only essential for exocrine pancreas homeostasis: while dispensable for pancreatic development after e10.5, it is required for complete acinar differentiation, for establishment of polarity and for the maintenance of acinar cells in the adult. Gata6 regulates directly the promoter of genes coding for digestive enzymes and the transcription factors Rbpjl and Mist1. Upon pancreas-selective Gata6 inactivation, massive loss of acinar cells and fat replacement take place. This is accompanied by increased acinar apoptosis and proliferation, acinar-to-ductal metaplasia and adipocyte transdifferentiation. By contrast, the endocrine pancreas is spared. Our data show that Gata6 is required for the complete differentiation of acinar cells through multiple transcriptional regulatory mechanisms. In addition, it is required for the maintenance of the adult acinar cell compartment. Our studies suggest that GATA6 alterations may contribute to diseases of the human adult exocrine pancreas.

  7. Mechanisms Involved in Injury and Repair of the Murine Lacrimal Gland: Role of Programmed Cell Death and Mesenchymal Stem Cells

    PubMed Central

    Zoukhri, Driss

    2011-01-01

    The non-keratinized epithelia of the ocular surface are constantly challenged by environmental insults, such as smoke, dust, and airborne pathogens. Tears are the sole physical protective barrier for the ocular surface. Production of tears in inadequate quantity or of inadequate quality results in constant irritation of the ocular surface, leading to dry eye disease, also referred to as keratoconjunctivitis sicca (KCS). Inflammation of the lacrimal gland, such as occurs in Sjögren’s syndrome, sarcoidosis, chronic graft versus-host disease, and other pathological conditions, results in inadequate secretion of the aqueous layer of the tear film, and is a leading cause of dry eye disease. The hallmarks of lacrimal gland inflammation are the presence of immune cell infiltrates, loss of acinar epithelial cells (the secreting cells), and increased production of proinflammatory cytokines. To date, the mechanisms leading to acinar cell loss and the associated decline in lacrimal gland secretion are still poorly understood. It is also not understood why the remaining lacrimal gland cells are unable to proliferate in order to regenerate a functioning lacrimal gland. This article reviews recent advances in exocrine tissue injury and repair, with emphasis on the roles of programmed cell death and stem/progenitor cells. PMID:20427009

  8. Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

    PubMed

    Freedman, S D; Scheele, G A

    1994-03-23

    The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

  9. Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell

    PubMed Central

    1995-01-01

    Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell

  10. Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

    PubMed

    Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

    The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

  11. Hydrogen sulfide: a novel gaseous signaling molecule and intracellular Ca2+ regulator in rat parotid acinar cells.

    PubMed

    Moustafa, Amira; Habara, Yoshiaki

    2015-10-01

    In addition to nitric oxide (NO), hydrogen sulfide (H2S) is recognized as a crucial gaseous messenger that exerts many biological actions in various tissues. An attempt was made to assess the roles and underlying mechanisms of both gases in isolated rat parotid acinar cells. Ductal cells and some acinar cells were found to express NO and H2S synthases. Cevimeline, a muscarinic receptor agonist upregulated endothelial NO synthase in parotid tissue. NO and H2S donors increased the intracellular Ca(2+) concentration ([Ca(2+)]i). This was not affected by inhibitors of phospholipase C and inositol 1,4,5-trisphosphate receptors, but was decreased by blockers of ryanodine receptors (RyRs), soluble guanylyl cyclase, and protein kinase G. The H2S donor evoked NO production, which was decreased by blockade of NO synthases or phosphoinositide 3-kinase or by hypotaurine, an H2S scavenger. The H2S donor-induced [Ca(2+)]i increase was diminished by a NO scavenger or the NO synthases blocker. These results suggest that NO and H2S play important roles in regulating [Ca(2+)]i via soluble guanylyl cyclase-cGMP-protein kinase G-RyRs, but not via inositol 1,4,5-trisphosphate receptors. The effect of H2S may be partially through NO produced via phosphoinositide 3-kinase-Akt-endothelial NO synthase. It was concluded that both gases regulate [Ca(2+)]i in a synergistic way, mainly via RyRs in rat parotid acinar cells. Copyright © 2015 the American Physiological Society.

  12. Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas.

    PubMed

    Furukawa, Toru; Sakamoto, Hitomi; Takeuchi, Shoko; Ameri, Mitra; Kuboki, Yuko; Yamamoto, Toshiyuki; Hatori, Takashi; Yamamoto, Masakazu; Sugiyama, Masanori; Ohike, Nobuyuki; Yamaguchi, Hiroshi; Shimizu, Michio; Shibata, Noriyuki; Shimizu, Kyoko; Shiratori, Keiko

    2015-03-06

    Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we identified recurrent mutations of BRCA2 and FAT genes. BRCA2 showed somatic or germline premature termination mutations, with loss of the wild-type allele in 3 of 7 tumors. FAT1, FAT3, and FAT4 showed somatic or germline missense mutations in 4 of 7 tumors. The germline FAT mutations were with loss of the wild-type allele. Loss of BRCA2 expression was observed in 5 of 11 tumors. One patient with a BRCA2-mutated tumor experienced complete remission of liver metastasis following cisplatinum chemotherapy. In conclusion, acinar cell carcinomas show a distinct mutation pattern and often harbor somatic or germline mutations of BRCA2 and FAT genes. This result may warrant assessment of BRCA2 abrogation in patients with the carcinoma to determine their sensitivity to chemotherapy.

  13. Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development.

    PubMed

    Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

    2012-01-15

    During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development

    PubMed Central

    Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

    2011-01-01

    SUMMARY During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3+ progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3CreERT/+) and Neurog3-deficient (Neurog3CreERT/CreERT) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition endocrine progenitor cells arise from single bipotent progenitor already committed to the duct/endocrine lineages and not from domain of cells having both potentialities. PMID:22056785

  15. Case report: primary acinar cell carcinoma of the liver treated with multimodality therapy

    PubMed Central

    Basturk, Olca; Shia, Jinru; Klimstra, David S.; Alago, William; D’Angelica, Michael I.; Abou-Alfa, Ghassan K.; O’Reilly, Eileen M.; Lowery, Maeve A.

    2017-01-01

    We describe a case of primary acinar cell carcinoma (ACC) originating in the liver in a 54-year-old female, diagnosed following persistent abnormal elevated liver function. Imaging revealed two masses, one dominant lesion in the right hepatic lobe and another in segment IVA. A right hepatectomy was performed to remove the larger lesion, while the mass in segment IVA was unresectable due to its proximity to the left hepatic vein. Immunohistochemical staining showed positivity for trypsin and chymotrypsin. Postoperatively the patient underwent hepatic arterial embolization of the other unresectable lesion followed by FOLFOX chemotherapy. At 20 months from diagnosis the patient is currently under observation with a decreasing necrotic mass and no other disease evident. Based on histology, immunohistochemistry and radiological findings a diagnosis of primary ACC of the liver was made. Genomic assessment of somatic mutations within the patient’s tumor was also performed through next generation sequencing and findings were consistent with an acinar malignancy. This case highlights a rare tumor subtype treated with a combination of therapeutic modalities through a multidisciplinary approach. PMID:29184698

  16. Pancreatic Fat Accumulation, Fibrosis, and Acinar Cell Injury in the Zucker Diabetic Fatty Rat Fed a Chronic High-Fat Diet

    PubMed Central

    Matsuda, Akiko; Makino, Naohiko; Tozawa, Tomohiro; Shirahata, Nakao; Honda, Teiichiro; Ikeda, Yushi; Sato, Hideyuki; Ito, Miho; Kakizaki, Yasuharu; Akamatsu, Manabu; Ueno, Yoshiyuki; Kawata, Sumio

    2014-01-01

    Objective The histological alteration of the exocrine pancreas in obesity has not been clarified. In the present study, we investigated biochemical and histological changes in the exocrine pancreas of obese model rats. Methods Zucker lean rats were fed a standard diet, and Zucker diabetic fatty (ZDF) rats were divided into 2 groups fed a standard diet and a high-fat diet, respectively. These experimental groups were fed each of the diets from 6 weeks until 12, 18, 24 weeks of age. We performed blood biochemical assays and histological analysis of the pancreas. Results In the ZDF rats fed a high-fat diet, the ratio of accumulated pancreatic fat area relative to exocrine gland area was increased significantly at 18 weeks of age in comparison with the other 2 groups (P < 0.05), and lipid droplets were observed in acinar cells. Subsequently, at 24 weeks of age in this group, pancreatic fibrosis and the serum exocrine pancreatic enzyme levels were increased significantly relative to the other 2 groups (P < 0.01). Conclusions In ZDF rats fed a chronic high-fat diet, fat accumulates in pancreatic acinar cells, and this fatty change seems to be related to subsequent pancreatic fibrosis and acinar cell injury. PMID:24717823

  17. The exocrine pancreas: the acinar-ductal tango in physiology and pathophysiology.

    PubMed

    Hegyi, Peter; Petersen, Ole H

    2013-01-01

    There are many reviews of pancreatic acinar cell function and also of pancreatic duct function, but there is an almost total absence of synthetic reviews bringing the integrated functions of these two vitally and mutually interdependent cells together. This is what we have attempted to do in this chapter. In the first part, we review the normal integrated function of the acinar-ductal system, with particular emphasis on how regulation of one type of cell also influences the other cell type. In the second part, we review a range of pathological processes, particularly those involved in acute pancreatitis (AP), an often-fatal human disease in which the pancreas digests itself, in order to explore how malfunction of one of the cell types adversely affects the function of the other.

  18. Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

    PubMed

    Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

    2014-09-05

    Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. Themore » cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.« less

  20. Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar-to-ductal metaplasia.

    PubMed

    Bombardo, Marta; Saponara, Enrica; Malagola, Ermanno; Chen, Rong; Seleznik, Gitta M; Haumaitre, Cecile; Quilichini, Evans; Zabel, Anja; Reding, Theresia; Graf, Rolf; Sonda, Sabrina

    2017-11-01

    Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease. We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants. HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis. These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease. © 2017 The British Pharmacological Society.

  1. Ethanol exerts dual effects on calcium homeostasis in CCK-8-stimulated mouse pancreatic acinar cells.

    PubMed

    Fernández-Sánchez, Marcela; del Castillo-Vaquero, Angel; Salido, Ginés M; González, Antonio

    2009-10-30

    A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells. Changes in [Ca2+]i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H2DCFDA, respectively. Ethanol, in the concentration range from 1 to 50 mM, evoked an oscillatory pattern in [Ca2+]i. In addition, ethanol evoked reactive oxygen species generation (ROS) production. Stimulation of cells with 1 nM or 20 pM CCK-8, respectively led to a transient change and oscillations in [Ca2+]i. In the presence of ethanol a transformation of 20 pM CCK-8-evoked physiological oscillations into a single transient increase in [Ca2+]i in the majority of cells was observed. Whereas, in response to 1 nM CCK-8, the total Ca2+ mobilization was significantly increased by ethanol pre-treatment. Preincubation of cells with 1 mM 4-MP, an inhibitor of alcohol dehydrogenase, or 10 microM of the antioxidant cinnamtannin B-1, reverted the effect of ethanol on total Ca2+ mobilization evoked by 1 nM CCK-8. Cinnamtannin B-1 blocked ethanol-evoked ROS production. ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.

  2. Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

    PubMed

    Weber, Heike; Jonas, Ludwig; Wakileh, Michael; Krüger, Burkhard

    2014-03-01

    The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

  3. Pancreatic acinar cells: ionic dependence of acetylcholine-induced membrane potential and resistance change.

    PubMed Central

    Nishiyama, A; Petersen, O H

    1975-01-01

    1. Intracellular recordings of membrane potential, input resistance and time constant have been made in vitro from the exocrine acinar cells of the mouse pancreas using glass micro-electrodes. The acinar cells were stimulated by acetylcholine (ACh). In some cases ACh was simply directly added to the tissue superfusion bath, in other experiments ACh was applied locally to pancreatic acini by micro-iontophoresis. 2. Current-voltage relations were investigated by injecting rectangular de- or hyperpolarizing current pulses through the recording micro-electrode. Within a relatively wide range (-20 to -70 mV) there was a linear relation between injected current and change in membrane potential. The slope of such linear curves corresponded to an input resistance of about 3-8 M omega. The membrane time constant was about 5-10 msec. 3. ACh depolarized the cell membrane and caused a marked reduction of input resistance and time constant. The minimum latency of the ACh-induced depolarization (microiontophoretic application) was 100-300 msec. Maximal depolarization was about 20 mV. The effect of this local ACh application was abolished by atropine (1-4 x 10-6 M). The blocking effect of atropine was fully reversible. 4. Stimulating with ACh during the passage of large depolarizing current pulses made it possible simultaneously to observe the effect of ACh at two different levels of resting potential (RP). At the spontaneous RP of about minus 40 mV ACh evoked a depolarization of usual magnitude (15-20 mV) while at the artificially displaced level of about -10 mV a small hyperpolarization (about 5 mV) was observed. It therefore appears that the reversal potential of the transmitter equilibrium potential is about -20 mV. 5. Replacement of the superfusion fluid C1 by sulphate or methylsulphate caused an initial short-lasting depolarization, thereafter the normal resting potential was reassumed... PMID:1142124

  4. Prostate specific antigen and acinar density: a new dimension, the "Prostatocrit".

    PubMed

    Robinson, Simon; Laniado, Marc; Montgomery, Bruce

    2017-01-01

    Prostate-specific antigen densities have limited success in diagnosing prostate cancer. We emphasise the importance of the peripheral zone when considered with its cellular constituents, the "prostatocrit". Using zonal volumes and asymmetry of glandular acini, we generate a peripheral zone acinar volume and density. With the ratio to the whole gland, we can better predict high grade and all grade cancer. We can model the gland into its acinar and stromal elements. This new "prostatocrit" model could offer more accurate nomograms for biopsy. 674 patients underwent TRUS and biopsy. Whole gland and zonal volumes were recorded. We compared ratio and acinar volumes when added to a "clinic" model using traditional PSA density. Univariate logistic regression was used to find significant predictors for all and high grade cancer. Backwards multiple logistic regression was used to generate ROC curves comparing the new model to conventional density and PSA alone. Prediction of all grades of prostate cancer: significant variables revealed four significant "prostatocrit" parameters: log peripheral zone acinar density; peripheral zone acinar volume/whole gland acinar volume; peripheral zone acinar density/whole gland volume; peripheral zone acinar density. Acinar model (AUC 0.774), clinic model (AUC 0.745) (P=0.0105). Prediction of high grade prostate cancer: peripheral zone acinar density ("prostatocrit") was the only significant density predictor. Acinar model (AUC 0.811), clinic model (AUC 0.769) (P=0.0005). There is renewed use for ratio and "prostatocrit" density of the peripheral zone in predicting cancer. This outperforms all traditional density measurements. Copyright® by the International Brazilian Journal of Urology.

  5. Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

    PubMed

    Sugiya, H; Hara-Yokoyama, M; Furuyama, S

    1992-03-30

    When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.

  6. Role of alveolar topology on acinar flows and convective mixing.

    PubMed

    Hofemeier, Philipp; Sznitman, Josué

    2014-06-01

    Due to experimental challenges, computational simulations are often sought to quantify inhaled aerosol transport in the pulmonary acinus. Commonly, these are performed using generic alveolar topologies, including spheres, toroids, and polyhedra, to mimic the complex acinar morphology. Yet, local acinar flows and ensuing particle transport are anticipated to be influenced by the specific morphological structures. We have assessed a range of acinar models under self-similar breathing conditions with respect to alveolar flow patterns, convective flow mixing, and deposition of fine particles (1.3 μm diameter). By tracking passive tracers over cumulative breathing cycles, we find that irreversible flow mixing correlates with the location and strength of the recirculating vortex inside the cavity. Such effects are strongest in proximal acinar generations where the ratio of alveolar to ductal flow rates is low and interalveolar disparities are most apparent. Our results for multi-alveolated acinar ducts highlight that fine 1 μm inhaled particles subject to alveolar flows are sensitive to the alveolar topology, underlining interalveolar disparities in particle deposition patterns. Despite the simplicity of the acinar models investigated, our findings suggest that alveolar topologies influence more significantly local flow patterns and deposition sites of fine particles for upper generations emphasizing the importance of the selected acinar model. In distal acinar generations, however, the alveolar geometry primarily needs to mimic the space-filling alveolar arrangement dictated by lung morphology.

  7. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

    PubMed

    Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

    2015-04-01

    The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Megamitochondria in the serous acinar cells of the submandibular gland of the neotropical fruit bat, Artibeus obscurus.

    PubMed

    Tandler, B; Nagato, T; Phillips, C J

    1997-05-01

    As part of a continuing investigation of the comparative ultrastructure of chiropteran salivary glands, we examined the submandibular glands of eight species of neotropical fruit bats in the genus Artibeus. We previously described secretory granules of unusual substructure in the seromucous demilunar cells of this organ in some species in this genus. In the present study, we turned our attention to the serous acinar cells in the same glands. Specimens of eight species of Artibeus were collected in neotropical localities. Salivary glands were extirpated in the field and thin slices were fixed by immersion in triple aldehyde-DMSO or in modified half-strength Karnovsky's fixative. Tissues were further processed for electron microscopy by conventional means. In contrast to seromucous cells, which exhibit species-specific diversification in bats of this genus, the secretory apparatus and secretory granules in the serous acinar cells are highly conserved across all seven species. The single exception involves the mitochondria in one species. In this instance, some of the serous cell mitochondria in Artibeus obscurus are modified into megamitochondria. Such organelles usually have short, peripheral cristae; a laminar inclusion is present in the matrix compartment of every outsized organelle. Inclusions of this nature never are present in normal-size mitochondria in the serous cells. None of the megamitochondria were observed in the process of degeneration. The giant mitochondria in A. obscurus have a matrical structure that is radically different from that of the only other megamitochondria reported to occur in bat salivary glands. The factors that lead to variation in megamitochondrial substructure in different species, as well as the functional capacities of such giant organelles, are unknown.

  9. The role of anisotropic expansion for pulmonary acinar aerosol deposition

    PubMed Central

    Hofemeier, Philipp; Sznitman, Josué

    2016-01-01

    Lung deformations at the local pulmonary acinar scale are intrinsically anisotropic. Despite progress in imaging modalities, the true heterogeneous nature of acinar expansion during breathing remains controversial, where our understanding of inhaled aerosol deposition still widely emanates from studies under self-similar, isotropic wall motions. Building on recent 3D models of multi-generation acinar networks, we explore in numerical simulations how different hypothesized scenarios of anisotropic expansion influence deposition outcomes of inhaled aerosols in the acinar depths. While the broader range of particles acknowledged to reach the acinar region (dp = 0.005–5.0 μm) are largely unaffected by the details of anisotropic expansion under tidal breathing, our results suggest nevertheless that anisotropy modulates the deposition sites and fractions for a narrow band of sub-micron particles (dp ~ 0.5–0.75 μm), where the fate of aerosols is greatly intertwined with local convective flows. Our findings underscore how intrinsic aerosol motion (i.e. diffusion, sedimentation) undermines the role of anisotropic wall expansion that is often attributed in determining aerosol mixing and acinar deposition. PMID:27614613

  10. Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling.

    PubMed

    Yago, Maria D; Diaz, Ricardo J; Ramirez, Rolando; Martinez, Maria A; Mañas, Mariano; Martinez-Victoria, Emilio

    2004-02-01

    The effects of dietary lipids on the fatty acid composition of rat pancreatic membranes and acinar cell function were investigated. Weaning rats were fed for 8 weeks on one of two diets which contained 100 g virgin olive oil (OO) or sunflower-seed oil (SO)/kg. Pancreatic plasma membranes were isolated and fatty acids determined. Amylase secretion and cytosolic concentrations of Ca(2+) and Mg(2+) were measured in pancreatic acini. Membrane fatty acids were profoundly affected by the diets; the rats fed OO had higher levels of 18 : 1n-9 (42.86 (sem 1.99) %) and total MUFA compared with the animals fed SO (25.37 (sem 1.11) %). Reciprocally, the SO diet resulted in greater levels of total and n-6 PUFA than the OO diet. The most striking effect was observed for 18 : 2n-6 (SO 17.88 (sem 1.32) %; OO 4.45 (sem 0.60) %), although the levels of 20 : 4n-6 were also different. The proportion of total saturated fatty acids was similar in both groups, and there was only a slight, not significant (P=0.098), effect on the unsaturation index. Compared with the OO group, acinar cells from the rats fed SO secreted more amylase at rest but less in response to cholecystokinin octapeptide, and this was paralleled by reduced Ca(2+) responses to the secretagogue. The results confirm that rat pancreatic cell membranes are strongly influenced by the type of dietary fat consumed and this is accompanied by a modulation of the secretory activity of pancreatic acinar cells that involves, at least in part, Ca(2+) signalling.

  11. Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

    PubMed

    Al-Adsani, Amani; Burke, Zoë D; Eberhard, Daniel; Lawrence, Katherine L; Shen, Chia-Ning; Rustgi, Anil K; Sakaue, Hiroshi; Farrant, J Mark; Tosh, David

    2010-10-27

    The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

  12. IL-1 beta and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release.

    PubMed

    Tanda, N; Ohyama, H; Yamakawa, M; Ericsson, M; Tsuji, T; McBride, J; Elovic, A; Wong, D T; Login, G R

    1998-01-01

    Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1 beta and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10(-5) M norepinephrine at 37 degrees C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1 beta and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1 beta (P < 0.03) and IL-6 (P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1 beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1 beta and IL-6 and release these cytokines from their granules after alpha- and beta-adrenergic stimulation.

  13. The role of anisotropic expansion for pulmonary acinar aerosol deposition.

    PubMed

    Hofemeier, Philipp; Sznitman, Josué

    2016-10-03

    Lung deformations at the local pulmonary acinar scale are intrinsically anisotropic. Despite progress in imaging modalities, the true heterogeneous nature of acinar expansion during breathing remains controversial, where our understanding of inhaled aerosol deposition still widely emanates from studies under self-similar, isotropic wall motions. Building on recent 3D models of multi-generation acinar networks, we explore in numerical simulations how different hypothesized scenarios of anisotropic expansion influence deposition outcomes of inhaled aerosols in the acinar depths. While the broader range of particles acknowledged to reach the acinar region (d p =0.005-5.0μm) are largely unaffected by the details of anisotropic expansion under tidal breathing, our results suggest nevertheless that anisotropy modulates the deposition sites and fractions for a narrow band of sub-micron particles (d p ~0.5-0.75μm), where the fate of aerosols is greatly intertwined with local convective flows. Our findings underscore how intrinsic aerosol motion (i.e. diffusion, sedimentation) undermines the role of anisotropic wall expansion that is often attributed in determining aerosol mixing and acinar deposition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Tumour necrosis factor α secretion induces protease activation and acinar cell necrosis in acute experimental pancreatitis in mice.

    PubMed

    Sendler, Matthias; Dummer, Annegret; Weiss, Frank U; Krüger, Burkhard; Wartmann, Thomas; Scharffetter-Kochanek, Karin; van Rooijen, Nico; Malla, Sudarshan Ravi; Aghdassi, Ali; Halangk, Walter; Lerch, Markus M; Mayerle, Julia

    2013-03-01

    Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases. Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 μg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies. Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini. The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings

  15. Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

    PubMed Central

    Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

    2016-01-01

    The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

  16. Dictyostelium cell death

    PubMed Central

    Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L.; Golstein, Pierre

    2003-01-01

    Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of “paddle” cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells. PMID:12654899

  17. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    PubMed

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  18. Mixed acinar-endocrine carcinoma of the pancreas: new clinical and pathological features in a contemporary series.

    PubMed

    Yu, Run; Jih, Lily; Zhai, Jing; Nissen, Nicholas N; Colquhoun, Steven; Wolin, Edward; Dhall, Deepti

    2013-04-01

    The objective of this study was to characterize the novel clinical and pathological features of mixed acinar-endocrine carcinoma of the pancreas. This was a retrospective review of medical records and surgical pathology specimens of patients with a diagnosis of mixed acinar-endocrine carcinoma of the pancreas at Cedars-Sinai Medical Center between 2005 and 2011. Additional immunohistochemistry was performed on the specimens of some patients. Five patients were identified. The median age at presentation was 74 years (range, 59-89 years), and all patients were male. The presenting symptoms were all related to tumor mass effects. The median size of the tumor was 10 cm (range, 3.9-16 cm). Preoperative clinical diagnosis aided by fine-needle aspiration biopsy was incorrect in all 5 cases. Most tumors (3/5) exhibited predominantly endocrine differentiation without hormonal production. Only 10% to 30% of cells were truly amphicrine, whereas most were differentiated into either endocrine or acinar phenotype. The clinical behavior ranged from moderate to aggressive with postoperative survival from 2.5 months to more than 3 years. Four patients received neoadjuvant or adjuvant chemotherapy with variable responses. Mixed acinar-endocrine carcinoma of the pancreas appears to be not uncommon in men, may harbor predominantly endocrine component, is often misdiagnosed by cytology, and exhibits variable clinical behavior. Mixed acinar-endocrine carcinoma of the pancreas should be considered in older patients with sizable pancreatic mass and may warrant aggressive surgical resection and chemotherapy.

  19. Murine pulmonary acinar mechanics during quasi-static inflation using synchrotron refraction-enhanced computed tomography.

    PubMed

    Sera, Toshihiro; Yokota, Hideo; Tanaka, Gaku; Uesugi, Kentaro; Yagi, Naoto; Schroter, Robert C

    2013-07-15

    We visualized pulmonary acini in the core regions of the mouse lung in situ using synchrotron refraction-enhanced computed tomography (CT) and evaluated their kinematics during quasi-static inflation. This CT system (with a cube voxel of 2.8 μm) allows excellent visualization of not just the conducting airways, but also the alveolar ducts and sacs, and tracking of the acinar shape and its deformation during inflation. The kinematics of individual alveoli and alveolar clusters with a group of terminal alveoli is influenced not only by the connecting alveolar duct and alveoli, but also by the neighboring structures. Acinar volume was not a linear function of lung volume. The alveolar duct diameter changed dramatically during inflation at low pressures and remained relatively constant above an airway pressure of ∼8 cmH2O during inflation. The ratio of acinar surface area to acinar volume indicates that acinar distension during low-pressure inflation differed from that during inflation over a higher pressure range; in particular, acinar deformation was accordion-like during low-pressure inflation. These results indicated that the alveoli and duct expand differently as total acinar volume increases and that the alveolar duct may expand predominantly during low-pressure inflation. Our findings suggest that acinar deformation in the core regions of the lung is complex and heterogeneous.

  20. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

    PubMed

    Galluzzi, Lorenzo; Vitale, Ilio; Aaronson, Stuart A; Abrams, John M; Adam, Dieter; Agostinis, Patrizia; Alnemri, Emad S; Altucci, Lucia; Amelio, Ivano; Andrews, David W; Annicchiarico-Petruzzelli, Margherita; Antonov, Alexey V; Arama, Eli; Baehrecke, Eric H; Barlev, Nickolai A; Bazan, Nicolas G; Bernassola, Francesca; Bertrand, Mathieu J M; Bianchi, Katiuscia; Blagosklonny, Mikhail V; Blomgren, Klas; Borner, Christoph; Boya, Patricia; Brenner, Catherine; Campanella, Michelangelo; Candi, Eleonora; Carmona-Gutierrez, Didac; Cecconi, Francesco; Chan, Francis K-M; Chandel, Navdeep S; Cheng, Emily H; Chipuk, Jerry E; Cidlowski, John A; Ciechanover, Aaron; Cohen, Gerald M; Conrad, Marcus; Cubillos-Ruiz, Juan R; Czabotar, Peter E; D'Angiolella, Vincenzo; Dawson, Ted M; Dawson, Valina L; De Laurenzi, Vincenzo; De Maria, Ruggero; Debatin, Klaus-Michael; DeBerardinis, Ralph J; Deshmukh, Mohanish; Di Daniele, Nicola; Di Virgilio, Francesco; Dixit, Vishva M; Dixon, Scott J; Duckett, Colin S; Dynlacht, Brian D; El-Deiry, Wafik S; Elrod, John W; Fimia, Gian Maria; Fulda, Simone; García-Sáez, Ana J; Garg, Abhishek D; Garrido, Carmen; Gavathiotis, Evripidis; Golstein, Pierre; Gottlieb, Eyal; Green, Douglas R; Greene, Lloyd A; Gronemeyer, Hinrich; Gross, Atan; Hajnoczky, Gyorgy; Hardwick, J Marie; Harris, Isaac S; Hengartner, Michael O; Hetz, Claudio; Ichijo, Hidenori; Jäättelä, Marja; Joseph, Bertrand; Jost, Philipp J; Juin, Philippe P; Kaiser, William J; Karin, Michael; Kaufmann, Thomas; Kepp, Oliver; Kimchi, Adi; Kitsis, Richard N; Klionsky, Daniel J; Knight, Richard A; Kumar, Sharad; Lee, Sam W; Lemasters, John J; Levine, Beth; Linkermann, Andreas; Lipton, Stuart A; Lockshin, Richard A; López-Otín, Carlos; Lowe, Scott W; Luedde, Tom; Lugli, Enrico; MacFarlane, Marion; Madeo, Frank; Malewicz, Michal; Malorni, Walter; Manic, Gwenola; Marine, Jean-Christophe; Martin, Seamus J; Martinou, Jean-Claude; Medema, Jan Paul; Mehlen, Patrick; Meier, Pascal; Melino, Sonia; Miao, Edward A; Molkentin, Jeffery D; Moll, Ute M; Muñoz-Pinedo, Cristina; Nagata, Shigekazu; Nuñez, Gabriel; Oberst, Andrew; Oren, Moshe; Overholtzer, Michael; Pagano, Michele; Panaretakis, Theocharis; Pasparakis, Manolis; Penninger, Josef M; Pereira, David M; Pervaiz, Shazib; Peter, Marcus E; Piacentini, Mauro; Pinton, Paolo; Prehn, Jochen H M; Puthalakath, Hamsa; Rabinovich, Gabriel A; Rehm, Markus; Rizzuto, Rosario; Rodrigues, Cecilia M P; Rubinsztein, David C; Rudel, Thomas; Ryan, Kevin M; Sayan, Emre; Scorrano, Luca; Shao, Feng; Shi, Yufang; Silke, John; Simon, Hans-Uwe; Sistigu, Antonella; Stockwell, Brent R; Strasser, Andreas; Szabadkai, Gyorgy; Tait, Stephen W G; Tang, Daolin; Tavernarakis, Nektarios; Thorburn, Andrew; Tsujimoto, Yoshihide; Turk, Boris; Vanden Berghe, Tom; Vandenabeele, Peter; Vander Heiden, Matthew G; Villunger, Andreas; Virgin, Herbert W; Vousden, Karen H; Vucic, Domagoj; Wagner, Erwin F; Walczak, Henning; Wallach, David; Wang, Ying; Wells, James A; Wood, Will; Yuan, Junying; Zakeri, Zahra; Zhivotovsky, Boris; Zitvogel, Laurence; Melino, Gerry; Kroemer, Guido

    2018-03-01

    Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

  1. Cell Death and Cell Death Responses in Liver Disease: Mechanisms and Clinical Relevance

    PubMed Central

    Luedde, Tom; Kaplowitz, Neil; Schwabe, Robert F.

    2015-01-01

    Summary Hepatocellular death is present in almost all types of human liver disease and is used as a sensitive parameter for the detection of acute and chronic liver disease of viral, toxic, metabolic, or autoimmune origin. Clinical data and animal models suggest that hepatocyte death is the key trigger of liver disease progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Modes of hepatocellular death differ substantially between liver diseases. Different modes of cell death such as apoptosis, necrosis, and necroptosis trigger specific cell death responses and promote progression of liver disease through distinct mechanisms. In this review, we first discuss molecular mechanisms by which different modes of cell death, damage-associated molecular patterns, and specific cell death responses contribute to the development of liver disease. We then review the clinical relevance of cell death, focusing on biomarkers; the contribution of cell death to drug-induced, viral, and fatty liver disease and liver cancer; and evidence for cell death pathways as therapeutic targets. PMID:25046161

  2. Cell death proteomics database: consolidating proteomics data on cell death.

    PubMed

    Arntzen, Magnus Ø; Bull, Vibeke H; Thiede, Bernd

    2013-05-03

    Programmed cell death is a ubiquitous process of utmost importance for the development and maintenance of multicellular organisms. More than 10 different types of programmed cell death forms have been discovered. Several proteomics analyses have been performed to gain insight in proteins involved in the different forms of programmed cell death. To consolidate these studies, we have developed the cell death proteomics (CDP) database, which comprehends data from apoptosis, autophagy, cytotoxic granule-mediated cell death, excitotoxicity, mitotic catastrophe, paraptosis, pyroptosis, and Wallerian degeneration. The CDP database is available as a web-based database to compare protein identifications and quantitative information across different experimental setups. The proteomics data of 73 publications were integrated and unified with protein annotations from UniProt-KB and gene ontology (GO). Currently, more than 6,500 records of more than 3,700 proteins are included in the CDP. Comparing apoptosis and autophagy using overrepresentation analysis of GO terms, the majority of enriched processes were found in both, but also some clear differences were perceived. Furthermore, the analysis revealed differences and similarities of the proteome between autophagosomal and overall autophagy. The CDP database represents a useful tool to consolidate data from proteome analyses of programmed cell death and is available at http://celldeathproteomics.uio.no.

  3. Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

    PubMed

    Fujiwara, Sachiko; Matsui, Tsubasa S; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

    2018-01-01

    Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

  4. Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells

    PubMed Central

    Matsui, Tsubasa S.; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

    2018-01-01

    Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330–1057) of Solo binds to the C-terminal region (1451–1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization. PMID:29672603

  5. Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

    PubMed Central

    Ramnath, Raina Devi; Sun, Jia; Adhikari, Sharmila; Bhatia, Madhav

    2007-01-01

    Abstract Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini. PMID:18205703

  6. Quantitative characterization of the protein contents of the exocrine pancreatic acinar cell by soft x-ray microscopy and advanced digital imaging methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Loo, Jr., Billy W.

    2000-06-01

    The study of the exocrine pancreatic acinar cell has been central to the development of models of many cellular processes, especially of protein transport and secretion. Traditional methods used to examine this system have provided a wealth of qualitative information from which mechanistic models have been inferred. However they have lacked the ability to make quantitative measurements, particularly of the distribution of protein in the cell, information critical for grounding of models in terms of magnitude and relative significance. This dissertation describes the development and application of new tools that were used to measure the protein content of the majormore » intracellular compartments in the acinar cell, particularly the zymogen granule. Soft x-ray microscopy permits image formation with high resolution and contrast determined by the underlying protein content of tissue rather than staining avidity. A sample preparation method compatible with x-ray microscopy was developed and its properties evaluated. Automatic computerized methods were developed to acquire, calibrate, and analyze large volumes of x-ray microscopic images of exocrine pancreatic tissue sections. Statistics were compiled on the protein density of several organelles, and on the protein density, size, and spatial distribution of tens of thousands of zymogen granules. The results of these measurements, and how they compare to predictions of different models of protein transport, are discussed.« less

  7. Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression.

    PubMed

    Ferreira, Rute M M; Sancho, Rocio; Messal, Hendrik A; Nye, Emma; Spencer-Dene, Bradley; Stone, Richard K; Stamp, Gordon; Rosewell, Ian; Quaglia, Alberto; Behrens, Axel

    2017-10-24

    The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs), duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development. Copyright © 2017 Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

  8. Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer.

    PubMed

    Haffner, Michael C; Guner, Gunes; Taheri, Diana; Netto, George J; Palsgrove, Doreen N; Zheng, Qizhi; Guedes, Liana Benevides; Kim, Kunhwa; Tsai, Harrison; Esopi, David M; Lotan, Tamara L; Sharma, Rajni; Meeker, Alan K; Chinnaiyan, Arul M; Nelson, William G; Yegnasubramanian, Srinivasan; Luo, Jun; Mehra, Rohit; Antonarakis, Emmanuel S; Drake, Charles G; De Marzo, Angelo M

    2018-06-01

    Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  9. Experimental evidence of age-related adaptive changes in human acinar airways

    PubMed Central

    Quirk, James D.; Sukstanskii, Alexander L.; Woods, Jason C.; Lutey, Barbara A.; Conradi, Mark S.; Gierada, David S.; Yusen, Roger D.; Castro, Mario

    2015-01-01

    The progressive decline of lung function with aging is associated with changes in lung structure at all levels, from conducting airways to acinar airways (alveolar ducts and sacs). While information on conducting airways is becoming available from computed tomography, in vivo information on the acinar airways is not conventionally available, even though acini occupy 95% of lung volume and serve as major gas exchange units of the lung. The objectives of this study are to measure morphometric parameters of lung acinar airways in living adult humans over a broad range of ages by using an innovative MRI-based technique, in vivo lung morphometry with hyperpolarized 3He gas, and to determine the influence of age-related differences in acinar airway morphometry on lung function. Pulmonary function tests and MRI with hyperpolarized 3He gas were performed on 24 healthy nonsmokers aged 19-71 years. The most significant age-related difference across this population was a 27% loss of alveolar depth, h, leading to a 46% increased acinar airway lumen radius, hence, decreased resistance to acinar air transport. Importantly, the data show a negative correlation between h and the pulmonary function measures forced expiratory volume in 1 s and forced vital capacity. In vivo lung morphometry provides unique information on age-related changes in lung microstructure and their influence on lung function. We hypothesize that the observed reduction of alveolar depth in subjects with advanced aging represents a remodeling process that might be a compensatory mechanism, without which the pulmonary functional decline due to other biological factors with advancing age would be significantly larger. PMID:26542518

  10. Activated macrophages create lineage-specific microenvironments for pancreatic acinar- and β-cell regeneration in mice.

    PubMed

    Criscimanna, Angela; Coudriet, Gina M; Gittes, George K; Piganelli, Jon D; Esni, Farzad

    2014-11-01

    Although the cells that contribute to pancreatic regeneration have been widely studied, little is known about the mediators of this process. During tissue regeneration, infiltrating macrophages debride the site of injury and coordinate the repair response. We investigated the role of macrophages in pancreatic regeneration in mice. We used a saporin-conjugated antibody against CD11b to reduce the number of macrophages in mice following diphtheria toxin receptor-mediated cell ablation of pancreatic cells, and evaluated the effects on pancreatic regeneration. We analyzed expression patterns of infiltrating macrophages after cell-specific injury or from the pancreas of nonobese diabetic mice. We developed an in vitro culture system to study the ability of macrophages to induce cell-specific regeneration. Depletion of macrophages impaired pancreatic regeneration. Macrophage polarization, as assessed by expression of tumor necrosis factor-α, interleukin 6, interleukin 10, and CD206, depended on the type of injury. The signals provided by polarized macrophages promoted lineage-specific generation of acinar or endocrine cells. Macrophage from nonobese diabetic mice failed to provide signals necessary for β-cell generation. Macrophages produce cell type-specific signals required for pancreatic regeneration in mice. Additional study of these processes and signals might lead to new approaches for treating type 1 diabetes or pancreatitis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  11. HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion.

    PubMed

    Lee, Robert J; Harlow, Janice M; Limberis, Maria P; Wilson, James M; Foskett, J Kevin

    2008-07-01

    Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3

  12. Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium-driven bile uptake.

    PubMed

    Ferdek, Pawel E; Jakubowska, Monika A; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

    2016-11-01

    uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca 2+ and significantly reduced in the absence of Na + , showing that bile-dependent cell death was a downstream event of Ca 2+ signals. Finally, combined application of TLC-S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC-S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells. © 2016 The Authors The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

  13. Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

    PubMed Central

    Jakubowska, Monika A.; Gerasimenko, Julia V.; Gerasimenko, Oleg V.; Petersen, Ole H.

    2016-01-01

    +‐dependent bile acid uptake mechanism in stellate cells. Bile acid treatment caused necrosis predominantly in stellate cells, which was abolished by removal of extracellular Ca2+ and significantly reduced in the absence of Na+, showing that bile‐dependent cell death was a downstream event of Ca2+ signals. Finally, combined application of TLC‐S and the inflammatory mediator bradykinin caused more extensive necrosis in both stellate and acinar cells than TLC‐S alone. Our findings shed new light on the mechanism by which bile acids promote pancreatic pathology. This involves not only signalling in acinar cells but also in stellate cells. PMID:27406326

  14. EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

    PubMed Central

    Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

    2013-01-01

    The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

  15. Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells.

    PubMed

    Yago, María D; Díaz, Ricardo J; Martínez, María A; Audi, Nama'a; Naranjo, José A; Martínez-Victoria, Emilio; Mañas, Mariano

    2006-04-01

    Olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated. This study aimed to clarify the mechanism of pancreatic adaptation to dietary fat. For this purpose, we examined whether dietary-induced modification of pancreatic membranes affects acinar cell function in response to the secretagogue acetylcholine (ACh). Weaning male Wistar rats were assigned to one of two experimental groups and fed for 8 weeks with a commercial chow (C) or a semisynthetic diet containing virgin olive oil as dietary fat (OO). The fatty acid composition of pancreatic plasma membranes was determined by gas-liquid chromatography. For assessment of secretory function, viable acini were incubated with ACh and amylase of supernatant was further assayed with a substrate reagent. Changes in cytosolic Ca(2+) concentration in response to ACh were measured by fura-2 AM fluorimetry. Compared to C rats, pancreatic cell membranes of OO rats had a higher level of monounsaturated fatty acids and a lower level of both saturated and polyunsaturated fatty acids, thus, reflecting the type of dietary fat given. Net amylase secretion in response to ACh was greatly enhanced after OO feeding, although this was not paralleled by enhancement of ACh-evoked Ca(2+) peak increases. In conclusion, chronic intake of diets that differ in the fat type influences not only the fatty acid composition of rat pancreatic membranes but also the responsiveness of acinar cells to ACh. This mechanism may be, at least in part, responsible for the adaptation of the exocrine pancreas to the type of fat available.

  16. Dead Cert: Measuring Cell Death.

    PubMed

    Crowley, Lisa C; Marfell, Brooke J; Scott, Adrian P; Boughaba, Jeanne A; Chojnowski, Grace; Christensen, Melinda E; Waterhouse, Nigel J

    2016-12-01

    Many cells in the body die at specific times to facilitate healthy development or because they have become old, damaged, or infected. Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer. The ability to objectively measure cell death in a laboratory setting is therefore essential to understanding and investigating the causes and treatments of many human diseases and disorders. Often, it is sufficient to know the extent of cell death in a sample; however, the mechanism of death may also have implications for disease progression, treatment, and the outcomes of experimental investigations. There are a myriad of assays available for measuring the known forms of cell death, including apoptosis, necrosis, autophagy, necroptosis, anoikis, and pyroptosis. Here, we introduce a range of assays for measuring cell death in cultured cells, and we outline basic techniques for distinguishing healthy cells from apoptotic or necrotic cells-the two most common forms of cell death. We also provide personal insight into where these assays may be useful and how they may or may not be used to distinguish apoptotic cell death from other death modalities. © 2016 Cold Spring Harbor Laboratory Press.

  17. Deletion Of XIAP reduces the severity of acute pancreatitis via regulation of cell death and nuclear factor-κB activity

    PubMed Central

    Liu, Yong; Chen, Xiao-Dong; Yu, Jiang; Chi, Jun-Lin; Long, Fei-Wu; Yang, Hong-Wei; Chen, Ke-Ling; Lv, Zhao-Ying; Zhou, Bin; Peng, Zhi-Hai; Sun, Xiao-Feng; Li, Yuan; Zhou, Zong-Guang

    2017-01-01

    Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that

  18. HCO3− Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

    PubMed Central

    Lee, Robert J.; Harlow, Janice M.; Limberis, Maria P.; Wilson, James M.; Foskett, J. Kevin

    2008-01-01

    Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl− secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl− secretion was accompanied by secretion of HCO3−, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 ± 0.01 in physiological medium (5% CO2–25 mM HCO3−). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl− content revealed by cell shrinkage, reflecting Cl− secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2–HCO3−-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3− efflux by ion substitution or exposure to the Cl− channel inhibitor niflumic acid (100 μM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3− during Ca2+-evoked fluid

  19. A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland

    PubMed Central

    Catalán, Marcelo A.; Kondo, Yusuke; Peña-Munzenmayer, Gaspar; Jaramillo, Yasna; Liu, Frances; Choi, Sooji; Crandall, Edward; Borok, Zea; Flodby, Per; Shull, Gary E.; Melvin, James E.

    2015-01-01

    Activation of an apical Ca2+-activated Cl− channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl− current and Cl− efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl− channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A−/−). Ca2+-dependent salivation was abolished in Tmem16A−/− mice, demonstrating that Tmem16A is obligatory for Ca2+-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A−/− mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr∆F508/∆F508) or ClC-2 (Clcn2−/−) Cl− channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl− channel. Indeed, Cl− channel blockers abolished fluid secretion, indicating that Cl− channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic–induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A−/− mice identify Tmem16A as the Cl− channel essential for muscarinic, Ca2+-dependent fluid secretion in adult mouse salivary glands. PMID:25646474

  20. Altered coupling of muscarinic acetylcholine receptors in pancreatic acinar carcinoma of rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chien, J.L.; Warren, J.R.

    The structure and function of muscarinic acetylcholine receptors (mAChR) in acinar carcinoma cells have been compared to mAChR in normal pancreatic acinar cells. Similar 80 kD proteins identified by SDS-PAGE of tumor and normal mAChR affinity-labeled with the muscarinic antagonist /sup 3/H-propylbenzilyl-choline mustards, and identical binding of the antagonist N-methylscopolamine to tumor and normal cells (K/sub D/approx.4x10/sup -10/ M), indicate conservation of mAChR proteins in carcinoma cells. Carcinoma mAChR display homogeneous binding of the agonists carbamylcholine (CCh), K/sub D/approx.3x10/sup -5/ M, and oxotremorine (Oxo), K/sub D/approx.x10/sup -6/ M, whereas normal cells display heterogeneous binding, with a minor component of highmore » affinity interactions for CCh, K/sub D/approx.3x10/sup -6/ M, and Oxo, K/sub D/approx.2x/sup -17/ M, and a major component of low affinity interactions for CCh, K/sub D/approx.1x10/sup -4/ M, and Oxo, K/sub D/approx.2x10/sup -5/ M. Both carcinoma and normal cells exhibit concentration-dependent CCh-stimulated increase in cytosolic free Ca/sup 2 +/, as measured by intracellular Quin 2 fluorescence and /sup 45/Ca/sup 2 +/ efflux. However, carcinoma cells demonstrate 50% maximal stimulation of intracellular Ca/sup 2 +/ release at a CCh concentration (EC/sub 50/approx.6x10/sup -7/ M) one log below that observed for normal cells. The authors propose an altered coupling of mAChR to intracellular Ca/sup 2 +/ homeostasis in carcinoma cells, which is manifest as a single activated receptor state for agonist binding, and increased sensitivity to muscarinic receptor stimulation of Ca/sup 2 +/ release.« less

  1. Acinar Cell Carcinoma of the Pancreas: Overview of Clinicopathologic Features and Insights into the Molecular Pathology.

    PubMed

    La Rosa, Stefano; Sessa, Fausto; Capella, Carlo

    2015-01-01

    Acinar cell carcinomas (ACCs) of the pancreas are rare pancreatic neoplasms accounting for about 1-2% of pancreatic tumors in adults and about 15% in pediatric subjects. They show different clinical symptoms at presentation, different morphological features, different outcomes, and different molecular alterations. This heterogeneous clinicopathological spectrum may give rise to difficulties in the clinical and pathological diagnosis with consequential therapeutic and prognostic implications. The molecular mechanisms involved in the onset and progression of ACCs are still not completely understood, although in recent years, several attempts have been made to clarify the molecular mechanisms involved in ACC biology. In this paper, we will review the main clinicopathological and molecular features of pancreatic ACCs of both adult and pediatric subjects to give the reader a comprehensive overview of this rare tumor type.

  2. c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

    PubMed

    La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

    2018-05-02

    The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

  3. Necroptosis: a potential, promising target and switch in acute pancreatitis.

    PubMed

    Wang, Gang; Qu, Feng-Zhi; Li, Le; Lv, Jia-Chen; Sun, Bei

    2016-02-01

    Pancreatic acinar cell death is the major pathophysiological change in early acute pancreatitis (AP), and the death modalities are important factors determining its progression and prognosis. During AP, acinar cells undergo two major modes of death, including necrosis and apoptosis. Acinar necrosis can lead to intensely local and systemic inflammatory responses, which both induce and aggravate the lesion. Necrosis has long been considered an unregulated, and passive cell death process. Since the effective interventions of necrosis are difficult to perform, its relevant studies have not received adequate attention. Necroptosis is a newly discovered cell death modality characterized by both necrosis and apoptosis, i.e., it is actively regulated by special genes, while has the typical morphological features of necrosis. Currently, necroptosis is gradually becoming an important topic in the fields of inflammatory diseases. The preliminary results from necroptosis in AP have confirmed the existence of acinar cell necroptosis, which may be a potential target for effectively regulating inflammatory injuries and improving its outcomes; however, the functional changes and mechanisms of necroptosis still require further investigation. This article reviewed the progress of necroptosis in AP to provide a reference for deeply understanding the pathogenic mechanisms of AP and identifying new therapeutic targets.

  4. Colourful death: six-parameter classification of cell death by flow cytometry--dead cells tell tales.

    PubMed

    Munoz, Luis E; Maueröder, Christian; Chaurio, Ricardo; Berens, Christian; Herrmann, Martin; Janko, Christina

    2013-08-01

    The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

  5. Cell Death in C. elegans Development.

    PubMed

    Malin, Jennifer Zuckerman; Shaham, Shai

    2015-01-01

    Cell death is a common and important feature of animal development, and cell death defects underlie many human disease states. The nematode Caenorhabditis elegans has proven fertile ground for uncovering molecular and cellular processes controlling programmed cell death. A core pathway consisting of the conserved proteins EGL-1/BH3-only, CED-9/BCL2, CED-4/APAF1, and CED-3/caspase promotes most cell death in the nematode, and a conserved set of proteins ensures the engulfment and degradation of dying cells. Multiple regulatory pathways control cell death onset in C. elegans, and many reveal similarities with tumor formation pathways in mammals, supporting the idea that cell death plays key roles in malignant progression. Nonetheless, a number of observations suggest that our understanding of developmental cell death in C. elegans is incomplete. The interaction between dying and engulfing cells seems to be more complex than originally appreciated, and it appears that key aspects of cell death initiation are not fully understood. It has also become apparent that the conserved apoptotic pathway is dispensable for the demise of the C. elegans linker cell, leading to the discovery of a previously unexplored gene program promoting cell death. Here, we review studies that formed the foundation of cell death research in C. elegans and describe new observations that expand, and in some cases remodel, this edifice. We raise the possibility that, in some cells, more than one death program may be needed to ensure cell death fidelity. © 2015 Elsevier Inc. All rights reserved.

  6. Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

    PubMed

    Fang, Shenglin; Yu, Xiaonan; Ding, Haoxuan; Han, Jianan; Feng, Jie

    2018-06-11

    Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. What cell death does in development.

    PubMed

    Zakeri, Zahra; Penaloza, Carlos G; Smith, Kyle; Ye, Yixia; Lockshin, Richard A

    2015-01-01

    Cell death is prominent in gametogenesis and shapes and sculpts embryos. In non-mammalian embryos one sees little or no cell death prior to the maternal-zygotic transition, but, in mammalian embryos, characteristic deaths of one or two cells occur at the end of compaction and are apparently necessary for the separation of the trophoblast from the inner cell mass. Considerable sculpting of the embryo occurs by cell deaths during organogenesis, and appropriate cell numbers, especially in the CNS and in the immune system, are generated by massive overproduction of cells and selection of a few, with death of the rest. The timing, identity, and genetic control of specific cells that die have been well documented in Caenorhabditis, but in other embryos the stochastic nature of the deaths limit our ability to do more than identify the regions in which cells will die. Complete disruption of the cell death machinery can be lethal, but many mutations of the regulatory machinery yield only modest or no phenotypes, indicating substantial redundancy and compensation of regulatory mechanisms. Most of the deaths are apoptotic and are identified by techniques used to recognize apoptosis, but techniques identifying lysosomes (whether in dying or involuting cells or in the phagocytes that invade the tissue) also reveal patterns of cell death. Aberrant cell deaths that produce known phenotypes are typically localized, indicating that the mechanism of activating a programmed death in a specific region, rather than the mechanism of death, is aberrant. These results lead us to conclude that we need to know much more about the conversations among cells that lead cells to commit suicide.

  8. Glutathione Efflux and Cell Death

    PubMed Central

    2012-01-01

    Abstract Significance: Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways. GSH depletion is often taken as a marker of oxidative stress and thus, as a consequence of its antioxidant properties scavenging reactive species of both oxygen and nitrogen (ROS/RNS). Recent Advances: There is increasing evidence demonstrating that GSH loss is an active phenomenon regulating the redox signaling events modulating cell death activation and progression. Critical Issues: In this work, we review the role of GSH depletion by its efflux, as an important event regulating alterations in the cellular redox balance during cell death independent from oxidative stress and ROS/RNS formation. We discuss the mechanisms involved in GSH efflux during cell death progression and the redox signaling events by which GSH depletion regulates the activation of the cell death machinery. Future Directions: The evidence summarized here clearly places GSH transport as a central mechanism mediating redox signaling during cell death progression. Future studies should be directed toward identifying the molecular identity of GSH transporters mediating GSH extrusion during cell death, and addressing the lack of sensitive approaches to quantify GSH efflux. Antioxid. Redox Signal. 17, 1694–1713. PMID:22656858

  9. Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

    PubMed

    Nagahara, Rei; Kimura, Masayuki; Itahashi, Megu; Sugahara, Go; Kawashima, Masashi; Murayama, Hirotada; Yoshida, Toshinori; Shibutani, Makoto

    2016-11-01

    Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas. © 2016 The Author(s).

  10. The deaths of a cell: how language and metaphor influence the science of cell death.

    PubMed

    Reynolds, Andrew S

    2014-12-01

    Multicellular development and tissue maintenance involve the regular elimination of damaged and healthy cells. The science of this genetically regulated cell death is particularly rich in metaphors: 'programmed cell death' or 'cell suicide' is considered an 'altruistic' act on the part of a cell for the benefit of the organism as a whole. It is also considered a form of 'social control' exerted by the body/organism over its component cells. This paper analyzes the various functions of these metaphors and critical discussion about them within the scientific community. Bodies such as the Nomenclature Committee on Cell Death (NCCD) have been charged with bringing order to the language of cell death to facilitate scientific progress. While the NCCD recommends adopting more objective biochemical terminology to describe the mechanisms of cell death, the metaphors in question retain an important function by highlighting the broader context within which cell death occurs. Scientific metaphors act as conceptual 'tools' which fulfill various roles, from highlighting a phenomenon as of particular interest, situating it in a particular context, or suggesting explanatory causal mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Programmed cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    The purpose of this conference to provide a multidisciplinary forum for exchange of state-of-the-art information on the role programmed cell death plays in normal development and homeostasis of many organisms. This volume contains abstracts of papers in the following areas: invertebrate development; immunology/neurology; bcl-2 family; biochemistry; programmed cell death in viruses; oncogenesis; vertebrate development; and diseases.

  12. Water permeability of acinar cell membranes in the isolated perfused rabbit mandibular salivary gland.

    PubMed Central

    Steward, M C; Seo, Y; Rawlings, J M; Case, R M

    1990-01-01

    1. The diffusive water permeability of epithelial cell membranes in the perfused rabbit mandibular salivary gland was measured at 37 degrees C by a 1H nuclear magnetic resonance relaxation method using an extracellular relaxation reagent, gadolinium diethylenetriaminepentaacetic acid (Gd(DTPA)). 2. In glands perfused with a HEPES-buffered solution containing 10 mmol l-1 Gd(DTPA), the spin-lattice (T1) relaxation of the water protons showed two exponential components. The water compartment responsible for the slower component corresponded in magnitude to 71 +/- 5% of the wet weight of the gland, and was attributed to the exchangeable intracellular water of the acinar cells. 3. The rate constant for water efflux from the cells was estimated to be 4.1 +/- 0.1 s-1 which would be consistent with a diffusive membrane permeability (Pd) of approximately 3 x 10(-3) cm s-1. Stimulation with acetylcholine (10(-6) mol l-1) did not cause any detectable change in membrane water permeability. 4. Since the basolateral membrane probably provides the main pathway for water efflux, the osmotic water permeability of this barrier (expressed per gland) was estimated to be less than 6.2 cm3 s-1. This would be insufficient to account for the generation of a near-isosmotic fluid at the flow rates observed during secretion, and suggests that a substantial fraction of the flow of water occurs via a paracellular route. PMID:1966053

  13. Expression of alveolar type II cell markers in acinar adenocarcinomas and adenoid cystic carcinomas arising from segmental bronchi. A study in a heterotopic bronchogenic carcinoma model in dogs.

    PubMed Central

    TenHave-Opbroek, A. A.; Hammond, W. G.; Benfield, J. R.; Teplitz, R. L.; Dijkman, J. H.

    1993-01-01

    The type II alveolar epithelial cell is one of two pluripotential stem cell phenotypes in normal mammalian lung morphogenesis; cells manifesting this phenotype have been found to constitute bronchioloalveolar regions of canine adenocarcinomas. We now studied type II cell expression in canine acinar adenocarcinomas and adenoid cystic (bronchial gland) carcinomas, using the same bronchogenic carcinoma model (subcutaneous bronchial autografts treated with 3-methylcholanthrene). Distinctive features of type II cells are the approximately cuboid cell shape, large and roundish nucleus, immunofluorescent staining of the cytoplasm for the surfactant protein SP-A, and presence of multilamellar bodies or their precursory forms. Cells with these type II cell characteristics were found in the basal epithelial layer of all tumor lesions and in upper layers as far as the lumen, singly or in clusters; they were also found in early invasive carcinomatous lesions but not in bronchial glands or bronchial epithelium before carcinogen exposure. Immunoblots of tumor homogenates showed reactive proteins within size classes of SP-A (28 to 36 kd) or its dimeric form (56 to 72 kd). These findings and those previously reported are consistent with the concept that chemical carcinogenesis in the adult bronchial epithelium may lead to type II cell carcinomas of varying glandular (acinar, adenoidcystic or bronchioloalveolar) growth patterns. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 PMID:8386445

  14. Nonthermal-plasma-mediated animal cell death

    NASA Astrophysics Data System (ADS)

    Kim, Wanil; Woo, Kyung-Chul; Kim, Gyoo-Cheon; Kim, Kyong-Tai

    2011-01-01

    Animal cell death comprising necrosis and apoptosis occurred in a well-regulated manner upon specific stimuli. The physiological meanings and detailed molecular mechanisms of cell death have been continuously investigated over several decades. Necrotic cell death has typical morphological changes, such as cell swelling and cell lysis followed by DNA degradation, whereas apoptosis shows blebbing formation and regular DNA fragmentation. Cell death is usually adopted to terminate cancer cells in vivo. The current strategies against tumour are based on the induction of cell death by adopting various methods, including radiotherapy and chemotherapeutics. Among these, radiotherapy is the most frequently used treatment method, but it still has obvious limitations. Recent studies have suggested that the use of nonthermal air plasma can be a prominent method for inducing cancer cell death. Plasma-irradiated cells showed the loss of genomic integrity, mitochondrial dysfunction, plasma membrane damage, etc. Tumour elimination with plasma irradiation is an emerging concept in cancer therapy and can be accelerated by targeting certain tumour-specific proteins with gold nanoparticles. Here, some recent developments are described so that the mechanisms related to plasma-mediated cell death and its perspectives in cancer treatment can be understood.

  15. Glutathione in Cancer Cell Death

    PubMed Central

    Ortega, Angel L.; Mena, Salvador; Estrela, Jose M.

    2011-01-01

    Glutathione (L-γ-glutamyl-L-cysteinyl-glycine; GSH) in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, and some cytokines; DNA synthesis; and cell proliferation and death. The intracellular thiol redox state (controlled by GSH) is one of the endogenous effectors involved in regulating the mitochondrial permeability transition pore complex and, in consequence, thiol oxidation can be a causal factor in the mitochondrion-based mechanism that leads to cell death. Nevertheless GSH depletion is a common feature not only of apoptosis but also of other types of cell death. Indeed rates of GSH synthesis and fluxes regulate its levels in cellular compartments, and potentially influence switches among different mechanisms of death. How changes in gene expression, post-translational modifications of proteins, and signaling cascades are implicated will be discussed. Furthermore, this review will finally analyze whether GSH depletion may facilitate cancer cell death under in vivo conditions, and how this can be applied to cancer therapy. PMID:24212662

  16. LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

    PubMed Central

    Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

    2012-01-01

    Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW

  17. Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

    PubMed

    Ding, Li; Liou, Geou-Yarh; Schmitt, Daniel M; Storz, Peter; Zhang, Jin-San; Billadeau, Daniel D

    2017-09-01

    Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  18. Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

    PubMed Central

    Shapiro, John P; Komar, Hannah M; Hancioglu, Baris; Yu, Lianbo; Jin, Ming; Ogata, Yuko; Hart, Phil A; Cruz-Monserrate, Zobeida; Lesinski, Gregory B; Conwell, Darwin L

    2017-01-01

    Objectives: Chronic pancreatitis (CP) is characterized by inflammation and fibrosis of the pancreas, leading to pain, parenchymal damage, and loss of exocrine and endocrine function. There are currently no curative therapies; diagnosis remains difficult and aspects of pathogenesis remain unclear. Thus, there is a need to identify novel biomarkers to improve diagnosis and understand pathophysiology. We hypothesize that pancreatic acinar regions contain proteomic signatures relevant to disease processes, including secreted proteins that could be detected in biofluids. Methods: Acini from pancreata of mice injected with or without caerulein were collected using laser capture microdissection followed by mass spectrometry analysis. This protocol enabled high-throughput analysis that captured altered protein expression throughout the stages of CP. Results: Over 2,900 proteins were identified, whereas 331 were significantly changed ≥2-fold by mass spectrometry spectral count analysis. Consistent with pathogenesis, we observed increases in proteins related to fibrosis (e.g., collagen, P<0.001), several proteases (e.g., trypsin 1, P<0.001), and altered expression of proteins associated with diminished pancreas function (e.g., lipase, amylase, P<0.05). In comparison with proteomic data from a public data set of CP patients, a significant correlation was observed between proteomic changes in tissue from both the caerulein model and CP patients (r=0.725, P<0.001). CONCLUSIONS: This study illustrates the ability to characterize proteome changes of acinar cells isolated from pancreata of caerulein-treated mice and demonstrates a relationship between signatures from murine and human CP. PMID:28406494

  19. A bicarbonate- and weak acid-permeable chloride conductance controlled by cytosolic Ca2+ and ATP in rat submandibular acinar cells.

    PubMed

    Ishikawa, T

    1996-09-01

    A Ca(2+)-activated Cl- conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mM ATP and 1 microM free Ca2+ and bathed in N-methyl-D-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nM or less than 1 nM free Ca2+ strongly reduced the Cl- currents, indicating the currents were Ca(2+)-dependent. Relaxation analysis of the "on" currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl- channels was: NO3- (2.00) > I- (1.85) > or = Br- (1.69) > Cl- (1.00) > bicarbonate (0.77) > or = acetate (0.70) > propionate (0.41) > > glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mM, the Ca(2+)-dependency of the Cl- current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-gamma S (2 mM) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mM). The addition of the calmodulin inhibitors trifluoperazine (100 microM) or calmidazolium (25 microM) to the bath solution and the inclusion of KN-62 (1 microM), a specific inhibitor of calmodulin kinase, or staurosporin (10 nM), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca(2+)-activated Cl- currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca(2+)-activated Cl- currents. The outward Cl- currents at +69 mV were inhibited by NPPB (100 microM), IAA-94 (100 microM), DIDS (0.03-1 mM), 9-AC (300 microM and 1 mM) and DPC (1 mM), whereas the inward currents at -101 mV were not. These results demonstrate the presence of a

  20. Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells.

    PubMed

    Fukushi, Y

    1999-01-01

    We studied the heterologous desensitization of muscarinic receptors by ATP in fura-2-loaded rat parotid acinar cells. Exposure to ATP or 3'-o-(4-benzoyl) benzoyl-ATP shortened the duration and decreased the magnitude of acetylcholine-induced Ca2+ release from intracellular Ca2+ stores in a dose-dependent manner. The shortening was observed only in an early stage of desensitization (within 20 s), whereas the decrease in the magnitude of the response was dependent upon the time the cells were exposed to the nucleotides. Atropine induced a profound shortening during the progressive decrease in the magnitude of acetylcholine-induced Ca2+ release. 3'-o-(4-Benzoyl) benzoyl-ATP did not induce an increase in the cytosolic Ca2+ concentration when the cells were incubated in the Ca2+- and Na+-free medium, but it did induce a strong desensitization of muscarinic receptors. The specific protein kinase C inhibitor bisindoylmaleimide resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. Phorbol 12-myristate 13-acetate potentiated the desensitization of muscarinic receptors. Ceramides that prevent the activation of phospholipase D resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. These results suggest that ATP, acting through P2Z purinoceptor-mediated phospholipase D, may produce a Ca2+-independent protein kinase C. Heterologous desensitization of muscarinic receptors by protein kinase C may shorten the duration and decrease the magnitude of acetylcholine-induced Ca2+ release.

  1. Particle dynamics and deposition in true-scale pulmonary acinar models.

    PubMed

    Fishler, Rami; Hofemeier, Philipp; Etzion, Yael; Dubowski, Yael; Sznitman, Josué

    2015-09-11

    Particle transport phenomena in the deep alveolated airways of the lungs (i.e. pulmonary acinus) govern deposition outcomes following inhalation of hazardous or pharmaceutical aerosols. Yet, there is still a dearth of experimental tools for resolving acinar particle dynamics and validating numerical simulations. Here, we present a true-scale experimental model of acinar structures consisting of bifurcating alveolated ducts that capture breathing-like wall motion and ensuing respiratory acinar flows. We study experimentally captured trajectories of inhaled polydispersed smoke particles (0.2 to 1 μm in diameter), demonstrating how intrinsic particle motion, i.e. gravity and diffusion, is crucial in determining dispersion and deposition of aerosols through a streamline crossing mechanism, a phenomenon paramount during flow reversal and locally within alveolar cavities. A simple conceptual framework is constructed for predicting the fate of inhaled particles near an alveolus by identifying capture and escape zones and considering how streamline crossing may shift particles between them. In addition, we examine the effect of particle size on detailed deposition patterns of monodispersed microspheres between 0.1-2 μm. Our experiments underline local modifications in the deposition patterns due to gravity for particles ≥0.5 μm compared to smaller particles, and show good agreement with corresponding numerical simulations.

  2. Particle dynamics and deposition in true-scale pulmonary acinar models

    PubMed Central

    Fishler, Rami; Hofemeier, Philipp; Etzion, Yael; Dubowski, Yael; Sznitman, Josué

    2015-01-01

    Particle transport phenomena in the deep alveolated airways of the lungs (i.e. pulmonary acinus) govern deposition outcomes following inhalation of hazardous or pharmaceutical aerosols. Yet, there is still a dearth of experimental tools for resolving acinar particle dynamics and validating numerical simulations. Here, we present a true-scale experimental model of acinar structures consisting of bifurcating alveolated ducts that capture breathing-like wall motion and ensuing respiratory acinar flows. We study experimentally captured trajectories of inhaled polydispersed smoke particles (0.2 to 1 μm in diameter), demonstrating how intrinsic particle motion, i.e. gravity and diffusion, is crucial in determining dispersion and deposition of aerosols through a streamline crossing mechanism, a phenomenon paramount during flow reversal and locally within alveolar cavities. A simple conceptual framework is constructed for predicting the fate of inhaled particles near an alveolus by identifying capture and escape zones and considering how streamline crossing may shift particles between them. In addition, we examine the effect of particle size on detailed deposition patterns of monodispersed microspheres between 0.1–2 μm. Our experiments underline local modifications in the deposition patterns due to gravity for particles ≥0.5 μm compared to smaller particles, and show good agreement with corresponding numerical simulations. PMID:26358580

  3. STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells

    PubMed Central

    Guo, Liang; Lichten, Louis A.; Ryu, Moon-Suhn; Liuzzi, Juan P.; Wang, Fudi; Cousins, Robert J.

    2010-01-01

    The exocrine pancreas plays an important role in endogenous zinc loss by regulating excretion into the intestinal tract and hence influences the dietary zinc requirement. The present experiments show that the zinc transporter ZnT2 (Slc30a2) is localized to the zymogen granules and that dietary zinc restriction in mice decreased the zinc concentration of zymogen granules and ZnT2 expression. Excess zinc given orally increased ZnT2 expression and was associated with increased pancreatic zinc accumulation. Rat AR42J acinar cells when induced into a secretory phenotype, using the glucocorticoid analog dexamethasone (DEX), exhibited increased ZnT2 expression and labile zinc as measured with a fluorophore. DEX administrated to mice also induced ZnT2 expression that accompanied a reduction of the pancreatic zinc content. ZnT2 promoter analyses identified elements required for responsiveness to zinc and DEX. Zinc regulation was traced to a MRE located downstream from the ZnT2 transcription start site. Responsiveness to DEX is produced by two upstream STAT5 binding sites that require the glucocorticoid receptor for activation. ZnT2 knockdown in the AR42J cells using siRNA resulted in increased cytoplasmic zinc and decreased zymogen granule zinc that further demonstrated that ZnT2 may mediate the sequestration of zinc into zymogen granules. We conclude, based upon experiments with intact mice and pancreatic acinar cells in culture, that ZnT2 participates in zinc transport into pancreatic zymogen granules through a glucocorticoid pathway requiring glucocorticoid receptor and STAT5, and zinc-regulated signaling pathways requiring MTF-1. The ZnT2 transporter appears to function in a physiologically responsive manner involving entero-pancreatic zinc trafficking. PMID:20133611

  4. Effects of cardiac oscillations and lung volume on acinar gas mixing during apnea

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mackenzie, C.F.; Skacel, M.; Barnas, G.M.

    1990-05-01

    We evaluated the importance of cardiogenic gas mixing in the acini of 13 dogs during 2 min of apnea. 133Xe (1-2 mCi in 4 ml of saline) was injected into an alveolar region through an occluded pulmonary artery branch, and washout was measured by gamma scintillation scanning during continued occlusion or with blood flow reinstated. The monoexponential rate constant for Xe washout (XeW) was -0.4 +/- 0.08 (SE) min-1 at functional residual capacity (FRC) with no blood flow in the injected region. It decreased by more than half at lung volumes 500 ml above and 392 ml below FRC. Withmore » intact pulmonary blood flow, XeW was -1.0 +/- 0.08 (SE) min-1 at FRC, and it increased with decreasing lung volume. However, if calculated Xe uptake by the blood was subtracted from the XeW measured with blood flow intact, resulting values at FRC and at FRC + 500 ml were not different from XeW with no blood flow. Reasonable calculation of Xe blood uptake at 392 ml below FRC was not possible because airway closure, increased shunt, and other factors affect XeW. After death, no significant XeW could be measured, which suggests that XeW caused by molecular diffusion was small. We conclude that (1) the effect of heart motion on the lung parenchyma increases acinar gas mixing during apnea, (2) this effect diminishes above or below FRC, and (3) there is probably no direct effect of pulmonary vascular pulsatility on acinar gas mixing.« less

  5. Programmed Cell Death During Caenorhabditis elegans Development

    PubMed Central

    Conradt, Barbara; Wu, Yi-Chun; Xue, Ding

    2016-01-01

    Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general. PMID:27516615

  6. [Methuosis: a novel type of cell death].

    PubMed

    Cai, Hongbing; Liu, Jinkun; Fan, Qin; Li, Xin

    2013-12-01

    Cell death is a major physiological or pathological phenomenon in life activities. The classic forms of cell death include apoptosis, necrosis, and autophagy. Recently, a novel type of cell death has been observed and termed as methuosis, in which excessive stimuli can induce cytoplasmic uptake and accumulation of small bubbles that gradually merge into giant vacuoles, eventually leading to decreased cellular metabolic activity, cell membrane rupture and cell death. In this article, we describe the nomenclature, morphological characteristics and underlying mechanisms of methuosis, compare methuosis with autophagy, oncosis and paraptosis, and review the related researches.

  7. Morphological classification of plant cell deaths.

    PubMed

    van Doorn, W G; Beers, E P; Dangl, J L; Franklin-Tong, V E; Gallois, P; Hara-Nishimura, I; Jones, A M; Kawai-Yamada, M; Lam, E; Mundy, J; Mur, L A J; Petersen, M; Smertenko, A; Taliansky, M; Van Breusegem, F; Wolpert, T; Woltering, E; Zhivotovsky, B; Bozhkov, P V

    2011-08-01

    Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.

  8. Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

    PubMed

    Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

    1993-03-10

    Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

  9. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

  10. Arabidopsis ACCELERATED CELL DEATH2 Modulates Programmed Cell DeathW⃞

    PubMed Central

    Yao, Nan; Greenberg, Jean T.

    2006-01-01

    The Arabidopsis thaliana chloroplast protein ACCELERATED CELL DEATH2 (ACD2) modulates the amount of programmed cell death (PCD) triggered by Pseudomonas syringae and protoporphyrin IX (PPIX) treatment. In vitro, ACD2 can reduce red chlorophyll catabolite, a chlorophyll derivative. We find that ACD2 shields root protoplasts that lack chlorophyll from light- and PPIX-induced PCD. Thus, chlorophyll catabolism is not obligatory for ACD2 anti-PCD function. Upon P. syringae infection, ACD2 levels and localization change in cells undergoing PCD and in their close neighbors. Thus, ACD2 shifts from being largely in chloroplasts to partitioning to chloroplasts, mitochondria, and, to a small extent, cytosol. ACD2 protects cells from PCD that requires the early mitochondrial oxidative burst. Later, the chloroplasts of dying cells generate NO, which only slightly affects cell viability. Finally, the mitochondria in dying cells have dramatically altered movements and cellular distribution. Overproduction of both ACD2 (localized to mitochondria and chloroplasts) and ascorbate peroxidase (localized to chloroplasts) greatly reduces P. syringae–induced PCD, suggesting a pro-PCD role for mitochondrial and chloroplast events. During infection, ACD2 may bind to and/or reduce PCD-inducing porphyrin-related molecules in mitochondria and possibly chloroplasts that generate reactive oxygen species, cause altered organelle behavior, and activate a cascade of PCD-inducing events. PMID:16387834

  11. Mitochondrial fission proteins regulate programmed cell death in yeast.

    PubMed

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  12. Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells.

    PubMed

    Kubisch, Constanze H; Logsdon, Craig D

    2007-06-01

    Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV-180 (10 nM-10 microM) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas.

  13. Live-cell visualization of gasdermin D-driven pyroptotic cell death.

    PubMed

    Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M; Yang, Jie; Xiao, Tsan S; Dubyak, George R; Huang, Alex Y; Abbott, Derek W

    2017-09-01

    Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells.

    PubMed

    Pal, Anupama; Kleer, Celina G

    2014-04-25

    Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior(1). However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system(2,3). Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D(4). 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved(3). One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D(6,7). Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during

  15. Cell death and cell lysis are separable events during pyroptosis

    PubMed Central

    DiPeso, Lucian; Ji, Daisy X; Vance, Russell E; Price, Jordan V

    2017-01-01

    Although much insight has been gained into the mechanisms by which activation of the inflammasome can trigger pyroptosis in mammalian cells, the precise kinetics of the end stages of pyroptosis have not been well characterized. Using time-lapse fluorescent imaging to analyze the kinetics of pyroptosis in individual murine macrophages, we observed distinct stages of cell death and cell lysis. Our data demonstrate that cell membrane permeability resulting from gasdermin D pore formation is coincident with the cessation of cell movement, loss of mitochondrial activity, and cell swelling, events that can be uncoupled from cell lysis. We propose a model of pyroptosis in which cell death can occur independently of cell lysis. The uncoupling of cell death from cell lysis may allow for better control of cytosolic contents upon activation of the inflammasome. PMID:29147575

  16. Acinar cell carcinoma of the pancreas presenting as diffuse pancreatic enlargement: Two case reports and literature review.

    PubMed

    Luo, Yaping; Hu, Guilan; Ma, Yanru; Guo, Ning; Li, Fang

    2017-09-01

    Pancreatic acinar cell carcinoma (ACC) is a rare malignant tumor of exocrine pancreas. It is typically a well-marginated large solid mass arising in a certain aspect of the pancreas. Diffuse involvement of ACC in the pancreas is very rare, and may simulate pancreatitis in radiological findings. We report 2 cases of ACC presenting as diffuse enlargement of the pancreas due to tumor involvement without formation of a distinct mass. The patients consisted of a 41-year-old man with weight loss and a 77-year-old man who was asymptomatic. Computed tomography (CT) and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT showed diffuse enlargement of the pancreas forming a sausage-like shape with homogenously increased FDG activity. Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) biopsy of the pancreatic lesion was performed. Histopathology results from the pancreas confirmed the diagnosis of pancreatic ACC. Because diffuse enlargement of the pancreas is a common imaging feature of pancreatitis, recognition of this rare morphologic pattern of ACC is important for radiological diagnosis of this tumor.

  17. Cytoplasmic vacuolization in cell death and survival

    PubMed Central

    Komissarov, Alexey A.; Rafieva, Lola M.; Kostrov, Sergey V.

    2016-01-01

    Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival. PMID:27331412

  18. Curcumin induces autophagic cell death in Spodoptera frugiperda cells.

    PubMed

    Veeran, Sethuraman; Shu, Benshui; Cui, Gaofeng; Fu, Shengjiao; Zhong, Guohua

    2017-06-01

    The increasing interest in the role of autophagy (type II cell death) in the regulation of insect toxicology has propelled study of investigating autophagic cell death pathways. Turmeric, the rhizome of the herb Curcuma longa (Mañjaḷ in Tamil, India and Jiānghuáng in Chinese) have been traditionally used for the pest control either alone or combination with other botanical pesticides. However, the mechanisms by which Curcuma longa or curcumin exerts cytotoxicity in pests are not well understood. In this study, we investigated the potency of Curcuma longa (curcumin) as a natural pesticide employing Sf9 insect line. Autophagy induction effect of curcumin on Spodoptera frugiperda (Sf9) cells was investigated using various techniques including cell proliferation assay, morphology analysis with inverted phase contrast microscope and Transmission Electron Microscope (TEM) analysis. Autophagy was evaluated using the fluorescent dye monodansylcadaverine (MDC). Cell death measurement was examined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) within the concentrations of 5-15μg/mL. Curcumin inhibited the growth of the Sf9 cells and induced autophagic cell death in a time and dose dependent manner. Staining the cells with MDC showed the presence of autophagic vacuoles while increased in a dose and time dependent manner. At the ultrastructural level transmission electron microscopy, cells revealed massive autophagy vacuole accumulation and absence of chromatin condensation. Protein expression levels of ATG8-I and ATG8-II, well-established markers of autophagy related protein were elevated in a time dependent manner after curcumin treatment. The present study proves that curcumin induces autophagic cell death in Sf9 insect cell line and this is the first report of cytotoxic effect of curcumin in insect cells and that will be utilized as natural pesticides in future. Copyright © 2017. Published by Elsevier Inc.

  19. Methods for assessing autophagy and autophagic cell death.

    PubMed

    Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

    2008-01-01

    Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

  20. Action of cholecystokinin and cholinergic agents on calcium transport in isolated pancreatic acinar cells.

    PubMed Central

    Gardner, J D; Conlon, T P; Kleveman, H L; Adams, T D; Ondetti, M A

    1975-01-01

    COOH-terminal octapeptide of cholecystokinin (CCK-octapeptide) and the cholinergic agent carbamylcholine each produced a fourfold stimulation of calcium outflux in guinea pig isolated pancreatic acinar cells. Neither agent altered calcium influx. Stimulation of calcium outflux was rapid and specific, was abolished by reducing the incubation temperature to 4 degrees C, and was a saturable function of the secretagogue concentration. The concentrations of CCK-octapeptide and carbamylcholine that produced half-maximal stimulation of calcium outflux were 3.1 x 10(-10) M and 4.9 x 10(-5) M, respectively. The cholinergic antagonist antropine competitively inhibited carbamylcholine stimulation of calcium outflux but did not alter stimulation produced by CCK-octapeptide. Stimulation of calcium outflux by maximal concentrations of carbamycholine plus CCK-octapeptide was the same as that produced by a maximal concentration of either agent alone.Calcium outflux became refractory to stimulation by secretagogues, and incubation with either CCK-ostapeptide or carbamylcholine produced a refractoriness to both agents. The relative potencies with CCK and its related fragments stimulated calcium outflux were CCK-octapeptide greater than heptapeptide greater than CCK greater than hexapeptide = gastrin. Secretin, glucagon, and vasoactive intestinal peptide, at concentrations as high as 10(-5) M, failed to alter calcium outflux and did not affect stimulation by CCK-octapeptide or by carbamycholine. Images PMID:1150877

  1. Ferroptosis and Cell Death Analysis by Flow Cytometry.

    PubMed

    Chen, Daishi; Eyupoglu, Ilker Y; Savaskan, Nicolai

    2017-01-01

    Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

  2. Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

    PubMed

    Wiman, K G; Zhivotovsky, B

    2017-05-01

    Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

  3. Dictyostelium cell death: early emergence and demise of highly polarized paddle cells.

    PubMed

    Levraud, Jean-Pierre; Adam, Myriam; Luciani, Marie-Françoise; de Chastellier, Chantal; Blanton, Richard L; Golstein, Pierre

    2003-03-31

    Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.

  4. Steady streaming: A key mixing mechanism in low-Reynolds-number acinar flows

    PubMed Central

    Kumar, Haribalan; Tawhai, Merryn H.; Hoffman, Eric A.; Lin, Ching-Long

    2011-01-01

    Study of mixing is important in understanding transport of submicron sized particles in the acinar region of the lung. In this article, we investigate transport in view of advective mixing utilizing Lagrangian particle tracking techniques: tracer advection, stretch rate and dispersion analysis. The phenomenon of steady streaming in an oscillatory flow is found to hold the key to the origin of kinematic mixing in the alveolus, the alveolar mouth and the alveolated duct. This mechanism provides the common route to folding of material lines and surfaces in any region of the acinar flow, and has no bearing on whether the geometry is expanding or if flow separates within the cavity or not. All analyses consistently indicate a significant decrease in mixing with decreasing Reynolds number (Re). For a given Re, dispersion is found to increase with degree of alveolation, indicating that geometry effects are important. These effects of Re and geometry can also be explained by the streaming mechanism. Based on flow conditions and resultant convective mixing measures, we conclude that significant convective mixing in the duct and within an alveolus could originate only in the first few generations of the acinar tree as a result of nonzero inertia, flow asymmetry, and large Keulegan–Carpenter (KC) number. PMID:21580803

  5. Die another way – non-apoptotic mechanisms of cell death

    PubMed Central

    Tait, Stephen W. G.; Ichim, Gabriel; Green, Douglas R.

    2014-01-01

    ABSTRACT Regulated, programmed cell death is crucial for all multicellular organisms. Cell death is essential in many processes, including tissue sculpting during embryogenesis, development of the immune system and destruction of damaged cells. The best-studied form of programmed cell death is apoptosis, a process that requires activation of caspase proteases. Recently it has been appreciated that various non-apoptotic forms of cell death also exist, such as necroptosis and pyroptosis. These non-apoptotic cell death modalities can be either triggered independently of apoptosis or are engaged should apoptosis fail to execute. In this Commentary, we discuss several regulated non-apoptotic forms of cell death including necroptosis, autophagic cell death, pyroptosis and caspase-independent cell death. We outline what we know about their mechanism, potential roles in vivo and define outstanding questions. Finally, we review data arguing that the means by which a cell dies actually matters, focusing our discussion on inflammatory aspects of cell death. PMID:24833670

  6. Patterns of cell death in the perinatal mouse forebrain.

    PubMed

    Mosley, Morgan; Shah, Charisma; Morse, Kiriana A; Miloro, Stephen A; Holmes, Melissa M; Ahern, Todd H; Forger, Nancy G

    2017-01-01

    The importance of cell death in brain development has long been appreciated, but many basic questions remain, such as what initiates or terminates the cell death period. One obstacle has been the lack of quantitative data defining exactly when cell death occurs. We recently created a "cell death atlas," using the detection of activated caspase-3 (AC3) to quantify apoptosis in the postnatal mouse ventral forebrain and hypothalamus, and found that the highest rates of cell death were seen at the earliest postnatal ages in most regions. Here we have extended these analyses to prenatal ages and additional brain regions. We quantified cell death in 16 forebrain regions across nine perinatal ages from embryonic day (E) 17 to postnatal day (P) 11 and found that cell death peaks just after birth in most regions. We found greater cell death in several regions in offspring delivered vaginally on the day of parturition compared with those of the same postconception age but still in utero at the time of collection. We also found massive cell death in the oriens layer of the hippocampus on P1 and in regions surrounding the anterior crossing of the corpus callosum on E18 as well as the persistence of large numbers of cells in those regions in adult mice lacking the pro-death Bax gene. Together these findings suggest that birth may be an important trigger of neuronal cell death and identify transient cell groups that may undergo wholesale elimination perinatally. J. Comp. Neurol. 525:47-64, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Cell death induced by hydroxyapatite on L929 fibroblast cells.

    PubMed

    Inayat-Hussain, S H; Rajab, N F; Roslie, H; Hussin, A A; Ali, A M; Annuar, B O

    2004-05-01

    Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

  8. Cell death at the intestinal epithelial front line.

    PubMed

    Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

    2016-07-01

    The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

  9. β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas.

    PubMed

    Kim, Hyo-Sup; Lee, Moon-Kyu

    2016-05-01

    Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β-cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β-cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin-producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β-cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β-cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin-producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin-producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin-producing cells, especially duct and acinar cells.

  10. Amino acid sequence and the cellular location of the Na(+)-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell.

    PubMed Central

    Tarpey, P S; Wood, I S; Shirazi-Beechey, S P; Beechey, R B

    1995-01-01

    The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7492327

  11. The slow cell death response when screening chemotherapeutic agents.

    PubMed

    Blois, Joseph; Smith, Adam; Josephson, Lee

    2011-09-01

    To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay. With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry. Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72 h. Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72 h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.

  12. Immunological consequences of kidney cell death.

    PubMed

    Sarhan, Maysa; von Mässenhausen, Anne; Hugo, Christian; Oberbauer, Rainer; Linkermann, Andreas

    2018-01-25

    Death of renal cells is central to the pathophysiology of acute tubular necrosis, autoimmunity, necrotizing glomerulonephritis, cystic kidney disease, urosepsis, delayed graft function and transplant rejection. By means of regulated necrosis, immunogenic damage-associated molecular patterns (DAMPs) and highly reactive organelles such as lysosomes, peroxisomes and mitochondria are released from the dying cells, thereby causing an overwhelming immunologic response. The rupture of the plasma membrane exhibits the "point of no return" for the immunogenicity of regulated cell death, explaining why apoptosis, a highly organized cell death subroutine with long-lasting plasma membrane integrity, elicits hardly any immune response. Ferroptosis, an iron-dependent necrotic type cell death, results in the release of DAMPs and large amounts of lipid peroxides. In contrast, anti-inflammatory cytokines are actively released from cells that die by necroptosis, limiting the DAMP-induced immune response to a surrounding microenvironment, whereas at the same time, inflammasome-associated caspases drive maturation of intracellularly expressed interleukin-1β (IL-1β). In a distinct setting, additionally interleukin-18 (IL-18) is expressed during pyroptosis, initiated by gasdermin-mediated plasma membrane rupture. As all of these pathways are druggable, we provide an overview of regulated necrosis in kidney diseases with a focus on immunogenicity and potential therapeutic interventions.

  13. Can deaths in police cells be prevented? Experience from Norway and death rates in other countries.

    PubMed

    Aasebø, Willy; Orskaug, Gunnar; Erikssen, Jan

    2016-01-01

    To describe the changes in death rates and causes of deaths in Norwegian police cells during the last 2 decades. To review reports on death rates in police cells that have been published in medical journals and elsewhere, and discuss the difficulties of comparing death rates between countries. Data on deaths in Norwegian police cells were collected retrospectively in 2002 and 2012 for two time periods: 1993-2001 (period 1) and 2003-2012 (period 2). Several databases were searched to find reports on deaths in police cells from as many countries as possible. The death rates in Norwegian police cells reduced significantly from 0.83 deaths per year per million inhabitants (DYM) in period 1 to 0.22 DYM in period 2 (p < 0.05). The most common cause of death in period 1 was alcohol intoxication including intracranial bleeding in persons with high blood alcohol levels, and the number declined from 16 persons in period 1 to 1 person in period 2 (p = 0.032). The median death rate in the surveyed Western countries was 0.44 DYM (range: 0.14-1.46 DYM). The number of deaths in Norwegian police cells reduced by about 75% over a period of approximately 10 years. This is probably mainly due to individuals with severe alcohol intoxication no longer being placed in police cells. However, there remain large methodology difficulties in comparing deaths rates between countries. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  14. Inhibition of caspases prevents ototoxic and ongoing hair cell death

    NASA Technical Reports Server (NTRS)

    Matsui, Jonathan I.; Ogilvie, Judith M.; Warchol, Mark E.

    2002-01-01

    Sensory hair cells die after acoustic trauma or ototoxic insults, but the signal transduction pathways that mediate hair cell death are not known. Here we identify several important signaling events that regulate the death of vestibular hair cells. Chick utricles were cultured in media supplemented with the ototoxic antibiotic neomycin and selected pharmacological agents that influence signaling molecules in cell death pathways. Hair cells that were treated with neomycin exhibited classically defined apoptotic morphologies such as condensed nuclei and fragmented DNA. Inhibition of protein synthesis (via treatment with cycloheximide) increased hair cell survival after treatment with neomycin, suggesting that hair cell death requires de novo protein synthesis. Finally, the inhibition of caspases promoted hair cell survival after neomycin treatment. Sensory hair cells in avian vestibular organs also undergo continual cell death and replacement throughout mature life. It is unclear whether the loss of hair cells stimulates the proliferation of supporting cells or whether the production of new cells triggers the death of hair cells. We examined the effects of caspase inhibition on spontaneous hair cell death in the chick utricle. Caspase inhibitors reduced the amount of ongoing hair cell death and ongoing supporting cell proliferation in a dose-dependent manner. In isolated sensory epithelia, however, caspase inhibitors did not affect supporting cell proliferation directly. Our data indicate that ongoing hair cell death stimulates supporting cell proliferation in the mature utricle.

  15. Cell death pathways associated with PDT

    NASA Astrophysics Data System (ADS)

    Kessel, David; Reiners, John J., Jr.

    2006-02-01

    Photodynamic therapy leads to both direct and indirect tumor cell death. The latter also involves the consequences of vascular shut-down and immunologic effects. While these factors are a major factor in tumor eradication, there is usually an element of direct cell killing that can reduce the cell population by as much as 2-3 logs. Necrosis was initially believed to represent the predominant PDT death mechanism. An apoptotic response to PDT was first reported by Oleinick in 1991, using a sensitizer that targets the anti-apoptotic protein Bcl-2. Apoptosis leads to fragmentation of DNA and of cells into apoptotic bodies that are removed by phagocytosis. Inflammatory effects are minimized, and the auto- catalytic elements of the process can amplify the death signal. In this study, we examined consequences of Bcl-2 photodamage by a porphycene sensitizer that targets the ER and causes photodamage to the anti-apoptotic protein Bcl-2. Death patterns after Bcl-2 inactivation by a small-molecular antagonist were also assessed. In addition to apoptosis, we also characterized a hitherto undescribed PDT effect, the initiation of autophagy. Autophagy was initially identified as a cell survival pathway, allowing the recycling of components as nutrients become scarce. We propose that autophagy can also represent both a potential survival pathway after PDT damage to cellular organelles, as well as a cell-death pathway. Recent literature reports indicate that autophagy, as well as apoptosis, can be evoked after down-regulation of Bcl-2, a result consistent with results reported here.

  16. BID links ferroptosis to mitochondrial cell death pathways.

    PubMed

    Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

    2017-08-01

    Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

    PubMed

    Pizato, Nathalia; Luzete, Beatriz Christina; Kiffer, Larissa Fernanda Melo Vasconcelos; Corrêa, Luís Henrique; de Oliveira Santos, Igor; Assumpção, José Antônio Fagundes; Ito, Marina Kiyomi; Magalhães, Kelly Grace

    2018-01-31

    The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1β secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.

  18. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Hengwen; Yang, Shana; Li, Jianhua

    Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. The aim of radiotherapy is to eradicate cancer cells with ionizing radiation. Except for the caspase-dependent mechanism, several lines of evidence demonstrated that caspase-independent mechanism is directly involved in the cell death responding to irradiation. For this reason, defining the contribution of caspase-independent molecular mechanisms represents the main goal in radiotherapy. In this study, we focused on the role of apoptosis-inducing factor (AIF), the caspase-independent molecular, in ionizing radiation induced hepatocellular carcinoma cell line (HepG2) cell death. We found that ionizing radiation has no function on AIF expressionmore » in HepG2 cells, but could induce AIF release from the mitochondria and translocate into nuclei. Inhibition of AIF could reduce ionizing radiation induced HepG2 cell death. These studies strongly support a direct relationship between AIF nuclear translocation and radiation induced cell death. What's more, AIF nuclear translocation is caspase-independent manner, but not caspase-dependent manner, in this process. These new findings add a further attractive point of investigation to better define the complex interplay between caspase-independent cell death and radiation therapy. - Highlights: • AIF nuclear translocation is involved in ionizing radiation induced hepatocellular carcinoma cell line HepG2 cell death. • AIF mediated cell death induced by ionizing radiation is caspase-independent. • Caspase-independent pathway is involved in ionzing radiation induced HepG2 cell death.« less

  19. Death of mitochondria during programmed cell death of leaf mesophyll cells.

    PubMed

    Selga, Tūrs; Selga, Maija; Pāvila, Vineta

    2005-12-01

    The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.

  20. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    PubMed Central

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-01-01

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS).We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. PMID:25034532

  1. Cell death pathways of particulate matter toxicity.

    PubMed

    Peixoto, Milena Simões; de Oliveira Galvão, Marcos Felipe; Batistuzzo de Medeiros, Silvia Regina

    2017-12-01

    Humans are exposed to various complex mixtures of particulate matter (PM) from different sources. Long-term exposure to high levels of these particulates has been linked to a diverse range of respiratory and cardiovascular diseases that have resulted in hospital admission. The evaluation of the effects of PM exposure on the mechanisms related to cell death has been a challenge for many researchers. Therefore, in this review, we have discussed the effects of airborne PM exposure on mechanisms related to cell death. For this purpose, we have compiled literature data on PM sources, the effects of exposure, and the assays and models used for evaluation, in order to establish comparisons between various studies. The analysis of this collected data suggested divergent responses to PM exposure that resulted in different cell death types (apoptosis, autophagy, and necrosis). In addition, PM induced oxidative stress within cells, which appeared to be an important factor in the determination of cell fate. When the levels of reactive oxygen species were overpowering, the cellular fate was directed toward cell death. This may be the underlying mechanism of the development or exacerbation of respiratory diseases, such as emphysema and chronic obstructive pulmonary diseases. In addition, PM was shown to cause DNA damage and the resulting mutations increased the risk of cancer. Furthermore, several conditions should be considered in the assessment of cell death in PM-exposed models, including the cell culture line, PM composition, and the interaction of the different cells types in in vivo models. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

    PubMed

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-09

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

  3. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    PubMed Central

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-01

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

  4. Photoreceptor cell death and rescue in retinal detachment and degenerations

    PubMed Central

    Murakami, Yusuke; Notomi, Shoji; Hisatomi, Toshio; Nakazawa, Toru; Ishibashi, Tatsuro; Miller, Joan W.; Vavvas, Demetrios G.

    2013-01-01

    Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss. PMID:23994436

  5. Mechanism of cell death resulting from DNA interstrand cross-linking in mammalian cells

    PubMed Central

    Osawa, T; Davies, D; Hartley, J A

    2011-01-01

    DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death. PMID:21814285

  6. Morphodynamics of a growing microbial colony driven by cell death

    NASA Astrophysics Data System (ADS)

    Ghosh, Pushpita; Levine, Herbert

    2017-11-01

    Bacterial cells can often self-organize into multicellular structures with complex spatiotemporal morphology. In this work, we study the spatiotemporal dynamics of a growing microbial colony in the presence of cell death. We present an individual-based model of nonmotile bacterial cells which grow and proliferate by consuming diffusing nutrients on a semisolid two-dimensional surface. The colony spreads by growth forces and sliding motility of cells and undergoes cell death followed by subsequent disintegration of the dead cells in the medium. We model cell death by considering two possible situations: In one of the cases, cell death occurs in response to the limitation of local nutrients, while the other case corresponds to an active death process, known as apoptotic or programmed cell death. We demonstrate how the colony morphology is influenced by the presence of cell death. Our results show that cell death facilitates transitions from roughly circular to highly branched structures at the periphery of an expanding colony. Interestingly, our results also reveal that for the colonies which are growing in higher initial nutrient concentrations, cell death occurs much earlier compared to the colonies which are growing in lower initial nutrient concentrations. This work provides new insights into the branched patterning of growing bacterial colonies as a consequence of complex interplay among the biochemical and mechanical effects.

  7. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells

    PubMed Central

    Alzaharna, Mazen; Alqouqa, Iyad; Cheung, Hon-Yeung

    2017-01-01

    Andrographolide (Andro) has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi) has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS)-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α) decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP) plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death. PMID:28182713

  8. Sorafenib-induced defective autophagy promotes cell death by necroptosis.

    PubMed

    Kharaziha, Pedram; Chioureas, Dimitris; Baltatzis, George; Fonseca, Pedro; Rodriguez, Patricia; Gogvadze, Vladimir; Lennartsson, Lena; Björklund, Ann-Charlotte; Zhivotovsky, Boris; Grandér, Dan; Egevad, Lars; Nilsson, Sten; Panaretakis, Theocharis

    2015-11-10

    Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

  9. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

    PubMed Central

    Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

    2015-01-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  10. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

    PubMed

    Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

    2015-06-01

    Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

  11. Multiple Modes of Cell Death Discovered in a Prokaryotic (Cyanobacterial) Endosymbiont

    PubMed Central

    Zheng, Weiwen; Rasmussen, Ulla; Zheng, Siping; Bao, Xiaodong; Chen, Bin; Gao, Yuan; Guan, Xiong; Larsson, John; Bergman, Birgitta

    2013-01-01

    Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom. PMID:23822984

  12. Ayanin diacetate-induced cell death is amplified by TRAIL in human leukemia cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marrero, Maria Teresa; Estevez, Sara; Negrin, Gledy

    Highlights: Black-Right-Pointing-Pointer Ayanin diacetate as apoptotic inducer in leukemia cells. Black-Right-Pointing-Pointer Cell death was prevented by caspase inhibitors and by the overexpression of Bcl-x{sub L}. Black-Right-Pointing-Pointer The intrinsic and the extrinsic pathways are involved in the mechanism of action. Black-Right-Pointing-Pointer Death receptors are up-regulated and TRAIL enhances apoptotic cell death. -- Abstract: Here we demonstrate that the semi-synthetic flavonoid ayanin diacetate induces cell death selectively in leukemia cells without affecting the proliferation of normal lymphocytes. Incubation of human leukemia cells with ayanin diacetate induced G{sub 2}-M phase cell cycle arrest and apoptosis which was prevented by the non-specific caspase inhibitormore » z-VAD-fmk and reduced by the overexpression of Bcl-x{sub L}. Ayanin diacetate-induced cell death was found to be associated with: (i) loss of inner mitochondrial membrane potential, (ii) the release of cytochrome c, (iii) the activation of multiple caspases, (iv) cleavage of poly(ADP-ribose) polymerase and (v) the up-regulation of death receptors for TRAIL, DR4 and DR5. Moreover, the combined treatment with ayanin diacetate and TRAIL amplified cell death, compared to single treatments. These results provide a basis for further exploring the potential applications of this combination for the treatment of cancer.« less

  13. Comparison of Types of Cell Death: Apoptosis and Necrosis.

    ERIC Educational Resources Information Center

    Manning, Francis; Zuzel, Katherine

    2003-01-01

    Cell death is an essential factor in many biological processes including development. Discusses two types of cell death: (1) necrosis (induced by sodium azide); and (2) apoptosis (induced by sodium chromate). Illustrates key features that differ between these two types of cells death including loss of membrane integrity and internucleosomal DNA…

  14. Ferroptosis is Involved in Acetaminophen Induced Cell Death.

    PubMed

    Lőrincz, Tamás; Jemnitz, Katalin; Kardon, Tamás; Mandl, József; Szarka, András

    2015-09-01

    The recently described form of programmed cell death, ferroptosis can be induced by agents causing GSH depletion or the inhibition of GPX4. Ferroptosis clearly shows distinct morphologic, biochemical and genetic features from apoptosis, necrosis and autophagy. Since NAPQI the highly reactive metabolite of the widely applied analgesic and antipyretic, acetaminophen induces a cell death which can be characterized by GSH depletion, GPX inhibition and caspase independency the involvement of ferroptosis in acetaminophen induced cell death has been investigated. The specific ferroptosis inhibitor ferrostatin-1 failed to elevate the viability of acetaminophen treated HepG2 cells. It should be noticed that these cells do not form NAPQI due to the lack of phase I enzyme expression therefore GSH depletion cannot be observed. However in the case of acetaminophen treated primary mouse hepatocytes the significant elevation of cell viability could be observed upon ferrostatin-1 treatment. Similar to ferrostatin-1 treatment, the addition of the RIP1 kinase inhibitor necrostatin-1 could also elevate the viability of acetaminophen treated primary hepatocytes. Ferrostatin-1 has no influence on the expression of CYP2E1 or on the cellular GSH level which suggest that the protective effect of ferrostatin-1 in APAP induced cell death is not based on the reduced metabolism of APAP to NAPQI or on altered NAPQI conjugation by cellular GSH. Our results suggest that beyond necroptosis and apoptosis a third programmed cell death, ferroptosis is also involved in acetaminophen induced cell death in primary hepatocytes.

  15. TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease.

    PubMed

    Wang, Tao; Lao, Uyen; Edgar, Bruce A

    2009-09-07

    Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.

  16. Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

    PubMed Central

    Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

    2011-01-01

    Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

  17. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Dae-Hee, E-mail: leedneo@gmail.com; Kim, Dong-Wook; Jung, Chang-Hwa

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We alsomore » found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.« less

  18. Crystalline structure of pulverized dental calculus induces cell death in oral epithelial cells.

    PubMed

    Ziauddin, S M; Yoshimura, A; Montenegro Raudales, J L; Ozaki, Y; Higuchi, K; Ukai, T; Kaneko, T; Miyazaki, T; Latz, E; Hara, Y

    2018-06-01

    Dental calculus is a mineralized deposit attached to the tooth surface. We have shown that cellular uptake of dental calculus triggers nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation, leading to the processing of the interleukin-1β precursor into its mature form in mouse and human phagocytes. The activation of the NLRP3 inflammasome also induced a lytic form of programmed cell death, pyroptosis, in these cells. However, the effects of dental calculus on other cell types in periodontal tissue have not been investigated. The aim of this study was to determine whether dental calculus can induce cell death in oral epithelial cells. HSC-2 human oral squamous carcinoma cells, HOMK107 human primary oral epithelial cells and immortalized mouse macrophages were exposed to dental calculus or 1 of its components, hydroxyapatite crystals. For inhibition assays, the cells were exposed to dental calculus in the presence or absence of cytochalasin D (endocytosis inhibitor), z-YVAD-fmk (caspase-1 inhibitor) or glyburide (NLRP3 inflammasome inhibitor). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release and staining with propidium iodide. Tumor necrosis factor-α production was quantified by enzyme-linked immunosorbent assay. Oral epithelial barrier function was examined by permeability assay. Dental calculus induced cell death in HSC-2 cells, as judged by LDH release and propidium iodide staining. Dental calculus also induced LDH release from HOMK107 cells. Following heat treatment, dental calculus lost its capacity to induce tumor necrosis factor-α in mouse macrophages, but could induce LDH release in HSC-2 cells, indicating a major role of inorganic components in cell death. Hydroxyapatite crystals also induced cell death in both HSC-2 and HOMK107 cells, as judged by LDH release, indicating the capacity of crystal particles to induce cell death. Cell death induced by dental

  19. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sakamoto, C.; Matozaki, T.; Nagao, M.

    1987-09-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate (Gpp(NH)p)>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg/sup 2 +/. When pancreatic acini were treated withmore » 1 ..mu..g/ml pertussis toxin for 4 h, subsequent /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor.« less

  20. Programmed cell death as a defence against infection

    PubMed Central

    Jorgensen, Ine; Rayamajhi, Manira; Miao, Edward A.

    2017-01-01

    Eukaryotic cells can die from physical trauma, resulting in necrosis. Alternately, they can die via programmed cell death upon stimulation of specific signalling pathways. Here we discuss the utility of four cell death pathways in innate immune defence against bacterial and viral infection: apoptosis, necroptosis, pyroptosis and NETosis. We describe the interactions that interweave different programmed cell death pathways, which create complex signalling networks that cross-guard each other in the evolutionary arms race with pathogens. Finally, we describe how the resulting cell corpses — apoptotic bodies, pore-induced intracellular traps (PITs) and neutrophil extracellular traps (NETs) — promote clearance of infection. PMID:28138137

  1. Increasing RpoS expression causes cell death in Borrelia burgdorferi.

    PubMed

    Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

    2013-01-01

    RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

  2. Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells.

    PubMed

    Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

    2016-10-25

    Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

  3. Host-Cell Survival and Death During Chlamydia Infection

    PubMed Central

    Ying, Songmin; Pettengill, Matthew; Ojcius, David M.; Häcker, Georg

    2008-01-01

    Different Chlamydia trachomatis strains are responsible for prevalent bacterial sexually-transmitted disease and represent the leading cause of preventable blindness worldwide. Factors that predispose individuals to disease and mechanisms by which chlamydiae cause inflammation and tissue damage remain unclear. Results from recent studies indicate that prolonged survival and subsequent death of infected cells and their effect on immune effector cells during chlamydial infection may be important in determining the outcome. Survival of infected cells is favored at early times of infection through inhibition of the mitochondrial pathway of apoptosis. Death at later times displays features of both apoptosis and necrosis, but pro-apoptotic caspases are not involved. Most studies on chlamydial modulation of host-cell death until now have been performed in cell lines. The consequences for pathogenesis and the immune response will require animal models of chlamydial infection, preferably mice with targeted deletions of genes that play a role in cell survival and death. PMID:18843378

  4. High mobility group box 1 induces the activation of the Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic acinar cells in rats, while AG490 and rapamycin inhibit their activation.

    PubMed

    Wang, Guoliang; Zhang, Jingchao; Dui, Danhua; Ren, Haoyuan; Liu, Jin

    2016-11-10

    The pathogenesis of severe acute pancreatitis (SAP) remains unclear. The Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway is important for various cytokines and growth factors. This study investigated the effect of the late inflammatory factor high mobility group box 1 (HMGB1) on the activation of JAK2/STAT3 in pancreatic acinar cells and the inhibitory effects of AG490 (a JAK2 inhibitor) and rapamycin (a STAT3 inhibitor) on this pathway. Rat pancreatic acinar cells were randomly divided into the control, HMGB1, AG490, and rapamycin groups. The mRNA levels of JAK2 and STAT3 at 10, 30, 60, and 120 minutes were detected using reverse transcription polymerase chain reaction (RT-PCR). The protein levels of JAK2 and STAT3 at 60 and 120 minutes were observed using Western blotting. Compared with the control group, the HMGB1 group exhibited significantly increased levels of JAK2 mRNA at each time point; STAT3 mRNA at 30, 60, and 120 minutes; and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). Compared with the HMGB1 group, the AG490 and rapamycin groups both exhibited significantly decreased levels of JAK2 mRNA at each time point (p < 0.05); STAT3 mRNA at 30, 60, and 120 minutes (p < 0.01); and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). HMGB1 induces the activation of the JAK2/STAT3 signaling pathway in rat pancreatic acinar cells, and this activation can be inhibited by AG490 and rapamycin. The results of this study may provide new insights for the treatment of SAP.

  5. UV-Induced cell death in plants.

    PubMed

    Nawkar, Ganesh M; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

    2013-01-14

    Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400-700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280-320 nm) and UV-A (320-390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD).

  6. Further considerations on in vitro skeletal muscle cell death

    PubMed Central

    Battistelli, Michela; Salucci, Sara; Burattini, Sabrina; Falcieri, Elisabetta

    2013-01-01

    Summary The present review discusses the apoptotic behavior induced by chemical and physical triggers in C2C12 skeletal muscle cells, comparing myoblast to myotube sensitivity, and investigating it by means of morphological, biochemical and cytofluorimetric analyses. After all treatments, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, in cells exposed to staurosporine, etoposide and UVB radiation, apoptotic and normal nuclei within the same fibercould be revealed. The presence of nuclear-dependent “territorial” death domains in the syncytium could explain a delayed cell death of myotubes compared to mononucleated cells. Moreover, autophagic granules abundantly appeared in myotubes after each treatment. Autophagy could protect muscle cell integrity against chemical and physical stimuli, making C2C12 myotubes, more resistant to cell death induction. PMID:24596689

  7. Two programmed cell death systems in Escherichia coli: an apoptotic-like death is inhibited by the mazEF-mediated death pathway.

    PubMed

    Erental, Ariel; Sharon, Idith; Engelberg-Kulka, Hanna

    2012-01-01

    In eukaryotes, the classical form of programmed cell death (PCD) is apoptosis, which has as its specific characteristics DNA fragmentation and membrane depolarization. In Escherichia coli a different PCD system has been reported. It is mediated by the toxin-antitoxin system module mazEF. The E. coli mazEF module is one of the most thoroughly studied toxin-antitoxin systems. mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. mazEF-mediated cell death is a population phenomenon requiring the quorum-sensing pentapeptide NNWNN designated Extracellular Death Factor (EDF). mazEF is triggered by several stressful conditions, including severe damage to the DNA. Here, using confocal microscopy and FACS analysis, we show that under conditions of severe DNA damage, the triggered mazEF-mediated cell death pathway leads to the inhibition of a second cell death pathway. The latter is an apoptotic-like death (ALD); ALD is mediated by recA and lexA. The mazEF-mediated pathway reduces recA mRNA levels. Based on these results, we offer a molecular model for the maintenance of an altruistic characteristic in cell populations. In our model, the ALD pathway is inhibited by the altruistic EDF-mazEF-mediated death pathway.

  8. Contribution of TMEM16F to pyroptotic cell death.

    PubMed

    Ousingsawat, Jiraporn; Wanitchakool, Podchanart; Schreiber, Rainer; Kunzelmann, Karl

    2018-02-20

    Pyroptosis is a highly inflammatory form of programmed cell death that is caused by infection with intracellular pathogens and activation of canonical or noncanonical inflammasomes. The purinergic receptor P2X 7 is activated by the noncanonical inflammasome and contributes essentially to pyroptotic cell death. The Ca 2+ activated phospholipid scramblase and ion channel TMEM16F has been shown earlier to control cellular effects downstream of purinergic P2X 7 receptors that ultimately lead to cell death. As pyroptotic cell death is accompanied by an increases in intracellular Ca 2+ , we asked whether TMEM16F is activated during pyroptosis. The N-terminal cleavage product of gasdermin D (GD-N) is an executioner of pyroptosis by forming large plasma membrane pores. Expression of GD-N enhanced basal Ca 2+ levels and induced cell death. We observed that GD-N induced cell death in HEK293 and HAP1 cells, which was depending on expression of endogenous TMEM16F. GD-N activated large whole cell currents that were suppressed by knockdown or inhibition of TMEM16F. The results suggest that whole cell currents induced by the pore forming domain of gasdermin-D, are at least in part due to activation of TMEM16F. Knockdown of other TMEM16 paralogues expressed in HAP1 cells suggest TMEM16F as a crucial element during pyroptosis and excluded a role of other TMEM16 proteins. Thus TMEM16F supports pyroptosis and other forms of inflammatory cell death such as ferroptosis. Its potent inhibition by tannic acid may be part of the anti-inflammatory effects of flavonoids.

  9. Die Another Day: Inhibition of Cell Death Pathways by Cytomegalovirus.

    PubMed

    Brune, Wolfram; Andoniou, Christopher E

    2017-09-02

    Multicellular organisms have evolved multiple genetically programmed cell death pathways that are essential for homeostasis. The finding that many viruses encode cell death inhibitors suggested that cellular suicide also functions as a first line of defence against invading pathogens. This theory was confirmed by studying viral mutants that lack certain cell death inhibitors. Cytomegaloviruses, a family of species-specific viruses, have proved particularly useful in this respect. Cytomegaloviruses are known to encode multiple death inhibitors that are required for efficient viral replication. Here, we outline the mechanisms used by the host cell to detect cytomegalovirus infection and discuss the methods employed by the cytomegalovirus family to prevent death of the host cell. In addition to enhancing our understanding of cytomegalovirus pathogenesis we detail how this research has provided significant insights into the cross-talk that exists between the various cell death pathways.

  10. Mechanical Stress Promotes Cisplatin-Induced Hepatocellular Carcinoma Cell Death

    PubMed Central

    Riad, Sandra; Bougherara, Habiba

    2015-01-01

    Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death). PMID:25685789

  11. Guidelines and recommendations on yeast cell death nomenclature.

    PubMed

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J; Breitenbach, Michael; Burhans, William C; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W; Grant, Chris M; Greenwood, Michael T; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D; Outeiro, Tiago F; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F; Sharon, Amir; Sigrist, Stephan J; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B; Tuite, Mick; Vögtle, F-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J; Zhao, Richard Y; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.

  12. Guidelines and recommendations on yeast cell death nomenclature

    PubMed Central

    Carmona-Gutierrez, Didac; Bauer, Maria Anna; Zimmermann, Andreas; Aguilera, Andrés; Austriaco, Nicanor; Ayscough, Kathryn; Balzan, Rena; Bar-Nun, Shoshana; Barrientos, Antonio; Belenky, Peter; Blondel, Marc; Braun, Ralf J.; Breitenbach, Michael; Burhans, William C.; Büttner, Sabrina; Cavalieri, Duccio; Chang, Michael; Cooper, Katrina F.; Côrte-Real, Manuela; Costa, Vítor; Cullin, Christophe; Dawes, Ian; Dengjel, Jörn; Dickman, Martin B.; Eisenberg, Tobias; Fahrenkrog, Birthe; Fasel, Nicolas; Fröhlich, Kai-Uwe; Gargouri, Ali; Giannattasio, Sergio; Goffrini, Paola; Gourlay, Campbell W.; Grant, Chris M.; Greenwood, Michael T.; Guaragnella, Nicoletta; Heger, Thomas; Heinisch, Jürgen; Herker, Eva; Herrmann, Johannes M.; Hofer, Sebastian; Jiménez-Ruiz, Antonio; Jungwirth, Helmut; Kainz, Katharina; Kontoyiannis, Dimitrios P.; Ludovico, Paula; Manon, Stéphen; Martegani, Enzo; Mazzoni, Cristina; Megeney, Lynn A.; Meisinger, Chris; Nielsen, Jens; Nyström, Thomas; Osiewacz, Heinz D.; Outeiro, Tiago F.; Park, Hay-Oak; Pendl, Tobias; Petranovic, Dina; Picot, Stephane; Polčic, Peter; Powers, Ted; Ramsdale, Mark; Rinnerthaler, Mark; Rockenfeller, Patrick; Ruckenstuhl, Christoph; Schaffrath, Raffael; Segovia, Maria; Severin, Fedor F.; Sharon, Amir; Sigrist, Stephan J.; Sommer-Ruck, Cornelia; Sousa, Maria João; Thevelein, Johan M.; Thevissen, Karin; Titorenko, Vladimir; Toledano, Michel B.; Tuite, Mick; Vögtle, F.-Nora; Westermann, Benedikt; Winderickx, Joris; Wissing, Silke; Wölfl, Stefan; Zhang, Zhaojie J.; Zhao, Richard Y.; Zhou, Bing; Galluzzi, Lorenzo; Kroemer, Guido; Madeo, Frank

    2018-01-01

    Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research. PMID:29354647

  13. Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

    PubMed Central

    Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

    2015-01-01

    Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

  14. Transglutaminase induction by various cell death and apoptosis pathways.

    PubMed

    Fesus, L; Madi, A; Balajthy, Z; Nemes, Z; Szondy, Z

    1996-10-31

    Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.

  15. UV-Induced Cell Death in Plants

    PubMed Central

    Nawkar, Ganesh M.; Maibam, Punyakishore; Park, Jung Hoon; Sahi, Vaidurya Pratap; Lee, Sang Yeol; Kang, Chang Ho

    2013-01-01

    Plants are photosynthetic organisms that depend on sunlight for energy. Plants respond to light through different photoreceptors and show photomorphogenic development. Apart from Photosynthetically Active Radiation (PAR; 400–700 nm), plants are exposed to UV light, which is comprised of UV-C (below 280 nm), UV-B (280–320 nm) and UV-A (320–390 nm). The atmospheric ozone layer protects UV-C radiation from reaching earth while the UVR8 protein acts as a receptor for UV-B radiation. Low levels of UV-B exposure initiate signaling through UVR8 and induce secondary metabolite genes involved in protection against UV while higher dosages are very detrimental to plants. It has also been reported that genes involved in MAPK cascade help the plant in providing tolerance against UV radiation. The important targets of UV radiation in plant cells are DNA, lipids and proteins and also vital processes such as photosynthesis. Recent studies showed that, in response to UV radiation, mitochondria and chloroplasts produce a reactive oxygen species (ROS). Arabidopsis metacaspase-8 (AtMC8) is induced in response to oxidative stress caused by ROS, which acts downstream of the radical induced cell death (AtRCD1) gene making plants vulnerable to cell death. The studies on salicylic and jasmonic acid signaling mutants revealed that SA and JA regulate the ROS level and antagonize ROS mediated cell death. Recently, molecular studies have revealed genes involved in response to UV exposure, with respect to programmed cell death (PCD). PMID:23344059

  16. Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii

    PubMed Central

    Yordanova, Zhenya P.; Woltering, Ernst J.; Kapchina-Toteva, Veneta M.; Iakimova, Elena T.

    2013-01-01

    Background and Aims Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. Methods Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. Key Results MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. Conclusions In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of

  17. A comprehensive computational human lung model incorporating inter-acinar dependencies: Application to spontaneous breathing and mechanical ventilation.

    PubMed

    Roth, Christian J; Ismail, Mahmoud; Yoshihara, Lena; Wall, Wolfgang A

    2017-01-01

    In this article, we propose a comprehensive computational model of the entire respiratory system, which allows simulating patient-specific lungs under different ventilation scenarios and provides a deeper insight into local straining and stressing of pulmonary acini. We include novel 0D inter-acinar linker elements to respect the interplay between neighboring alveoli, an essential feature especially in heterogeneously distended lungs. The model is applicable to healthy and diseased patient-specific lung geometries. Presented computations in this work are based on a patient-specific lung geometry obtained from computed tomography data and composed of 60,143 conducting airways, 30,072 acini, and 140,135 inter-acinar linkers. The conducting airways start at the trachea and end before the respiratory bronchioles. The acini are connected to the conducting airways via terminal airways and to each other via inter-acinar linkers forming a fully coupled anatomically based respiratory model. Presented numerical examples include simulation of breathing during a spirometry-like test, measurement of a quasi-static pressure-volume curve using a supersyringe maneuver, and volume-controlled mechanical ventilation. The simulations show that our model incorporating inter-acinar dependencies successfully reproduces physiological results in healthy and diseased states. Moreover, within these scenarios, a deeper insight into local pressure, volume, and flow rate distribution in the human lung is investigated and discussed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Natural Compounds As Modulators of Non-apoptotic Cell Death in Cancer Cells

    PubMed Central

    Guamán-Ortiz, Luis Miguel; Orellana, Maria Isabel Ramirez; Ratovitski, Edward A.

    2017-01-01

    Cell death is an innate capability of cells to be removed from microenvironment, if and when they are damaged by multiple stresses. Cell death is often regulated by multiple molecular pathways and mechanism, including apoptosis, autophagy, and necroptosis. The molecular network underlying these processes is often intertwined and one pathway can dynamically shift to another one acquiring certain protein components, in particular upon treatment with various drugs. The strategy to treat human cancer ultimately relies on the ability of anticancer therapeutics to induce tumor-specific cell death, while leaving normal adjacent cells undamaged. However, tumor cells often develop the resistance to the drug-induced cell death, thus representing a great challenge for the anticancer approaches. Numerous compounds originated from the natural sources and biopharmaceutical industries are applied today in clinics showing advantageous results. However, some exhibit serious toxic side effects. Thus, novel effective therapeutic approaches in treating cancers are continued to be developed. Natural compounds with anticancer activity have gained a great interest among researchers and clinicians alike since they have shown more favorable safety and efficacy then the synthetic marketed drugs. Numerous studies in vitro and in vivo have found that several natural compounds display promising anticancer potentials. This review underlines certain information regarding the role of natural compounds from plants, microorganisms and sea life forms, which are able to induce non-apoptotic cell death in tumor cells, namely autophagy and necroptosis. PMID:28367073

  19. Autophagy promotes caspase-dependent cell death during Drosophila development.

    PubMed

    Mohseni, Nilufar; McMillan, Stephanie C; Chaudhary, Roopali; Mok, Jane; Reed, Bruce H

    2009-04-01

    The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the downregulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, upregulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.

  20. TRO40303 Ameliorates Alcohol-Induced Pancreatitis Through Reduction of Fatty Acid Ethyl Ester–Induced Mitochondrial Injury and Necrotic Cell Death

    PubMed Central

    Javed, Muhammad Ahsan; Wen, Li; Awais, Muhammad; Latawiec, Diane; Huang, Wei; Chvanov, Michael; Schaller, Sophie; Bordet, Thierry; Michaud, Magali; Pruss, Rebecca; Tepikin, Alexei; Criddle, David; Sutton, Robert

    2018-01-01

    Objectives Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). Methods Changes in mitochondrial membrane potential (Δψm), cytosolic Ca2+ ([Ca2+]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. Results TRO40303 prevented loss of Δψm and necrosis induced by 100 μM palmitoleic acid ethyl ester or 500 μM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. Conclusions TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP. PMID:29200128

  1. Sickle cell trait and sudden death--bringing it home.

    PubMed Central

    Mitchell, Bruce L.

    2007-01-01

    Sickle cell trait continues to be the leading cause of sudden death for young African Americans in military basic training and civilian organized sports. The syndrome may have caused the death of up to 10 college football players since 1974 and, as recently as 2000, was suspected as the cause of death of three U.S. Army recruits. The penal military-style boot camps in the United States and the recent death of two teenagers with sickle cell trait merits renewed vigor in the education of athletic instructors, the military and the public about conditions associated with sudden death in individuals with sickle cell trait. Images Figure 1 Figure 2 PMID:17393956

  2. Necroptosis: an emerging type of cell death in liver diseases.

    PubMed

    Saeed, Waqar Khalid; Jun, Dae Won

    2014-09-21

    Cell death has been extensively evaluated for decades and it is well recognized that pharmacological interventions directed to inhibit cell death can prevent significant cell loss and can thus improve an organ's physiological function. For long, only apoptosis was considered as a sole form of programmed cell death. Recently necroptosis, a RIP1/RIP3-dependent programmed cell death, has been identified as an apoptotic backup cell death mechanism with necrotic morphology. The evidences of necroptosis and protective effects achieved by blocking necroptosis have been extensively reported in recent past. However, only a few studies reported the evidence of necroptosis and protective effects achieved by inhibiting necroptosis in liver related disease conditions. Although the number of necroptosis initiators is increasing; however, interestingly, it is still unclear that what actually triggers necroptosis in different liver diseases or if there is always a different necroptosis initiator in each specific disease condition followed by specific downstream signaling molecules. Understanding the precise mechanism of necroptosis as well as counteracting other cell death pathways in liver diseases could provide a useful insight towards achieving extensive therapeutic significance. By targeting necroptosis and/or other parallel death pathways, a significant cell loss and thus a decrement in an organ's physiological function can be prevented.

  3. K6 linked polyubiquitylation of FADD by CHIP prevents death inducing signaling complex formation suppressing cell death.

    PubMed

    Seo, Jinho; Lee, Eun-Woo; Shin, Jihye; Seong, Daehyeon; Nam, Young Woo; Jeong, Manhyung; Lee, Seon-Hyeong; Lee, Cheolju; Song, Jaewhan

    2018-05-23

    Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.

  4. C/EBPβ LIP augments cell death by inducing osteoglycin.

    PubMed

    Wassermann-Dozorets, Rina; Rubinstein, Menachem

    2017-04-06

    Many types of tumor cell are devoid of the extracellular matrix proteoglycan osteoglycin (Ogn), but its role in tumor biology is poorly studied. Here we show that RNAi of Ogn attenuates stress-triggered cell death, whereas its overexpression increases cell death. We found that the transcription factor C/EBPβ regulates the expression of Ogn. C/EBPβ is expressed as a full-length, active form (LAP) and as a truncated, dominant-negative form (LIP), and the LIP/LAP ratio is positively correlated with the extent of cell death under stress. For example, we reported that drug-resistant tumor cells lack LIP altogether, and its supplementation abolished their resistance to chemotherapy and to endoplasmic reticulum (ER) stress. Here we further show that elevated LIP/LAP ratio robustly increased Ogn expression and cell death under stress by modulating the mitogen-activated protein kinase/activator protein 1 pathway (MAPK/AP-1). Our findings suggest that LIP deficiency renders tumor cell resistant to ER stress by preventing the induction of Ogn.

  5. Diagnosis of Cell Death by Means of Infrared Spectroscopy

    PubMed Central

    Zelig, Udi; Kapelushnik, Joseph; Moreh, Raymond; Mordechai, Shaul; Nathan, Ilana

    2009-01-01

    Abstract Fourier transform infrared (FTIR) spectroscopy has been established as a fast spectroscopic method for biochemical analysis of cells and tissues. In this research we aimed to investigate FTIR's utility for identifying and characterizing different modes of cell death, using leukemic cell lines as a model system. CCRF-CEM and U937 leukemia cells were treated with arabinoside and doxorubicin apoptosis inducers, as well as with potassium cyanide, saponin, freezing-thawing, and H2O2 necrosis inducers. Cell death mode was determined by various gold standard biochemical methods in parallel with FTIR-microscope measurements. Both cell death modes exhibit large spectral changes in lipid absorbance during apoptosis and necrosis; however, these changes are similar and thus cannot be used to distinguish apoptosis from necrosis. In contrast to the above confounding factor, our results reveal that apoptosis and necrosis can still be distinguished by the degree of DNA opaqueness to infrared light. Moreover, these two cell death modes also can be differentiated by their infrared absorbance, which relates to the secondary structure of total cellular protein. In light of these findings, we conclude that, because of its capacity to monitor multiple biomolecular parameters, FTIR spectroscopy enables unambiguous and easy analysis of cell death modes and may be useful for biochemical and medical applications. PMID:19804743

  6. Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran

    The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119,more » WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.« less

  7. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    PubMed

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

    2015-11-01

    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

  8. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

    PubMed Central

    Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

    2015-01-01

    Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

  9. How Kidney Cell Death Induces Renal Necroinflammation.

    PubMed

    Mulay, Shrikant R; Kumar, Santhosh V; Lech, Maciej; Desai, Jyaysi; Anders, Hans-Joachim

    2016-05-01

    The nephrons of the kidney are independent functional units harboring cells of a low turnover during homeostasis. As such, physiological renal cell death is a rather rare event and dead cells are flushed away rapidly with the urinary flow. Renal cell necrosis occurs in acute kidney injuries such as thrombotic microangiopathies, necrotizing glomerulonephritis, or tubular necrosis. All of these are associated with intense intrarenal inflammation, which contributes to further renal cell loss, an autoamplifying process referred to as necroinflammation. But how does renal cell necrosis trigger inflammation? Here, we discuss the role of danger-associated molecular patterns (DAMPs), mitochondrial (mito)-DAMPs, and alarmins, as well as their respective pattern recognition receptors. The capacity of DAMPs and alarmins to trigger cytokine and chemokine release initiates the recruitment of leukocytes into the kidney that further amplify necroinflammation. Infiltrating neutrophils often undergo neutrophil extracellular trap formation associated with neutrophil death or necroptosis, which implies a release of histones, which act not only as DAMPs but also elicit direct cytotoxic effects on renal cells, namely endothelial cells. Proinflammatory macrophages and eventually cytotoxic T cells further drive kidney cell death and inflammation. Dissecting the molecular mechanisms of necroinflammation may help to identify the best therapeutic targets to limit nephron loss in kidney injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Non-apoptotic cell death in animal development.

    PubMed

    Kutscher, Lena M; Shaham, Shai

    2017-08-01

    Programmed cell death (PCD) is an important process in the development of multicellular organisms. Apoptosis, a form of PCD characterized morphologically by chromatin condensation, membrane blebbing, and cytoplasm compaction, and molecularly by the activation of caspase proteases, has been extensively investigated. Studies in Caenorhabditis elegans, Drosophila, mice, and the developing chick have revealed, however, that developmental PCD also occurs through other mechanisms, morphologically and molecularly distinct from apoptosis. Some non-apoptotic PCD pathways, including those regulating germ cell death in Drosophila, still appear to employ caspases. However, another prominent cell death program, linker cell-type death (LCD), is morphologically conserved, and independent of the key genes that drive apoptosis, functioning, at least in part, through the ubiquitin proteasome system. These non-apoptotic processes may serve as backup programs when caspases are inactivated or unavailable, or, more likely, as freestanding cell culling programs. Non-apoptotic PCD has been documented extensively in the developing nervous system, and during the formation of germline and somatic gonadal structures, suggesting that preservation of these mechanisms is likely under strong selective pressure. Here, we discuss our current understanding of non-apoptotic PCD in animal development, and explore possible roles for LCD and other non-apoptotic developmental pathways in vertebrates. We raise the possibility that during vertebrate development, apoptosis may not be the major PCD mechanism.

  11. Technological advances in real-time tracking of cell death

    PubMed Central

    Skommer, Joanna; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

    2010-01-01

    Cell population can be viewed as a quantum system, which like Schrödinger’s cat exists as a combination of survival- and death-allowing states. Tracking and understanding cell-to-cell variability in processes of high spatio-temporal complexity such as cell death is at the core of current systems biology approaches. As probabilistic modeling tools attempt to impute information inaccessible by current experimental approaches, advances in technologies for single-cell imaging and omics (proteomics, genomics, metabolomics) should go hand in hand with the computational efforts. Over the last few years we have made exciting technological advances that allow studies of cell death dynamically in real-time and with the unprecedented accuracy. These approaches are based on innovative fluorescent assays and recombinant proteins, bioelectrical properties of cells, and more recently also on state-of-the-art optical spectroscopy. Here, we review current status of the most innovative analytical technologies for dynamic tracking of cell death, and address the interdisciplinary promises and future challenges of these methods. PMID:20519963

  12. Chloroquine synergizes with FTS to enhance cell growth inhibition and cell death

    PubMed Central

    Schmukler, Eran; Wolfson, Eya; Haklai, Roni; Elad-Sfadia, Galit; Kloog, Yoel; Pinkas-Kramarski, Ronit

    2014-01-01

    The Ras family of small GTPases transmits extracellular signals that regulate cell growth, differentiation, motility and death. Ras signaling is constitutively active in a large number of human cancers. Ras can also regulate autophagy by affecting several signaling pathways including the mTOR pathway. Autophagy is a process that regulates the balance between protein synthesis and protein degradation. It is important for normal growth control, but may be defective in diseases. Previously, we have shown that Ras inhibition by FTS induces autophagy, which partially protects cancer cells and may limit the use of FTS as an anti-cancer drug. Since FTS is a non toxic drug we hypothesized that FTS and chloroquine (an autophagy inhibitor) will synergize in cell growth inhibition and cell death. Thus, in the present study, we explored the mechanism of each individual drug and their combined action. Our results demonstrate that in HCT-116 and in Panc-1 cells, FTS induces autophagy, which can be inhibited by chloroquine. Furthermore, the combined treatment synergistically decreased the number of viable cells. Interestingly, the combined treatment enhanced apoptotic cell death as indicated by increased sub-G1 cell population, increased Hoechst staining, activation of caspase 3, decrease in survivin expression and release of cytochrome c. Thus, chloroquine treatment may promote FTS-mediated inhibition of tumor cell growth and may stimulate apoptotic cell death. PMID:24368422

  13. Control of non-apoptotic nurse cell death by engulfment genes in Drosophila.

    PubMed

    Timmons, Allison K; Mondragon, Albert A; Meehan, Tracy L; McCall, Kimberly

    2017-04-03

    Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.

  14. Jasmonic acid signaling modulates ozone-induced hypersensitive cell death.

    PubMed

    Rao, M V; Lee, H; Creelman, R A; Mullet, J E; Davis, K R

    2000-09-01

    Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.

  15. Aeromonas hydrophila exotoxin induces cytoplasmic vacuolation and cell death in VERO cells.

    PubMed

    Di Pietro, Angela; Picerno, Isa; Visalli, Giuseppa; Chirico, Cristina; Spataro, Pasquale; Cannavò, Giuseppe; Scoglio, Maria E

    2005-07-01

    Many organisms are able to cause cell vacuolation, but it is unclear if this can be considered a step of apoptosis or necrosis, or a distinct form of cell death. In this study VERO cells were used to evaluate the relationship between vacuolation and cell death pattern caused by exotoxins produced by environmental strains of A. hydrophila. Cell damage has been evaluated morphologically as well as biochemically. Cytotoxic and vacuolating titres were strictly correlated and the vacuolation has to be considered an early indicator of cytotoxicity that causes cell apoptosis or necrosis in relation to the dose. Signs of apoptosis (chromatin condensation and blebbing) were observed at low concentration and TGase activity, referable to apoptosis induction, confirms morphological observations. In fact, putrescine incorporation was related both to cytotoxin concentration and time of incubation. Moreover, the observed doubling cells with necrotic features permit us to suppose that cell sensitivity and death pattern could change during the different phases of cellular cycle.

  16. Early induction of c-Myc is associated with neuronal cell death.

    PubMed

    Lee, Hyun-Pil; Kudo, Wataru; Zhu, Xiongwei; Smith, Mark A; Lee, Hyoung-gon

    2011-11-14

    Neuronal cell cycle activation has been implicated in neurodegenerative diseases such as Alzheimer's disease, while the initiating mechanism of cell cycle activation remains to be determined. Interestingly, our previous studies have shown that cell cycle activation by c-Myc (Myc) leads to neuronal cell death which suggests Myc might be a key regulator of cell cycle re-entry mediated neuronal cell death. However, the pattern of Myc expression in the process of neuronal cell death has not been addressed. To this end, we examined Myc induction by the neurotoxic agents camptothecin and amyloid-β peptide in a differentiated SH-SY5Y neuronal cell culture model. Myc expression was found to be significantly increased following either treatment and importantly, the induction of Myc preceded neuronal cell death suggesting it is an early event of neuronal cell death. Since ectopic expression of Myc in neurons causes the cell cycle activation and neurodegeneration in vivo, the current data suggest that induction of Myc by neurotoxic agents or other disease factors might be a key mediator in cell cycle activation and consequent cell death that is a feature of neurodegenerative diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. Detecting cell death with optical coherence tomography and envelope statistics

    NASA Astrophysics Data System (ADS)

    Farhat, Golnaz; Yang, Victor X. D.; Czarnota, Gregory J.; Kolios, Michael C.

    2011-02-01

    Currently no standard clinical or preclinical noninvasive method exists to monitor cell death based on morphological changes at the cellular level. In our past work we have demonstrated that quantitative high frequency ultrasound imaging can detect cell death in vitro and in vivo. In this study we apply quantitative methods previously used with high frequency ultrasound to optical coherence tomography (OCT) to detect cell death. The ultimate goal of this work is to use these methods for optically-based clinical and preclinical cancer treatment monitoring. Optical coherence tomography data were acquired from acute myeloid leukemia cells undergoing three modes of cell death. Significant increases in integrated backscatter were observed for cells undergoing apoptosis and mitotic arrest, while necrotic cells induced a decrease. These changes appear to be linked to structural changes observed in histology obtained from the cell samples. Signal envelope statistics were analyzed from fittings of the generalized gamma distribution to histograms of envelope intensities. The parameters from this distribution demonstrated sensitivities to morphological changes in the cell samples. These results indicate that OCT integrated backscatter and first order envelope statistics can be used to detect and potentially differentiate between modes of cell death in vitro.

  18. Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

    PubMed Central

    Schneider, Naira F. Z.; Cerella, Claudia; Lee, Jin-Young; Mazumder, Aloran; Kim, Kyung Rok; de Carvalho, Annelise; Munkert, Jennifer; Pádua, Rodrigo M.; Kreis, Wolfgang; Kim, Kyu-Won; Christov, Christo; Dicato, Mario; Kim, Hyun-Jung; Han, Byung Woo; Braga, Fernão C.; Simões, Cláudia M. O.; Diederich, Marc

    2018-01-01

    Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities. PMID:29545747

  19. Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

    PubMed

    Gröbner, Sabine; Fritz, Evelyn; Schoch, Friederike; Schaller, Martin; Berger, Alexander C; Bitzer, Michael; Autenrieth, Ingo B

    2010-10-01

    Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential DeltaPsi(m). Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.

  20. Cylindromatosis mediates neuronal cell death in vitro and in vivo.

    PubMed

    Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

    2018-01-19

    The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

  1. Modelling the balance between quiescence and cell death in normal and tumour cell populations.

    PubMed

    Spinelli, Lorenzo; Torricelli, Alessandro; Ubezio, Paolo; Basse, Britta

    2006-08-01

    When considering either human adult tissues (in vivo) or cell cultures (in vitro), cell number is regulated by the relationship between quiescent cells, proliferating cells, cell death and other controls of cell cycle duration. By formulating a mathematical description we see that even small alterations of this relationship may cause a non-growing population to start growing with doubling times characteristic of human tumours. Our model consists of two age structured partial differential equations for the proliferating and quiescent cell compartments. Model parameters are death rates from and transition rates between these compartments. The partial differential equations can be solved for the steady-age distributions, giving the distribution of the cells through the cell cycle, dependent on specific model parameter values. Appropriate formulas can then be derived for various population characteristic quantities such as labelling index, proliferation fraction, doubling time and potential doubling time of the cell population. Such characteristic quantities can be estimated experimentally, although with decreasing precision from in vitro, to in vivo experimental systems and to the clinic. The model can be used to investigate the effects of a single alteration of either quiescence or cell death control on the growth of the whole population and the non-trivial dependence of the doubling time and other observable quantities on particular underlying cell cycle scenarios of death and quiescence. The model indicates that tumour evolution in vivo is a sequence of steady-states, each characterised by particular death and quiescence rate functions. We suggest that a key passage of carcinogenesis is a loss of the communication between quiescence, death and cell cycle machineries, causing a defect in their precise, cell cycle dependent relationship.

  2. Stem cell death and survival in heart regeneration and repair.

    PubMed

    Abdelwahid, Eltyeb; Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

    2016-03-01

    Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function.

  3. Stem cell death and survival in heart regeneration and repair

    PubMed Central

    Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

    2016-01-01

    Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function. PMID:26687129

  4. Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

    PubMed Central

    Wang, Lihong; Liu, Liping; Shi, Yan; Cao, Hanwei; Chaturvedi, Rupesh; Calcutt, M. Wade; Hu, Tianhui; Ren, Xiubao; Wilson, Keith T.; Polk, D. Brent; Yan, Fang

    2012-01-01

    Berberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the mouse immorto-Min colonic epithelial (IMCE) cells carrying the Apc min mutation, and of normal colon epithelial cells, namely young adult mouse colonic epithelium (YAMC) cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth. PMID:22574158

  5. Vacuolar processing enzyme: an executor of plant cell death.

    PubMed

    Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

    2005-08-01

    Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

  6. Hemoglobins, programmed cell death and somatic embryogenesis.

    PubMed

    Hill, Robert D; Huang, Shuanglong; Stasolla, Claudio

    2013-10-01

    Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somatic embryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somatic embryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somatic embryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival. Copyright © 2013 Elsevier Ireland Ltd

  7. The engulfment receptor Draper is required for autophagy during cell death.

    PubMed

    McPhee, Christina K; Baehrecke, Eric H

    2010-11-01

    Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction: during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.

  8. Oxidative Stress and Programmed Cell Death in Yeast

    PubMed Central

    Farrugia, Gianluca; Balzan, Rena

    2012-01-01

    Yeasts, such as Saccharomyces cerevisiae, have long served as useful models for the study of oxidative stress, an event associated with cell death and severe human pathologies. This review will discuss oxidative stress in yeast, in terms of sources of reactive oxygen species (ROS), their molecular targets, and the metabolic responses elicited by cellular ROS accumulation. Responses of yeast to accumulated ROS include upregulation of antioxidants mediated by complex transcriptional changes, activation of pro-survival pathways such as mitophagy, and programmed cell death (PCD) which, apart from apoptosis, includes pathways such as autophagy and necrosis, a form of cell death long considered accidental and uncoordinated. The role of ROS in yeast aging will also be discussed. PMID:22737670

  9. Jasmonic Acid Signaling Modulates Ozone-Induced Hypersensitive Cell Death

    PubMed Central

    Rao, Mulpuri V.; Lee, Hyung-il; Creelman, Robert A.; Mullet, John E.; Davis, Keith R.

    2000-01-01

    Recent studies suggest that cross-talk between salicylic acid (SA)–, jasmonic acid (JA)–, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O3) exposure activates a hypersensitive response (HR)–like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O3-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O3-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O3-induced H2O2 content and SA concentrations and completely abolished O3-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O3 exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O3 of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O3-induced HR-like cell death. PMID:11006337

  10. BaxΔ2 sensitizes colorectal cancer cells to proteasome inhibitor-induced cell death

    PubMed Central

    Mañas, Adriana; Chen, Wenjing; Nelson, Adam; Yao, Qi; Xiang, Jialing

    2018-01-01

    Proteasome inhibitors, such as bortezomib and carfilzomib, are FDA approved for the treatment of hemopoietic cancers, but recent studies have shown their great potential for treatment of solid tumors. BaxΔ2, a unique proapoptotic Bax isoform, promotes non-mitochondrial cell death and sensitizes cancer cells to chemotherapy. However, endogenous BaxΔ2 proteins are unstable and susceptible to proteasomal degradation. Here, we screened a panel of proteasome inhibitors in colorectal cancer cells with different Bax statuses. We found that all proteasome inhibitors tested were able to block BaxΔ2 degradation without affecting the level of Baxα or Bcl-2 proteins. Among the inhibitors tested, only bortezomib and carfilzomib were able to induce differential cell death corresponding to the distinct Bax statuses. BaxΔ2-positive cells had a significantly higher level of cell death at low nanomolar concentrations than Baxα-positive or Bax-negative cells. Furthermore, bortezomib-induced cell death in BaxΔ2-positive cells was predominantly dependent on the caspase 8/3 pathway, consistent with our previous studies. These results imply that BaxΔ2 can selectively sensitize cancer cells to proteasome inhibitors, enhancing their potential to treat colon cancer and other solid tumors. PMID:29291406

  11. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Curry, Merril C.; Peters, Amelia A.; Kenny, Paraic A.

    Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levelsmore » of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.« less

  12. The synthetic purine reversine selectively induces cell death of cancer cells.

    PubMed

    Piccoli, Marco; Palazzolo, Giacomo; Conforti, Erika; Lamorte, Giuseppe; Papini, Nadia; Creo, Pasquale; Fania, Chiara; Scaringi, Raffaella; Bergante, Sonia; Tringali, Cristina; Roncoroni, Leda; Mazzoleni, Stefania; Doneda, Luisa; Galli, Rossella; Venerando, Bruno; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

    2012-10-01

    The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. Copyright © 2012 Wiley Periodicals, Inc.

  13. RIP3 attenuates the pancreatic damage induced by deletion of ATG7.

    PubMed

    Zhou, Xiaodong; Xie, Li; Xia, Leizhou; Bergmann, Frank; Büchler, Markus W; Kroemer, Guido; Hackert, Thilo; Fortunato, Franco

    2017-07-13

    Invalidation of pancreatic autophagy entails pancreatic atrophy, endocrine and exocrine insufficiency and pancreatitis. The aim of this study was to investigate whether depletion of Rip3, which is involved in necroptotic signaling, may attenuate the pancreatic atrophy and pancreatitis resulting from autophagy inhibition. Autophagy and necroptosis signaling were evaluated in mice lacking expression of Rip3 in all organs and Atg7 in the pancreas. Acinar cell death, inflammation and fibrosis were evaluated by using of a compendium of immunofluorescence methods and immunoblots. Mice deficient for pancreatic Atg7 developed acute pancreatitis, which progressed to chronic pancreatitis. This phenotype reduces autophagy, increase apoptosis and necroptosis, inflammation and fibrosis, as well as premature death of the animals. Knockout of Rip3 exacerbated the apoptotic death of acinar cells, increased tissue damage, reduced macrophage infiltration and further accelerated the death of the mice with Atg7-deficient pancreas. The pancreatic degeneration induced by autophagy inhibition was exacerbated by Rip3 deletion.

  14. Mechanisms underlying 3-bromopyruvate-induced cell death in colon cancer.

    PubMed

    Sun, Yiming; Liu, Zhe; Zou, Xue; Lan, Yadong; Sun, Xiaojin; Wang, Xiu; Zhao, Surong; Jiang, Chenchen; Liu, Hao

    2015-08-01

    3-Bromopyruvate (3BP) is an energy-depleting drug that inhibits Hexokinase II activity by alkylation during glycolysis, thereby suppressing the production of ATP and inducing cell death. As such, 3BP can potentially serve as an anti-tumorigenic agent. Our previous research showed that 3BP can induce apoptosis via AKT /protein Kinase B signaling in breast cancer cells. Here we found that 3BP can also induce colon cancer cell death by necroptosis and apoptosis at the same time and concentration in the SW480 and HT29 cell lines; in the latter, autophagy was also found to be a mechanism of cell death. In HT29 cells, combined treatment with 3BP and the autophagy inhibitor 3-methyladenine (3-MA) exacerbated cell death, while viability in 3BP-treated cells was enhanced by concomitant treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and the necroptosis inhibitor necrostatin (Nec)-1. Moreover, 3BP inhibited tumor growth in a SW480 xenograft mouse model. These results indicate that 3BP can suppress tumor growth and induce cell death by multiple mechanisms at the same time and concentration in different types of colon cancer cell by depleting cellular energy stores.

  15. Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

    PubMed

    Chou, Ya-Shuan; Young, Tai-Horng; Lou, Pei-Jen

    2015-11-01

    Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the

  16. Plasma membrane changes during programmed cell deaths

    PubMed Central

    Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai

    2018-01-01

    Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity. PMID:29076500

  17. Restricted diffusion in a model acinar labyrinth by NMR: Theoretical and numerical results

    NASA Astrophysics Data System (ADS)

    Grebenkov, D. S.; Guillot, G.; Sapoval, B.

    2007-01-01

    A branched geometrical structure of the mammal lungs is known to be crucial for rapid access of oxygen to blood. But an important pulmonary disease like emphysema results in partial destruction of the alveolar tissue and enlargement of the distal airspaces, which may reduce the total oxygen transfer. This effect has been intensively studied during the last decade by MRI of hyperpolarized gases like helium-3. The relation between geometry and signal attenuation remained obscure due to a lack of realistic geometrical model of the acinar morphology. In this paper, we use Monte Carlo simulations of restricted diffusion in a realistic model acinus to compute the signal attenuation in a diffusion-weighted NMR experiment. We demonstrate that this technique should be sensitive to destruction of the branched structure: partial removal of the interalveolar tissue creates loops in the tree-like acinar architecture that enhance diffusive motion and the consequent signal attenuation. The role of the local geometry and related practical applications are discussed.

  18. The importance of being dead: cell death mechanisms assessment in anti-sarcoma therapy.

    PubMed

    Rello-Varona, Santiago; Herrero-Martín, David; Lagares-Tena, Laura; López-Alemany, Roser; Mulet-Margalef, Núria; Huertas-Martínez, Juan; Garcia-Monclús, Silvia; García Del Muro, Xavier; Muñoz-Pinedo, Cristina; Tirado, Oscar Martínez

    2015-01-01

    Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called "autophagic cell death" or "mitotic catastrophe") have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review, our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

  19. Cell death sensitization of leukemia cells by opioid receptor activation

    PubMed Central

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  20. Polyoma small T antigen triggers cell death via mitotic catastrophe

    PubMed Central

    Fernando, Arun T Pores; Andrabi, Shaida; Cizmecioglu, Onur; Zhu, Cailei; Livingston, David M.; Higgins, Jonathan M.G; Schaffhausen, Brian S; Roberts, Thomas M

    2014-01-01

    Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST-expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, resulting in the activation of the Spindle Assembly Checkpoint (SAC). Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed that, PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors. PMID:24998850

  1. Cell death versus cell survival instructed by supramolecular cohesion of nanostructures

    NASA Astrophysics Data System (ADS)

    Newcomb, Christina J.; Sur, Shantanu; Ortony, Julia H.; Lee, One-Sun; Matson, John B.; Boekhoven, Job; Yu, Jeong Min; Schatz, George C.; Stupp, Samuel I.

    2014-02-01

    Many naturally occurring peptides containing cationic and hydrophobic domains have evolved to interact with mammalian cell membranes and have been incorporated into materials for non-viral gene delivery, cancer therapy or treatment of microbial infections. Their electrostatic attraction to the negatively charged cell surface and hydrophobic interactions with the membrane lipids enable intracellular delivery or cell lysis. Although the effects of hydrophobicity and cationic charge of soluble molecules on the cell membrane are well known, the interactions between materials with these molecular features and cells remain poorly understood. Here we report that varying the cohesive forces within nanofibres of supramolecular materials with nearly identical cationic and hydrophobic structure instruct cell death or cell survival. Weak intermolecular bonds promote cell death through disruption of lipid membranes, while materials reinforced by hydrogen bonds support cell viability. These findings provide new strategies to design biomaterials that interact with the cell membrane.

  2. How does metabolism affect cell death in cancer?

    PubMed

    Villa, Elodie; Ricci, Jean-Ehrland

    2016-07-01

    In cancer research, identifying a specificity of tumor cells compared with 'normal' proliferating cells for targeted therapy is often considered the Holy Grail for researchers and clinicians. Although diverse in origin, most cancer cells share characteristics including the ability to escape cell death mechanisms and the utilization of different methods of energy production. In the current paradigm, aerobic glycolysis is considered the central metabolic characteristic of cancer cells (Warburg effect). However, recent data indicate that cancer cells also show significant changes in other metabolic pathways. Indeed, it was recently suggested that Kreb's cycle, pentose phosphate pathway intermediates, and essential and nonessential amino acids have key roles. Renewed interest in the fact that cancer cells have to reprogram their metabolism in order to proliferate or resist treatment must take into consideration the ability of tumor cells to adapt their metabolism to the local microenvironment (low oxygen, low nutrients). This variety of metabolic sources might be either a strength, resulting in infinite possibilities for adaptation and increased ability to resist chemotherapy-induced death, or a weakness that could be targeted to kill cancer cells. Here, we discuss recent insights showing how energetic metabolism may regulate cell death and how this might be relevant for cancer treatment. © 2015 FEBS.

  3. A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants.

    PubMed

    Olvera-Carrillo, Yadira; Van Bel, Michiel; Van Hautegem, Tom; Fendrych, Matyáš; Huysmans, Marlies; Simaskova, Maria; van Durme, Matthias; Buscaill, Pierre; Rivas, Susana; Coll, Nuria S.; Coppens, Frederik; Maere, Steven; Nowack, Moritz K.

    2015-12-01

    A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta. © 2015 American Society of Plant Biologists. All Rights Reserved.

  4. Using natural products to promote caspase-8-dependent cancer cell death.

    PubMed

    Tewary, Poonam; Gunatilaka, A A Leslie; Sayers, Thomas J

    2017-02-01

    The selective killing of cancer cells without toxicity to normal nontransformed cells is an idealized goal of cancer therapy. Thus, there has been much interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that appears to selectively kill cancer cells. TRAIL has been reported to trigger apoptosis and under some circumstances, an alternate death signaling pathway termed necroptosis. The relative importance of necroptosis for cell death induction in vivo is under intensive investigation. Nonetheless, many cancer cells (particularly those freshly isolated from cancer patients) are highly resistant to TRAIL-mediated cell death. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. There are numerous reports in which combination of TRAIL with standard antineoplastic drugs has resulted in enhanced cancer cell death in vitro. However, many of these chemotherapeutic drugs are nonspecific and associated with adverse effects, which raise serious concerns for cancer therapy in patients. By contrast, natural products have been shown to be safer and efficacious alternatives. Recently, a number of studies have suggested that certain natural products when combined with TRAIL can enhance cancer cell death. In this review, we highlight molecular pathways that might be targeted by various natural products to promote cell death, and focus on our recent work with withanolides as TRAIL sensitizers. Finally, we will suggest synergistic approaches for combining active withanolides with various forms of immunotherapy to promote cancer cell death and an effective antitumor immune response.

  5. Independent controls for neocortical neuron production and histogenetic cell death

    NASA Technical Reports Server (NTRS)

    Verney, C.; Takahashi, T.; Bhide, P. G.; Nowakowski, R. S.; Caviness, V. S. Jr

    2000-01-01

    We estimated the proportion of cells eliminated by histogenetic cell death during the first 2 postnatal weeks in areas 1, 3 and 40 of the mouse parietal neocortex. For each layer and for the subcortical white matter in each neocortical area, the number of dying cells per mm(2) was calculated and the proportionate cell death for each day of the 2-week interval was estimated. The data show that cell death proceeds essentially uniformly across the neocortical areas and layers and that it does not follow either the spatiotemporal gradient of cell cycle progression in the pseudostratified ventricular epithelium of the cerebral wall, the source of neocortical neurons, or the 'inside-out' neocortical neuronogenetic sequence. Therefore, we infer that the control mechanisms of neocortical histogenetic cell death are independent of mechanisms controlling neuronogenesis or neuronal migration but may be associated with the ingrowth, expansion and a system-wide matching of neuronal connectivity. Copyright 2000 S. Karger AG, Basel.

  6. Sensory hair cell death and regeneration in fishes

    PubMed Central

    Monroe, Jerry D.; Rajadinakaran, Gopinath; Smith, Michael E.

    2015-01-01

    Sensory hair cells are specialized mechanotransductive receptors required for hearing and vestibular function. Loss of hair cells in humans and other mammals is permanent and causes reduced hearing and balance. In the early 1980’s, it was shown that hair cells continue to be added to the inner ear sensory epithelia in cartilaginous and bony fishes. Soon thereafter, hair cell regeneration was documented in the chick cochlea following acoustic trauma. Since then, research using chick and other avian models has led to great insights into hair cell death and regeneration. However, with the rise of the zebrafish as a model organism for studying disease and developmental processes, there has been an increased interest in studying sensory hair cell death and regeneration in its lateral line and inner ears. Advances derived from studies in zebrafish and other fish species include understanding the effect of ototoxins on hair cells and finding otoprotectants to mitigate ototoxin damage, the role of cellular proliferation vs. direct transdifferentiation during hair cell regeneration, and elucidating cellular pathways involved in the regeneration process. This review will summarize research on hair cell death and regeneration using fish models, indicate the potential strengths and weaknesses of these models, and discuss several emerging areas of future studies. PMID:25954154

  7. Dying dangerously: Necrotic cell death and chronic inflammation promote tumor growth.

    PubMed

    Lotze, Michael T; Demarco, Richard A

    2004-12-01

    Extract: We all shudder about untimely deaths or those that we were not prepared for. As such we perceive such "unscheduled" deaths as dangerous. Similarly, apoptotic death (literally falling leaves) or the programmed cell death of cells in multicellular organisms ranging from slime mold and simple worms through to mammals, has a level of tidiness and well-orchestrated activities with literally hundreds if not thousands of gene products employed with either the primary or secondary purpose of coordinating the orderly death of cells throughout life. During inflammation of any sort, driven by tissue damage or injury or infection by pathogens (virus, bacteria, and parasites), apoptotic death similarly serves to quickly rid the host of damaged cells, promote removal and digestion of the infected cell, and prepare the way for tissue remodeling and repair. When this goes awry, for example during periods of chronic inflammation, tissues are subjected to the contrasting needs of driving apoptotic death whilst maintaining the barrier function of the epithelia (such as skin cells) as well as the selective permeability of mucosal sites (i.e., areas where mucus is secreted to protect the cells from their surroundings, such as gut cells protecting themselves from the gastric acids). Prudently, they need to limit and husband local resources sufficiently for the maintenance of tissue integrity and renewal. It is our provocative and novel contention that cancer in adults (and not children) most often arises in a setting of chronic inflammation and disordered cell death rather than one associated primarily with disordered cell growth as it is popularly imagined by scientists, clinicians, and the general public.

  8. Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death.

    PubMed

    Ramírez-Barrantes, Ricardo; Córdova, Claudio; Gatica, Sebastian; Rodriguez, Belén; Lozano, Carlo; Marchant, Ivanny; Echeverria, Cesar; Simon, Felipe; Olivero, Pablo

    2018-01-01

    The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose-response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca 2+ and mitochondrial dysfunction participates in CAP-induced TRPV1-mediated cell death. By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca 2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca 2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca 2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.

  9. Targeting Programmed Cell Death Using Small-Molecule Compounds to Improve Potential Cancer Therapy.

    PubMed

    Ke, Bowen; Tian, Mao; Li, Jingjing; Liu, Bo; He, Gu

    2016-11-01

    Evasion of cell death is one of the hallmarks of cancer cells, beginning with long-established apoptosis and extending to other new forms of cell death. An elaboration of cell death pathways thus will contribute to a better understanding of cancer pathogenesis and therapeutics. With the recent substantial biochemical and genetic explorations of cell death subroutines, their classification has switched from primarily morphological to more molecular definitions. According to their measurable biochemical features and intricate mechanisms, cell death subroutines can be divided into apoptosis, autophagic cell death, mitotic catastrophe, necroptosis, parthanatos, ferroptosis, pyroptosis, pyronecrosis, anoikis, cornification, entosis, and NETosis. Supportive evidence has gradually revealed the prime molecular mechanisms of each subroutine and thus providing series of possible targets in cancer therapy, while the intricate relationships between different cell death subroutines still remain to be clarified. Over the past decades, cancer drug discovery has significantly benefited from the use of small-molecule compounds to target classical modalities of cell death such as apoptosis, while newly identified cell death subroutines has also emerging their potential for cancer drug discovery in recent years. In this review, we comprehensively focus on summarizing 12 cell death subroutines and discussing their corresponding small-molecule compounds in potential cancer therapy. Together, these inspiring findings may provide more evidence to fill in the gaps between cell death subroutines and small-molecule compounds to better develop novel cancer therapeutic strategies. © 2016 Wiley Periodicals, Inc.

  10. Regulated Forms of Cell Death in Fungi

    PubMed Central

    Gonçalves, A. Pedro; Heller, Jens; Daskalov, Asen; Videira, Arnaldo; Glass, N. Louise

    2017-01-01

    Cell death occurs in all domains of life. While some cells die in an uncontrolled way due to exposure to external cues, other cells die in a regulated manner as part of a genetically encoded developmental program. Like other eukaryotic species, fungi undergo programmed cell death (PCD) in response to various triggers. For example, exposure to external stress conditions can activate PCD pathways in fungi. Calcium redistribution between the extracellular space, the cytoplasm and intracellular storage organelles appears to be pivotal for this kind of cell death. PCD is also part of the fungal life cycle, in which it occurs during sexual and asexual reproduction, aging, and as part of development associated with infection in phytopathogenic fungi. Additionally, a fungal non-self-recognition mechanism termed heterokaryon incompatibility (HI) also involves PCD. Some of the molecular players mediating PCD during HI show remarkable similarities to major constituents involved in innate immunity in metazoans and plants. In this review we discuss recent research on fungal PCD mechanisms in comparison to more characterized mechanisms in metazoans. We highlight the role of PCD in fungi in response to exogenic compounds, fungal development and non-self-recognition processes and discuss identified intracellular signaling pathways and molecules that regulate fungal PCD. PMID:28983298

  11. Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

    PubMed

    Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

    2017-07-17

    Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

  12. Necroptosis: an alternative cell death program defending against cancer

    PubMed Central

    Chen, Dongshi; Yu, Jian; Zhang, Lin

    2016-01-01

    One of the hallmarks of cancer is resistance to programmed cell death, which maintains the survival of cells en route to oncogenic transformation and underlies therapeutic resistance. Recent studies demonstrate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a form of necrotic death governed by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL). Necroptosis serves as a critical cell-killing mechanism in response to severe stress and blocked apoptosis, and can be induced by inflammatory cytokines or chemotherapeutic drugs. Genetic or epigenetic alterations of necroptosis regulators such as RIP3 and cylindromatosis (CYLD), are frequently found in human tumors. Unlike apoptosis, necroptosis elicits a more robust immune response that may function as a defensive mechanism by eliminating tumor-causing mutations and viruses. Furthermore, several classes of anticancer agents currently under clinical development, such as SMAC and BH3 mimetics, can promote necroptosis in addition to apoptosis. A more complete understanding of the interplay among necroptosis, apoptosis, and other cell death modalities is critical for developing new therapeutic strategies to enhance killing of tumor cells. PMID:26968619

  13. Necroptosis: an alternative cell death program defending against cancer.

    PubMed

    Chen, Dongshi; Yu, Jian; Zhang, Lin

    2016-04-01

    One of the hallmarks of cancer is resistance to programmed cell death, which maintains the survival of cells en route to oncogenic transformation and underlies therapeutic resistance. Recent studies demonstrate that programmed cell death is not confined to caspase-dependent apoptosis, but includes necroptosis, a form of necrotic death governed by Receptor-Interacting Protein 1 (RIP1), RIP3, and Mixed Lineage Kinase Domain-Like (MLKL) protein. Necroptosis serves as a critical cell-killing mechanism in response to severe stress and blocked apoptosis, and can be induced by inflammatory cytokines or chemotherapeutic drugs. Genetic or epigenetic alterations of necroptosis regulators such as RIP3 and cylindromatosis (CYLD), are frequently found in human tumors. Unlike apoptosis, necroptosis elicits a more robust immune response that may function as a defensive mechanism by eliminating tumor-causing mutations and viruses. Furthermore, several classes of anticancer agents currently under clinical development, such as SMAC and BH3 mimetics, can promote necroptosis in addition to apoptosis. A more complete understanding of the interplay among necroptosis, apoptosis, and other cell death modalities is critical for developing new therapeutic strategies to enhance killing of tumor cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death.

    PubMed

    Chen, George G; Sin, Fanny L F; Leung, Billy C S; Ng, Ho K; Poon, Wai S

    2005-04-01

    DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli. 2004 Wiley-Liss, Inc.

  15. Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

    NASA Astrophysics Data System (ADS)

    Doitsh, Gilad; Galloway, Nicole L. K.; Geng, Xin; Yang, Zhiyuan; Monroe, Kathryn M.; Zepeda, Orlando; Hunt, Peter W.; Hatano, Hiroyu; Sowinski, Stefanie; Muñoz-Arias, Isa; Greene, Warner C.

    2014-01-01

    The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of `anti-AIDS' therapeutics targeting the host rather than the virus.

  16. Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium

    PubMed Central

    1989-01-01

    The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol- induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o- aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the

  17. Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

    PubMed Central

    Burugula, Balabharathi; Ganesh, Bhagyalaxmi S.

    2011-01-01

    Purpose. Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs. Methods. Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 μM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 μM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB). Results. Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression. Conclusions. The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions. PMID:21498608

  18. Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Ruochan; Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008; Fu, Sha

    High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis andmore » necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.« less

  19. Coordination of cell death and the cell cycle: linking proliferation to death through private and communal couplers.

    PubMed

    Abrams, John M; White, Michael A

    2004-12-01

    In development and in the adult, complex signaling pathways operate within and between cells to coordinate proliferation and cell death. These networks can be viewed as coupling devices that link engines driving the cell cycle and the initiation of apoptosis. We propose three simple frameworks for modeling the effects of proliferative drive on apoptotic propensity. This perspective offers a potentially useful foundation for predicting group behaviors of cells in normal and pathological settings.

  20. Therapeutic approaches to preventing cell death in Huntington disease.

    PubMed

    Kaplan, Anna; Stockwell, Brent R

    2012-12-01

    Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors-fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Therapeutic approaches to preventing cell death in Huntington disease

    PubMed Central

    Kaplan, Anna; Stockwell, Brent R.

    2012-01-01

    Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors—fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders. PMID:22967354

  2. Cytoprotective dibenzoylmethane derivatives protect cells from oxidative stress-induced necrotic cell death.

    PubMed

    Hegedűs, Csaba; Lakatos, Petra; Kiss-Szikszai, Attila; Patonay, Tamás; Gergely, Szabolcs; Gregus, Andrea; Bai, Péter; Haskó, György; Szabó, Éva; Virág, László

    2013-06-01

    Screening of a small in-house library of 1863 compounds identified 29 compounds that protected Jurkat cells from hydrogen peroxide-induced cytotoxicity. From the cytoprotective compounds eleven proved to possess antioxidant activity (ABTS radical scavenger effect) and two were found to inhibit poly(ADP-ribosyl)ation (PARylation), a cytotoxic pathway operating in severely injured cells. Four cytoprotective dibenzoylmethane (DBM) derivatives were investigated in more detail as they did not scavenge hydrogen peroxide nor did they inhibit PARylation. These compounds protected cells from necrotic cell death while caspase activation, a parameter of apoptotic cell death was not affected. Hydrogen peroxide activated extracellular signal regulated kinase (ERK1/2) and p38 MAP kinases but not c-Jun N-terminal kinase (JNK). The cytoprotective DBMs suppressed the activation of Erk1/2 but not that of p38. Cytoprotection was confirmed in another cell type (A549 lung epithelial cells), indicating that the cytoprotective effect is not cell type specific. In conclusion we identified DBM analogs as a novel class of cytoprotective compounds inhibiting ERK1/2 kinase and protecting from necrotic cell death by a mechanism independent of poly(ADP-ribose) polymerase inhibition. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. 6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells.

    PubMed

    Li, Ting-Yi; Chiang, Been-Huang

    2017-09-01

    6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6-shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6h, 6-shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

    PubMed

    Aits, Sonja; Gustafsson, Lotta; Hallgren, Oskar; Brest, Patrick; Gustafsson, Mattias; Trulsson, Maria; Mossberg, Ann-Kristin; Simon, Hans-Uwe; Mograbi, Baharia; Svanborg, Catharina

    2009-03-01

    HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3-I) to granular (LC3-II) staining in LC3-GFP-transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3-methyladenine. HAMLET also caused accumulation of LC3-II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin-1 and Atg5. Suppression of Beclin-1 and Atg5 improved the survival of HAMLET-treated tumor cells and inhibited the increase in granular LC3-GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET-induced tumor cell death.

  5. TP53 alterations in pancreatic acinar cell carcinoma: new insights into the molecular pathology of this rare cancer.

    PubMed

    La Rosa, Stefano; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Furlan, Daniela; Sahnane, Nora; Vanoli, Alessandro; Albarello, Luca; Zhang, Lizhi; Notohara, Kenji; Casnedi, Selenia; Chenard, Marie-Pierre; Adsay, Volkan; Asioli, Sofia; Capella, Carlo; Sessa, Fausto

    2016-03-01

    The molecular alterations of pancreatic acinar cell carcinomas (ACCs) are poorly understood and have been reported as being different from those in ductal adenocarcinomas. Loss of TP53 gene function in the pathogenesis of ACCs is controversial since contradictory findings have been published. A comprehensive analysis of the different possible genetic and epigenetic mechanisms leading to TP53 alteration in ACC has never been reported and hence the role of TP53 in the pathogenesis and/or progression of ACC remains unclear. We investigated TP53 alterations in 54 tumor samples from 44 patients, including primary and metastatic ACC, using sequencing analysis, methylation-specific multiplex ligation probe amplification, fluorescence in situ hybridization, and immunohistochemistry. TP53 mutations were found in 13 % of primary ACCs and in 31 % of metastases. Primary ACCs and metastases showed the same mutational profile, with the exception of one case, characterized by a wild-type sequence in the primary carcinoma and a mutation in the corresponding metastasis. FISH analysis revealed deletion of the TP53 region in 53 % of primary ACCs and in 50 % of metastases. Promoter hypermethylation was found in one case. The molecular alterations correlated well with the immunohistochemical findings. A statistically significant association was found between the combination of mutation of one allele and loss of the other allele of TP53 and worse survival.

  6. Inducible nitric oxide synthase in T cells regulates T cell death and immune memory

    PubMed Central

    Vig, Monika; Srivastava, Smita; Kandpal, Usha; Sade, Hadassah; Lewis, Virginia; Sarin, Apurva; George, Anna; Bal, Vineeta; Durdik, Jeannine M.; Rath, Satyajit

    2004-01-01

    The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS+/+ APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS–/– T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination. PMID:15199408

  7. Tritrichomonas foetus Induces Apoptotic Cell Death in Bovine Vaginal Epithelial Cells

    PubMed Central

    Singh, B. N.; Lucas, J. J.; Hayes, G. R.; Kumar, Ish; Beach, D. H.; Frajblat, Marcel; Gilbert, R. O.; Sommer, U.; Costello, C. E.

    2004-01-01

    Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISAPLUS assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter

  8. Zinc as a paracrine effector in pancreatic islet cell death.

    PubMed

    Kim, B J; Kim, Y H; Kim, S; Kim, J W; Koh, J Y; Oh, S H; Lee, M K; Kim, K W; Lee, M S

    2000-03-01

    Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.

  9. Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells.

    PubMed

    Morimoto, Satoshi; Tanaka, Yumi; Sasaki, Kaori; Tanaka, Hiroyuki; Fukamizu, Tomohide; Shoyama, Yoshinari; Shoyama, Yukihiro; Taura, Futoshi

    2007-07-13

    Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Delta(1)-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca(2+) and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

  10. Leucine Affects α-Amylase Synthesis through PI3K/Akt-mTOR Signaling Pathways in Pancreatic Acinar Cells of Dairy Calves.

    PubMed

    Guo, Long; Liang, Ziqi; Zheng, Chen; Liu, Baolong; Yin, Qingyan; Cao, Yangchun; Yao, Junhu

    2018-05-23

    Dietary nutrient utilization, particularly starch, is potentially limited by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine acts as an effective signal molecular in the mTOR signaling pathway, which regulates a series of biological processes, especially protein synthesis. It has been reported that leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were used as a model to determine the cellular signal of leucine influence on α-amylase synthesis. PA cells were isolated from newborn Holstein dairy bull calves and cultured in Dulbecco's modifed Eagle's medium/nutrient mixture F12 liquid media containing four leucine treatments (0, 0.23, 0.45, and 0.90 mM, respectively), following α-amylase activity, zymogen granule, and signal pathway factor expression detection. Rapamycin, a specific inhibitor of mTOR, was also applied to PA cells. Results showed that leucine increased ( p < 0.05) synthesis of α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1 while reduced ( p < 0.05) GCN2 expression. Inhibition of mTOR signaling downregulated the α-amylase synthesis. In addition, the extracellular leucine dosage significantly influenced intracellular metabolism of isoleucine ( p < 0.05). Overall, leucine regulates α-amylase synthesis through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway in PA cells of dairy calves. These pathways form the signaling network that controls the protein synthesis and metabolism. It would be of great interest in future studies to explore the function of leucine in ruminant nutrition.

  11. Long-term treatment of anterior pituitary cells with nitric oxide induces programmed cell death.

    PubMed

    Velardez, Miguel Omar; Poliandri, Ariel Hernán; Cabilla, Jimena Paula; Bodo, Cristian Carlos Armando; Machiavelli, Leticia Inés; Duvilanski, Beatriz Haydeé

    2004-04-01

    Nitric oxide (NO) plays a complex role in modulating programmed cell death. It can either protect the cell from apoptotic death or mediate apoptosis, depending on its concentration and the cell type and/or status. In this study, we demonstrate that long-term exposition to NO induces cell death of anterior pituitary cells from Wistar female rats. DETA NONOate (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, 1 mm], a NO donor that releases NO for an extended period of time, decreased cellular viability and prolactin release from primary cultures of anterior pituitary cells. Morphological studies showed an increase in the number of cells with chromatin condensation and nuclear fragmentation at 24 and 48 h after DETA/NO exposure. DNA internucleosomal fragmentation was also observed at the same time. Reversibility of the NO effect on cellular viability and prolactin release was observed only when the cells were incubated with DETA/NO for less than 6 h. Most apoptotic cells were immunopositive for prolactin, suggesting a high susceptibility of lactotrophs to the effect of NO. The cytotoxic effect of NO is dependent of caspase-9 and caspase-3, but seems to be independent of oxidative stress or nitrosative stress. Our results show that the exposition of anterior pituitary cells to NO for long periods induces programmed cell death of anterior pituitary cells.

  12. Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

    PubMed

    Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

    2016-09-01

    Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

  13. Programmed Cell Death-1/Programmed Death-ligand 1 Pathway: A New Target for Sepsis.

    PubMed

    Liu, Qiang; Li, Chun-Sheng

    2017-04-20

    Sepsis remains a leading cause of death in many Intensive Care Units worldwide. Immunosuppression has been a primary focus of sepsis research as a key pathophysiological mechanism. Given the important role of the negative costimulatory molecules programmed cell death-1 (PD-1) and programmed death-ligand 1 (PD-L1) in the occurrence of immunosuppression during sepsis, we reviewed literatures related to the PD-1/PD-L1 pathway to examine its potential as a new target for sepsis treatment. Studies of the association between PD-1/PD-L1 and sepsis published up to January 31, 2017, were obtained by searching the PubMed database. English language studies, including those based on animal models, clinical research, and reviews, with data related to PD-1/PD-L1 and sepsis, were evaluated. Immunomodulatory therapeutics could reverse the deactivation of immune cells caused by sepsis and restore immune cell activation and function. Blockade of the PD-1/PD-L1 pathway could reduce the exhaustion of T-cells and enhance the proliferation and activation of T-cells. The anti-PD-1/PD-L1 pathway shows promise as a new target for sepsis treatment. This review provides a basis for clinical trials and future studies aimed at revaluating the efficacy and safety of this targeted approach.

  14. Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

    PubMed

    Wu, Chenxi; Chen, Yujun; Wang, Feng; Chen, Changyan; Zhang, Shiping; Li, Chaojie; Li, Wenzhe; Wu, Shian; Xue, Lei

    2015-10-01

    Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

  15. Modulating cell-to-cell variability and sensitivity to death ligands by co-drugging

    NASA Astrophysics Data System (ADS)

    Flusberg, Deborah A.; Sorger, Peter K.

    2013-06-01

    TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) holds promise as an anti-cancer therapeutic but efficiently induces apoptosis in only a subset of tumor cell lines. Moreover, even in clonal populations of responsive lines, only a fraction of cells dies in response to TRAIL and individual cells exhibit cell-to-cell variability in the timing of cell death. Fractional killing in these cell populations appears to arise not from genetic differences among cells but rather from differences in gene expression states, fluctuations in protein levels and the extent to which TRAIL-induced death or survival pathways become activated. In this study, we ask how cell-to-cell variability manifests in cell types with different sensitivities to TRAIL, as well as how it changes when cells are exposed to combinations of drugs. We show that individual cells that survive treatment with TRAIL can regenerate the sensitivity and death-time distribution of the parental population, demonstrating that fractional killing is a stable property of cell populations. We also show that cell-to-cell variability in the timing and probability of apoptosis in response to treatment can be tuned using combinations of drugs that together increase apoptotic sensitivity compared to treatment with one drug alone. In the case of TRAIL, modulation of cell-to-cell variability by co-drugging appears to involve a reduction in the threshold for mitochondrial outer membrane permeabilization.

  16. Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation.

    PubMed

    Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

    2016-11-25

    Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Necroptosis-like Neuronal Cell Death Caused by Cellular Cholesterol Accumulation*

    PubMed Central

    Funakoshi, Takeshi; Aki, Toshihiko; Tajiri, Masateru; Unuma, Kana; Uemura, Koichi

    2016-01-01

    Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells. PMID:27756839

  18. Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

    PubMed

    Hallgren, Oskar; Aits, Sonja; Brest, Patrick; Gustafsson, Lotta; Mossberg, Ann-Kristin; Wullt, Björn; Svanborg, Catharina

    2008-01-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer. The mechanisms of tumor cell death remain unclear, however. Immediately after the encounter with tumor cells, HAMLET invades the cells and causes mitochondrial membrane depolarization, cytochrome c release, phosphatidyl serine exposure, and a low caspase response. A fraction of the cells undergoes morphological changes characteristic of apoptosis, but caspase inhibition does not rescue the cells and Bcl-2 overexpression or altered p53 status does not influence the sensitivity of tumor cells to HAMLET. HAMLET also creates a state of unfolded protein overload and activates 20S proteasomes, which contributes to cell death. In parallel, HAMLET translocates to tumor cell nuclei, where high-affinity interactions with histones cause chromatin disruption, loss of transcription, and nuclear condensation. The dying cells also show morphological changes compatible with macroautophagy, and recent studies indicate that macroautophagy is involved in the cell death response to HAMLET. The results suggest that HAMLET, like a hydra with many heads, may interact with several crucial cellular organelles, thereby activating several forms of cell death, in parallel. This complexity might underlie the rapid death response of tumor cells and the broad antitumor activity of HAMLET.

  19. N-Desmethyldauricine Induces Autophagic Cell Death in Apoptosis-Defective Cells via Ca2+ Mobilization.

    PubMed

    Law, Betty Y K; Mok, Simon W F; Chen, Juan; Michelangeli, Francesco; Jiang, Zhi-Hong; Han, Yu; Qu, Yuan Q; Qiu, Alena C L; Xu, Su-Wei; Xue, Wei-Wei; Yao, Xiao-Jun; Gao, Jia Y; Javed, Masood-Ul-Hassan; Coghi, Paolo; Liu, Liang; Wong, Vincent K W

    2017-01-01

    Resistance of cancer cells to chemotherapy remains a significant problem in oncology. Mechanisms regulating programmed cell death, including apoptosis, autophagy or necrosis, in the treatment of cancers have been extensively investigated over the last few decades. Autophagy is now emerging as an important pathway in regulating cell death or survival in cancer therapy. Recent studies demonstrated variety of natural small-molecules could induce autophagic cell death in apoptosis-resistant cancer cells, therefore, discovery of novel autophagic enhancers from natural products could be a promising strategy for treatment of chemotherapy-resistant cancer. By computational virtual docking analysis, biochemical assays, and advanced live-cell imaging techniques, we have identified N -desmethyldauricine (LP-4), isolated from rhizoma of Menispermum dauricum DC as a novel inducer of autophagy. LP-4 was shown to induce autophagy via the Ulk-1-PERK and Ca 2+ /Calmodulin-dependent protein kinase kinase β (CaMKKβ)-AMPK-mTOR signaling cascades, via mobilizing calcium release through inhibition of SERCA, and importantly, lead to autophagic cell death in a panel of cancer cells, apoptosis-defective and apoptosis-resistant cells. Taken together, this study provides detailed insights into the cytotoxic mechanism of a novel autophagic compound that targeting the apoptosis resistant cancer cells, and new implication on drug discovery from natural products for drug resistant cancer therapy.

  20. Cell Death Pathways and Phthalocyanine as an Efficient Agent for Photodynamic Cancer Therapy

    PubMed Central

    Mfouo-Tynga, Ivan; Abrahamse, Heidi

    2015-01-01

    The mechanisms of cell death can be predetermined (programmed) or not and categorized into apoptotic, autophagic and necrotic pathways. The process of Hayflick limits completes the execution of death-related mechanisms. Reactive oxygen species (ROS) are associated with oxidative stress and subsequent cytodamage by oxidizing and degrading cell components. ROS are also involved in immune responses, where they stabilize and activate both hypoxia-inducible factors and phagocytic effectors. ROS production and presence enhance cytodamage and photodynamic-induced cell death. Photodynamic cancer therapy (PDT) uses non-toxic chemotherapeutic agents, photosensitizer (PS), to initiate a light-dependent and ROS-related cell death. Phthalocyanines (PCs) are third generation and stable PSs with improved photochemical abilities. They are effective inducers of cell death in various neoplastic models. The metallated PCs localize in critical cellular organelles and are better inducers of cell death than other previous generation PSs as they favor mainly apoptotic cell death events. PMID:25955645

  1. Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

    PubMed

    Bol, M; Van Geyt, C; Baert, S; Decrock, E; Wang, N; De Bock, M; Gadicherla, A K; Randon, C; Evans, W H; Beele, H; Cornelissen, R; Leybaert, L

    2013-04-01

    Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability. Copyright © 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

  2. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

    PubMed

    Scarlatti, F; Maffei, R; Beau, I; Codogno, P; Ghidoni, R

    2008-08-01

    Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

  3. VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons.

    PubMed

    Tenn, Catherine C; Wang, Yushan

    2007-05-01

    Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.

  4. Zebrafish hair cell mechanics and physiology through the lens of noise-induced hair cell death

    NASA Astrophysics Data System (ADS)

    Coffin, Allison B.; Xu, Jie; Uribe, Phillip M.

    2018-05-01

    Hair cells are exquisitely sensitive to auditory stimuli, but also to damage from a variety of sources including noise trauma and ototoxic drugs. Mammals cannot regenerate cochlear hair cells, while non-mammalian vertebrates exhibit robust regenerative capacity. Our research group uses the lateral line system of larval zebrafish to explore the mechanisms underlying hair cell damage, identify protective therapies, and determine molecular drivers of innate regeneration. The lateral line system contains externally located sensory organs called neuromasts, each composed of ˜8-20 hair cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and share similar susceptibility to ototoxic damage. In the last decade, the lateral line has emerged as a powerful model system for understanding hair cell death mechanisms and for identifying novel protective compounds. Here we demonstrate that the lateral line is a tractable model for noise-induced hair cell death. We have developed a novel noise damage system capable of inducing over 50% loss of lateral line hair cells, with hair cell death occurring in a dose- and time-dependent manner. Cell death is greatest 72 hours post-exposure. However, early signs of hair cell damage, including changes in membrane integrity and reduced mechanotransduction, are apparent within hours of noise exposure. These features, early signs of damage followed by delayed hair cell death, are consistent with mammalian data, suggesting that noise acts similarly on zebrafish and mammalian hair cells. In our future work we will use our new model system to investigate noise damage events in real time, and to develop protective therapies for future translational research.

  5. Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

    PubMed

    Milutinovic, Snezana; Kashyap, Arun K; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O'Neil, Ryann; Kurtzman, Aaron L; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H; Diaz, Paul W; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R; Reed, John C

    2016-01-01

    Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. ©2015 American Association for Cancer Research.

  6. Dual agonist Surrobody™ simultaneously activates death receptors DR4 and DR5 to induce cancer cell death

    PubMed Central

    Milutinovic, Snezana; Kashyap, Arun K.; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O' Neil, Ryann; Kurtzman, Aaron L.; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H.; Diaz, Paul W.; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R.; Reed, John C.

    2015-01-01

    Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, TRAIL (Apo2L). Since most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody™ technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing pro-apoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 mono-specific antibodies. Taken together, Surrobody shows promising preclinical pro-apoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. PMID:26516157

  7. Streptococcus sanguinis induces foam cell formation and cell death of macrophages in association with production of reactive oxygen species.

    PubMed

    Okahashi, Nobuo; Okinaga, Toshinori; Sakurai, Atsuo; Terao, Yutaka; Nakata, Masanobu; Nakashima, Keisuke; Shintani, Seikou; Kawabata, Shigetada; Ooshima, Takashi; Nishihara, Tatsuji

    2011-10-01

    Streptococcus sanguinis, a normal inhabitant of the human oral cavity, is a common streptococcal species implicated in infective endocarditis. Herein, we investigated the effects of infection with S. sanguinis on foam cell formation and cell death of macrophages. Infection with S. sanguinis stimulated foam cell formation of THP-1, a human macrophage cell line. At a multiplicity of infection >100, S. sanguinis-induced cell death of the macrophages. Viable bacterial infection was required to trigger cell death because heat-inactivated S. sanguinis did not induce cell death. The production of cytokines interleukin-1β and tumor necrosis factor-α from macrophages was also stimulated during bacterial infection. Inhibition of the production of reactive oxygen species (ROS) resulted in reduced cell death, suggesting an association of ROS with cell death. Furthermore, S. sanguinis-induced cell death appeared to be independent of activation of inflammasomes, because cleavage of procaspase-1 was not evident in infected macrophages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  8. Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection

    PubMed Central

    Hayashi, Makoto T.; Cesare, Anthony J.; Rivera, Teresa; Karlseder, Jan

    2015-01-01

    Tumour formation is blocked by two barriers, replicative senescence and crisis1. Senescence is triggered by short telomeres and is bypassed by disruption of tumour suppressive pathways. After senescence bypass, cells undergo crisis, during which almost all of the cells in the population die. Cells that escape crisis harbor unstable genomes and other parameters of transformation. The mechanism of cell death during crisis remained elusive. We show that cells in crisis undergo spontaneous mitotic arrest, resulting in death during mitosis or in the following cell cycle. The phenotype was induced by loss of p53 function, and suppressed by telomerase overexpression. Telomere fusions triggered mitotic arrest in p53-compromised non-crisis cells, indicating such fusions as the underlying cause. Exacerbation of mitotic telomere deprotection by partial TRF2 knockdown2 increased the ratio of cells that died during mitotic arrest and sensitized cancer cells to mitotic poisons. We propose a crisis pathway wherein chromosome fusions induce mitotic arrest, resulting in mitotic telomere deprotection and cell death, thereby eliminating precancerous cells from the population. PMID:26108857

  9. Sulbutiamine counteracts trophic factor deprivation induced apoptotic cell death in transformed retinal ganglion cells.

    PubMed

    Kang, Kui Dong; Majid, Aman Shah Abdul; Kim, Kyung-A; Kang, Kyungsu; Ahn, Hong Ryul; Nho, Chu Won; Jung, Sang Hoon

    2010-11-01

    Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B(1) and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture.

  10. TORC1 is required to balance cell proliferation and cell death in planarians

    PubMed Central

    Tu, Kimberly C.; Pearson, Bret J.; Alvarado, Alejandro Sánchez

    2012-01-01

    Multicellular organisms are equipped with cellular mechanisms that enable them to replace differentiated cells lost to normal physiological turnover, injury, and for some such as planarians, even amputation. This process of tissue homeostasis is generally mediated by adult stem cells (ASCs), tissue-specific stem cells responsible for maintaining anatomical form and function. To do so, ASCs must modulate the balance between cell proliferation, i.e. in response to nutrients, and that of cell death, i.e. in response to starvation or injury. But how these two antagonistic processes are coordinated remains unclear. Here, we explore the role of the core components of the TOR pathway during planarian tissue homeostasis and regeneration and identified an essential function for TORC1 in these two processes. RNAi-mediated silencing of TOR in intact animals resulted in a significant increase in cell death, whereas stem cell proliferation and stem cell maintenance were unaffected. Amputated animals failed to increase stem cell proliferation after wounding and displayed defects in tissue remodeling. Together, our findings suggest two distinct roles for TORC1 in planarians. TORC1 is required to modulate the balance between cell proliferation and cell death during normal cell turnover and in response to nutrients. In addition, it is required to initiate appropriate stem cell proliferation during regeneration and for proper tissue remodeling to occur to maintain scale and proportion. PMID:22445864

  11. Ethylene Insensitivity Modulates Ozone-Induced Cell Death in Birch1

    PubMed Central

    Vahala, Jorma; Ruonala, Raili; Keinänen, Markku; Tuominen, Hannele; Kangasjärvi, Jaakko

    2003-01-01

    We have used genotypic variation in birch (Betula pendula Roth) to investigate the roles of ozone (O3)-induced ethylene (ET), jasmonic acid, and salicylic acid in the regulation of tissue tolerance to O3. Of these hormones, ET evolution correlated best with O3-induced cell death. Disruption of ET perception by transformation of birch with the dominant negative mutant allele etr1-1 of the Arabidopsis ET receptor gene ETR1 or blocking of ET perception with 1-methylcyclopropene reduced but did not completely prevent the O3-induced cell death, when inhibition of ET biosynthesis with aminooxyacetic acid completely abolished O3 lesion formation. This suggests the presence of an ET-signaling-independent but ET biosynthesis-dependent component in the ET-mediated stimulation of cell death in O3-exposed birch. Functional ET signaling was required for the O3 induction of the gene encoding β-cyanoalanine synthase, which catalyzes detoxification of the cyanide formed during ET biosynthesis. The results suggest that functional ET signaling is required to protect birch from the O3-induced cell death and that a decrease in ET sensitivity together with a simultaneous, high ET biosynthesis can potentially cause cell death through a deficient detoxification of cyanide. PMID:12746524

  12. Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia.

    PubMed

    Chen, Yongqiang; Henson, Elizabeth S; Xiao, Wenyan; Huang, Daniel; McMillan-Ward, Eileen M; Israels, Sara J; Gibson, Spencer B

    2016-06-02

    Autophagy is an intracellular lysosomal degradation pathway where its primary function is to allow cells to survive under stressful conditions. Autophagy is, however, a double-edge sword that can either promote cell survival or cell death. In cancer, hypoxic regions contribute to poor prognosis due to the ability of cancer cells to adapt to hypoxia in part through autophagy. In contrast, autophagy could contribute to hypoxia induced cell death in cancer cells. In this study, we showed that autophagy increased during hypoxia. At 4 h of hypoxia, autophagy promoted cell survival whereas, after 48 h of hypoxia, autophagy increased cell death. Furthermore, we found that the tyrosine phosphorylation of EGFR (epidermal growth factor receptor) decreased after 16 h in hypoxia. Furthermore, EGFR binding to BECN1 in hypoxia was significantly higher at 4 h compared to 72 h. Knocking down or inhibiting EGFR resulted in an increase in autophagy contributing to increased cell death under hypoxia. In contrast, when EGFR was reactivated by the addition of EGF, the level of autophagy was reduced which led to decreased cell death. Hypoxia led to autophagic degradation of the lipid raft protein CAV1 (caveolin 1) that is known to bind and activate EGFR in a ligand-independent manner during hypoxia. By knocking down CAV1, the amount of EGFR phosphorylation was decreased in hypoxia and amount of autophagy and cell death increased. This indicates that the activation of EGFR plays a critical role in the switch between cell survival and cell death induced by autophagy in hypoxia.

  13. Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells.

    PubMed

    La, Xiaoqin; Zhang, Lichao; Li, Zhuoyu; Yang, Peng; Wang, Yingying

    2017-03-28

    Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34.

  14. Myricetin Protects Against Cytokine-Induced Cell Death in RIN-m5f β Cells

    PubMed Central

    Ding, Ye; Zhang, Zhao-Feng; Dai, Xiao-Qian

    2012-01-01

    Abstract Cytokine-induced cell death is recognized as a major cause of progressive β-cell loss. Tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interferon γ (IFN-γ) in combination trigger a series of events that lead to β-cell death. In the past few decades, the use of myricetin as an anti-inflammatory and cytoprotective agent has gained much attention. The present study focused on the protective roles of myricetin against cytokine-induced cell death in insulin-secreting RIN-m5f β cells. The results showed that myricetin (especially at concentrations of 10 μM and 20 μM) increased cell viability and decreased cell apoptosis induced by the cytokine mixture of TNF-α (10 ng/mL), IL-1β (5 ng/mL), and IFN-γ (1000 IU/mL) for 3 days. Moreover, the cytokines increased the total and p65 subunit levels of nuclear factor κB, decreased inhibitor κB α levels, stimulated the accumulation of nitric oxide, increased cytochrome c release from mitochondria, and induced reactive oxygen species generation; myricetin (especially at the concentration of 20 μM) abolished all of these parameters. These results suggest that myricetin might have therapeutic value for preventing β-cell death. PMID:22846080

  15. DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA

    PubMed Central

    Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

    2018-01-01

    The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells

  16. DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

    PubMed

    Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

    2018-01-01

    The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells

  17. Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

    PubMed

    Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M; Chandel, Navdeep S; Vanden Hoek, Terry L; Schumacker, Paul T

    2010-12-15

    Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis

    PubMed Central

    Loor, Gabriel; Kondapalli, Jyothisri; Schriewer, Jacqueline M.; Chandel, Navdeep S.; Vanden Hoek, Terry L.; Schumacker, Paul T.

    2010-01-01

    Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, while other studies implicate activation of the mitochondrial permeability transition poreas the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, while it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetyl cysteine and exogenous glutathione (GSH), or by over-expression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells over-expressing Cu, Zn-SOD or MnSOD. Over-expression of antiapoptotic Bcl-XLprotected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochromec, Bax/Bak, caspase-9 and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them. PMID:20937380

  19. Metal-accelerated oxidation in plant cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Czuba, M.

    1993-05-01

    Cadmium and mercury toxicity is further enhanced by external oxidizing conditions O[sub 3] or inherent plant processes. Lepidium sativum L, Lycopersicon esculentum Mill., or Phaseolus vulgaris L, were grown inpeat-lite to maturity under continuous cadmium exposure followed by one oxidant (O[sub 3]-6 hr. 30 pphm) exposure, with or without foliar calcium pretreatments. In comparison, Daucus carota, L and other species grown in a 71-V suspension, with or without 2,4-D were exposed continuously to low levels of methylmercury during exponential growth and analyzed in aggregates of distinct populations. Proteins were extracted and analyzed. Mechanisms of toxicity and eventual cell death aremore » Ca-mediated and involve chloroplast, stomatal-water relations and changes in oxidant-anti-oxidant components in cells. Whether the metal-accelerated oxidative damage proceeds to cell death, depends on the species and its differential biotransformation system and cell association component.« less

  20. Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments

    PubMed Central

    Inoue, H; Tani, K

    2014-01-01

    Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to

  1. NADPH Oxidase Activation Contributes to Heavy Ion Irradiation–Induced Cell Death

    PubMed Central

    Wang, Yupei; Liu, Qing; Zhao, Weiping; Zhou, Xin; Miao, Guoying; Sun, Chao

    2017-01-01

    Increased oxidative stress plays an important role in heavy ion radiation–induced cell death. The mechanism involved in the generation of elevated reactive oxygen species (ROS) is not fully illustrated. Here we show that NADPH oxidase activation is closely related to heavy ion radiation–induced cell death via excessive ROS generation. Cell death and cellular ROS can be greatly reduced in irradiated cancer cells with the preincubation of diphenyleneiodium, an inhibitor of NADPH oxidase. Most of the NADPH oxidase (NOX) family proteins (NOX1, NOX2, NOX3, NOX4, and NOX5) showed increased expression after heavy ion irradiation. Meanwhile, the cytoplasmic subunit p47phox was translocated to the cell membrane and localized with NOX2 to form reactive NADPH oxidase. Our data suggest for the first time that ROS generation, as mediated by NADPH oxidase activation, could be an important contributor to heavy ion irradiation–induced cell death. PMID:28473742

  2. Extracellular acidification by lactic acid suppresses glucose deprivation-induced cell death and autophagy in B16 melanoma cells.

    PubMed

    Matsuo, Taisuke; Sadzuka, Yasuyuki

    2018-02-19

    In solid tumors, cancer cells survive and proliferate under conditions of microenvironment stress such as poor nutrients and hypoxia due to inadequate vascularization. These stress conditions in turn activate autophagy, which is important for cancer cell survival. However, autophagy has a contrary effect of inducing cell death in cancer cells cultured in vitro under conditions of glucose deprivation. In this study, we hypothesized that supplementation of lactic acid serves as a means of cell survival under glucose-deprived conditions. At neutral pH, cell death of B16 murine melanoma cells by autophagy under glucose-deprived conditions was observed. However, supplementation of lactic acid suppressed cell death and autophagy in B16 melanoma cells when cultured in glucose-deprived conditions. Sodium lactate, which does not change extracellular pH, did not inhibit cell death, while HCl-adjusted acidic pH suppressed cell death under glucose-deprived conditions. These results suggested that an acidic pH is crucial for cell survival under glucose-deprived conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  4. Is necroptosis a death pathway in aluminum-induced neuroblastoma cell demise?

    PubMed

    Zhang, Q L; Niu, Q; Ji, X L; Conti, P; Boscolo, P

    2008-01-01

    Besides being an aggravating factor secondary to major physiological alterations in degenerative diseases, aluminum has also been considered as a risk factor in the etiology. Although many in vivo and in vitro data are in favor of apoptosis and necrosis being involved in Al induced neurodegenerative processes, there is considerable evidence that very complex events may contribute to neural cell death. Necroptosis, a novel cell death pathway, was recently reported to contribute to ischemia brain injury. It is different from, but associated with, apoptosis and necrosis, the two common major pathways of cell demise. In the present study, SH-SY5Y cells were put under stress by Al, a potential degenerative cell death inducer. Nec-1, a specific inhibitor, was used to identify necroptosis. The characteristics observed in Nec-1 and Al treated SH-SY5Y cells showed that necrotic morphological changes were reduced, and a sharp decrease of necrotic rate was detected. Besides, there were Al-induced mitochondria membrane potential decreasing, reactive oxygen species remaining, and autophagosomes declining. The mechanism of Nec-1s effect on cell death may be related to caspases pathways. To our best knowledge, this is the pioneer report on necroptosis in mixed human neural cell death pathways, which might offer a novel therapeutic target for neurodegenerative diseases, and an extended window for neuroprotection.

  5. Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

    PubMed

    Garcia-Garcia, Aracely; Zavala-Flores, Laura; Rodriguez-Rocha, Humberto; Franco, Rodrigo

    2012-12-15

    Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

  6. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, Mu-Yun; Shen, Yuh-Chiang; National Research Institute of Chinese Medicine, Taipei, Taiwan

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ERmore » stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  7. Cell death and survival signalling in the cardiovascular system.

    PubMed

    Tucka, Joanna; Bennett, Martin; Littlewood, Trevor

    2012-01-01

    The loss of cells is an important factor in many diseases, including those of the cardiovascular system. Whereas apoptosis is an essential process in development and tissue homeostasis, its occurrence is often associated with various pathologies. Apoptosis of neurons that fail to make appropriate connections is essential for the selection of correct neural signalling in the developing embryo, but its appearance in adults is often associated with neurodegenerative disease. Similarly, in the cardiovascular system, remodeling of the mammalian outflow tract during the transition from a single to dual series circulation with four chambers is accompanied by a precise pattern of cell death, but apoptosis of cardiomyocytes contributes to ischemia-reperfusion injury in the heart. In many cases, it is unclear whether apoptosis represents a causative association or merely a consequence of the disease itself. There are many excellent reviews on cell death in the cardiovascular system (1-5); in this review we outline the critical signalling pathways that promote the survival of cardiovascular cells, and their relevance to both physiological cell death and disease.

  8. Trial watch: Immunogenic cell death induction by anticancer chemotherapeutics.

    PubMed

    Garg, Abhishek D; More, Sanket; Rufo, Nicole; Mece, Odeta; Sassano, Maria Livia; Agostinis, Patrizia; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2017-01-01

    The expression "immunogenic cell death" (ICD) refers to a functionally unique form of cell death that facilitates (instead of suppressing) a T cell-dependent immune response specific for dead cell-derived antigens. ICD critically relies on the activation of adaptive responses in dying cells, culminating with the exposure or secretion of immunostimulatory molecules commonly referred to as "damage-associated molecular patterns". Only a few agents can elicit bona fide ICD, including some clinically established chemotherapeutics such as doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide and oxaliplatin. In this Trial Watch, we discuss recent progress on the development of ICD-inducing chemotherapeutic regimens, focusing on studies that evaluate clinical efficacy in conjunction with immunological biomarkers.

  9. Berberine-induced autophagic cell death by elevating GRP78 levels in cancer cells

    PubMed Central

    Li, Zhuoyu; Yang, Peng; Wang, Yingying

    2017-01-01

    Berberine, an isoquinoline alkaloid extracted from Coptidis Rhizoma, has been shown to induce cancer cell autophagic death. Glucose regulated protein 78 (GRP78) is associated with stress-induced autophagy. However, the related mechanisms between berberine-induced cancer cell autophagy and GRP78 remain to be elucidated. Here, we report that berberine can induce autophagic cancer cell death by elevating levels of GRP78. These results further demonstrated that berberine enhanced GRP78 by suppression of ubiquitination / proteasomal degradation of GRP78 and activation of ATF6. Moreover, fluorescence spectrum assay revealed that berberine could bind to GRP78 and form complexes. Finally, co-IP analysis showed that the ability of GRP78 to bind to VPS34 was increased with berberine treatment. Taken together, our results suggest that berberine induces autophagic cancer cell death via enhancing GRP78 levels and the ability of GRP78 to bind to VPS34. PMID:28157699

  10. Fasting boosts sensitivity of human skin melanoma to cisplatin-induced cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Antunes, Fernanda; Corazzari, Marco; National Institute for Infectious Diseases IRCCS “Lazzaro Spallanzani”

    Melanoma is one of leading cause of tumor death worldwide. Anti-cancer strategy includes combination of different chemo-therapeutic agents as well as radiation; however these treatments have limited efficacy and induce significant toxic effects on healthy cells. One of most promising novel therapeutic approach to cancer therapy is the combination of anti-cancer drugs with calorie restriction. Here we investigated the effect Cisplatin (CDDP), one of the most potent chemotherapeutic agent used to treat tumors, in association with fasting in wild type and mutated BRAF{sup V600E} melanoma cell lines. Here we show that nutrient deprivation can consistently enhance the sensitivity of tumormore » cells to cell death induction by CDDP, also of those malignancies particularly resistant to any treatment, such as oncogenic BRAF melanomas. Mechanistic studies revealed that the combined therapy induced cell death is characterized by ROS accumulation and ATF4 in the absence of ER-stress. In addition, we show that autophagy is not involved in the enhanced sensitivity of melanoma cells to combined CDDP/EBSS-induced apoptosis. While, the exposure to 2-DG further enhanced the apoptotic rate observed in SK Mel 28 cells upon treatment with both CDDP and EBSS. - Highlights: • Calorie restriction associated to chemo-therapeutic drugs enhance cell death induction in many resistant malignancies • Cisplatin in association with starvation significantly increases cell death also in those high resistant melanoma cells bearing BRAF mutations • Combined treatment also including 2-DG results in similar cell death levels in both wild type and mutated BRAF cells.« less

  11. Cell death in the pathogenesis of systemic lupus erythematosus and lupus nephritis.

    PubMed

    Mistry, Pragnesh; Kaplan, Mariana J

    2017-12-01

    Nephritis is one of the most severe complications of systemic lupus erythematosus (SLE). One key characteristic of lupus nephritis (LN) is the deposition of immune complexes containing nucleic acids and/or proteins binding to nucleic acids and autoantibodies recognizing these molecules. A variety of cell death processes are implicated in the generation and externalization of modified nuclear autoantigens and in the development of LN. Among these processes, apoptosis, primary and secondary necrosis, NETosis, necroptosis, pyroptosis, and autophagy have been proposed to play roles in tissue damage and immune dysregulation. Cell death occurs in healthy individuals during conditions of homeostasis yet autoimmunity does not develop, at least in part, because of rapid clearance of dying cells. In SLE, accelerated cell death combined with a clearance deficiency may lead to the accumulation and externalization of nuclear autoantigens and to autoantibody production. In addition, specific types of cell death may modify autoantigens and alter their immunogenicity. These modified molecules may then become novel targets of the immune system and promote autoimmune responses in predisposed hosts. In this review, we examine various cell death pathways and discuss how enhanced cell death, impaired clearance, and post-translational modifications of proteins could contribute to the development of lupus nephritis. Published by Elsevier Inc.

  12. Cell Death and Cancer Therapy: Don't Forget to Kill the Cancer Cell!

    PubMed

    Letai, Anthony

    2015-11-15

    In our current age of targeted therapies, there is understandably considerable attention paid to the specific molecular targets of pharmaceutical intervention. For a targeted drug to work, it must bind to a target selectively and impair its function. Monitoring biomarkers of the impaired target function can provide vital in vivo pharmacodynamic information. Moreover, genetic changes to the target are often the source of resistance to targeted agents. However, for the treatment of cancer, it is necessary that the therapy not only provide efficient binding and inhibition of the target, but also that this intervention reliably kills the cancer cell. In this CCR Focus section, four articles make the connection between therapies that target T-cell activation, autophagy, IAP proteins, and BCL-2 and the commitment of cancer cells to cell death. Before addressing those exciting classes of targeted therapies, however, an overview is provided to discuss cell death induced by what is arguably still the most successful set of drugs in the history of medical oncology, conventional chemotherapy. See all articles in this CCR Focus section, "Cell Death and Cancer Therapy." ©2015 American Association for Cancer Research.

  13. Tributyltin induces Yca1p-dependent cell death of yeast Saccharomyces cerevisiae.

    PubMed

    Chahomchuen, Thippayarat; Akiyama, Koichi; Sekito, Takayuki; Sugimoto, Naoko; Okabe, Masaaki; Nishimoto, Sogo; Sugahara, Takuya; Kakinuma, Yoshimi

    2009-10-01

    Tributyltin chloride (TBT), an environmental pollutant, is toxic to a variety of eukaryotic and prokaryotic organisms. Although it has been reported that TBT induces apoptotic cell death in mammalian, the action of TBT on eukaryotic microorganisms has not yet been fully investigated. In this study we examined the mechanism involved in cell death caused by TBT exposure in Saccharomyces cerevisiae. The median lethal concentration of TBT was 10 microM for the parent strain BY4741 and 3 microM for the pdr5Delta mutant defective in a major multidrug transporter, respectively. Fluorescence microscopic observations revealed nuclear condensation and chromatin fragmentation in cells treated with TBT indicating that cells underwent an apoptosis-like cell dearth. TBT-induced cell death was suppressed by deletion of the yca1 gene encoding a homologue of the mammalian caspase. In parallel, reactive oxygen species (ROS) were produced by TBT. These results suggest that TBT induces apoptosis-like cell death in yeast via an Yca1p-dependent pathway possibly downstream of the ROS production. This is the first report on TBT-induced apoptotic cell death in yeast.

  14. Combined effects of starvation and butyrate on autophagy-dependent gingival epithelial cell death.

    PubMed

    Evans, M; Murofushi, T; Tsuda, H; Mikami, Y; Zhao, N; Ochiai, K; Kurita-Ochiai, T; Yamamoto, M; Otsuka, K; Suzuki, N

    2017-06-01

    Bacteria in the dental biofilm surrounding marginal gingival grooves cause periodontal diseases. Numerous bacteria within the biofilm consume nutrients from the gingival crevicular fluid. Furthermore, some gram-negative bacteria in mature dental biofilms produce butyrate. Thus, gingival epithelial cells in close proximity to mature dental biofilms are at risk of both starvation and exposure to butyrate. In the present study, we determined the combined effects of starvation and butyrate exposure on gingival epithelial cell death and the underlying mechanisms. The Ca9-22 cell line was used as an in vitro counterpart of gingival epithelial cells. Cell death was measured as the amount of total DNA in the dead cells using SYTOX Green dye, which penetrates through membranes of dead cells and emits fluorescence when it intercalates into double-stranded DNA. AMP-activated protein kinase (AMPK) activity, the amount of autophagy, and acetylation of histone H3 were determined using western blot. Gene expression levels of microtubule-associated protein 1 light chain 3b (lc3b) were determined using quantitative reverse transcription-polymerase chain reaction. Butyrate-induced cell death occurred in a dose-dependent manner whether cells were starved or fed. However, the induction of cell death was two to four times higher when cells were placed under starvation conditions compared to when they were fed. Moreover, both starvation and butyrate exposure induced AMPK activity and autophagy. While AMPK inactivation resulted in decreased autophagy and butyrate-induced cell death under conditions of starvation, AMPK activation resulted in butyrate-induced cell death when cells were fed. Combined with the results of our previous report, which demonstrated butyrate-induced autophagy-dependent cell death, the results of this study suggest that the combination of starvation and butyrate exposure activates AMPK inducing autophagy and subsequent cell death. Notably, this combination markedly

  15. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumormore » incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.« less

  16. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Sung Hun; Yoo, Chong Il; Medical Research Institute, College of Medicine, Pusan National University, Pusan, 602-739

    2006-09-01

    The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatmentmore » caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.« less

  17. Cell death monitoring using quantitative optical coherence tomography methods

    NASA Astrophysics Data System (ADS)

    Farhat, Golnaz; Yang, Victor X. D.; Kolios, Michael C.; Czarnota, Gregory J.

    2011-03-01

    Cell death is characterized by a series of predictable morphological changes, which modify the light scattering properties of cells. We present a multi-parametric approach to detecting changes in subcellular morphology related to cell death using optical coherence tomography (OCT). Optical coherence tomography data were acquired from acute myeloid leukemia (AML) cells undergoing apoptosis over a period of 48 hours. Integrated backscatter (IB) and spectral slope (SS) were computed from OCT backscatter spectra and statistical parameters were extracted from a generalized gamma (GG) distribution fit to OCT signal intensity histograms. The IB increased by 2-fold over 48 hours with significant increases observed as early as 4 hours. The SS increased in steepness by 2.5-fold with significant changes at 12 hours, while the GG parameters were sensitive to apoptotic changes at 24 to 48 hours. Histology slides indicated nuclear condensation and fragmentation at 24 hours, suggesting the late scattering changes could be related to nuclear structure. A second series of measurements from AML cells treated with cisplatin, colchicine or ionizing radiation suggested that the GG parameters could potentially differentiate between modes of cell death. Distinct cellular morphology was observed in histology slides obtained from cells treated under each condition.

  18. Differential Induction of Immunogenic Cell Death and Interferon Expression in Cancer Cells by Structured ssRNAs.

    PubMed

    Lee, Jaewoo; Lee, Youngju; Xu, Li; White, Rebekah; Sullenger, Bruce A

    2017-06-07

    Activation of the RNA-sensing pattern recognition receptor (PRR) in cancer cells leads to cell death and cytokine expression. This cancer cell death releases tumor antigens and damage-associated molecular patterns (DAMPs) that induce anti-tumor immunity. However, these cytokines and DAMPs also cause adverse inflammatory and thrombotic complications that can limit the overall therapeutic benefits of PRR-targeting anti-cancer therapies. To overcome this problem, we generated and evaluated two novel and distinct ssRNA molecules (immunogenic cell-killing RNA [ICR]2 and ICR4). ICR2 and ICR4 differentially stimulated cell death and PRR signaling pathways and induced different patterns of cytokine expression in cancer and innate immune cells. Interestingly, DAMPs released from ICR2- and ICR4-treated cancer cells had distinct patterns of stimulation of innate immune receptors and coagulation. Finally, ICR2 and ICR4 inhibited in vivo tumor growth as effectively as poly(I:C). ICR2 and ICR4 are potential therapeutic agents that differentially induce cell death, immune stimulation, and coagulation when introduced into tumors. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  19. Low-frequency quantitative ultrasound imaging of cell death in vivo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sadeghi-Naini, Ali; Falou, Omar; Czarnota, Gregory J.

    Purpose: Currently, no clinical imaging modality is used routinely to assess tumor response to cancer therapies within hours to days of the delivery of treatment. Here, the authors demonstrate the efficacy of ultrasound at a clinically relevant frequency to quantitatively detect changes in tumors in response to cancer therapies using preclinical mouse models.Methods: Conventional low-frequency and corresponding high-frequency ultrasound (ranging from 4 to 28 MHz) were used along with quantitative spectroscopic and signal envelope statistical analyses on data obtained from xenograft tumors treated with chemotherapy, x-ray radiation, as well as a novel vascular targeting microbubble therapy.Results: Ultrasound-based spectroscopic biomarkers indicatedmore » significant changes in cell-death associated parameters in responsive tumors. Specifically changes in the midband fit, spectral slope, and 0-MHz intercept biomarkers were investigated for different types of treatment and demonstrated cell-death related changes. The midband fit and 0-MHz intercept biomarker derived from low-frequency data demonstrated increases ranging approximately from 0 to 6 dBr and 0 to 8 dBr, respectively, depending on treatments administrated. These data paralleled results observed for high-frequency ultrasound data. Statistical analysis of ultrasound signal envelope was performed as an alternative method to obtain histogram-based biomarkers and provided confirmatory results. Histological analysis of tumor specimens indicated up to 61% cell death present in the tumors depending on treatments administered, consistent with quantitative ultrasound findings indicating cell death. Ultrasound-based spectroscopic biomarkers demonstrated a good correlation with histological morphological findings indicative of cell death (r{sup 2}= 0.71, 0.82; p < 0.001).Conclusions: In summary, the results provide preclinical evidence, for the first time, that quantitative ultrasound used at a clinically relevant

  20. Calpain Determines the Propensity of Adult Hippocampal Neural Stem Cells to Autophagic Cell Death Following Insulin Withdrawal.

    PubMed

    Chung, Kyung Min; Park, Hyunhee; Jung, Seonghee; Ha, Shinwon; Yoo, Seung-Jun; Woo, Hanwoong; Lee, Hyang Ju; Kim, Seong Who; Kim, Eun-Kyoung; Moon, Cheil; Yu, Seong-Woon

    2015-10-01

    Programmed cell death (PCD) has significant effects on the function of neural stem cells (NSCs) during brain development and degeneration. We have previously reported that adult rat hippocampal neural stem (HCN) cells underwent autophagic cell death (ACD) rather than apoptosis following insulin withdrawal despite their intact apoptotic capabilities. Here, we report a switch in the mode of cell death in HCN cells with calpain as a critical determinant. In HCN cells, calpain 1 expression was barely detectable while calpain 2 was predominant. Inhibition of calpain in insulin-deprived HCN cells further augmented ACD. In contrast, expression of calpain 1 switched ACD to apoptosis. The proteasome inhibitor lactacystin blocked calpain 2 degradation and elevated the intracellular Ca(2+) concentration. In combination, these effects potentiated calpain activity and converted the mode of cell death to apoptosis. Our results indicate that low calpain activity, due to absence of calpain 1 and degradation of calpain 2, results in a preference for ACD over apoptosis in insulin-deprived HCN cells. On the other hand, conditions leading to high calpain activity completely switch the mode of cell death to apoptosis. This is the first report on the PCD mode switching mechanism in NSCs. The dynamic change in calpain activity through the proteasome-mediated modulation of the calpain and intracellular Ca(2+) levels may be the critical contributor to the demise of NSCs. Our findings provide a novel insight into the complex mechanisms interconnecting autophagy and apoptosis and their roles in the regulation of NSC death. © 2015 AlphaMed Press.

  1. Esterification of 24S-OHC induces formation of atypical lipid droplet-like structures, leading to neuronal cell death[S

    PubMed Central

    Takabe, Wakako; Urano, Yasuomi; Vo, Diep-Khanh Ho; Shibuya, Kimiyuki; Tanno, Masaki; Kitagishi, Hiroaki; Fujimoto, Toyoshi; Noguchi, Noriko

    2016-01-01

    The 24(S)-hydroxycholesterol (24S-OHC), which plays an important role in maintaining brain cholesterol homeostasis, has been shown to possess neurotoxicity. We have previously reported that 24S-OHC esterification by ACAT1 and the resulting lipid droplet (LD) formation are responsible for 24S-OHC-induced cell death. In the present study, we investigate the functional roles of 24S-OHC esters and LD formation in 24S-OHC-induced cell death, and we identify four long-chain unsaturated fatty acids (oleic acid, linoleic acid, arachidonic acid, and DHA) with which 24S-OHC is esterified in human neuroblastoma SH-SY5Y cells treated with 24S-OHC. Here, we find that cotreatment of cells with 24S-OHC and each of these four unsaturated fatty acids increases prevalence of the corresponding 24S-OHC ester and exacerbates induction of cell death as compared with cell death induced by treatment with 24S-OHC alone. Using electron microscopy, we find in the present study that 24S-OHC induces formation of LD-like structures coupled with enlarged endoplasmic reticulum (ER) lumina, and that these effects are suppressed by treatment with ACAT inhibitor. Collectively, these results illustrate that ACAT1-catalyzed esterification of 24S-OHC with long-chain unsaturated fatty acid followed by formation of atypical LD-like structures at the ER membrane is a critical requirement for 24S-OHC-induced cell death. PMID:27647838

  2. RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death

    PubMed Central

    Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

    2016-01-01

    Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to

  3. RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

    PubMed

    Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

    2016-09-27

    Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to

  4. Zinc deficiency mediates alcohol-induced apoptotic cell death in the liver of rats through activating ER and mitochondrial cell death pathways

    PubMed Central

    Sun, Qian; Zhong, Wei; Zhang, Wenliang; Li, Qiong; Sun, Xiuhua; Tan, Xiaobing; Sun, Xinguo; Dong, Daoyin

    2015-01-01

    Hepatic zinc deficiency has been well documented in alcoholic patients, but the mechanisms by which zinc deficiency mediates cell death have not been well defined. The objectives of this study were to determine whether alcohol perturbs subcellular zinc homeostasis and how organelle zinc depletion may link with cell death pathways. Wistar rats were pair-fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure significantly reduced zinc level in isolated hepatic endoplasmic reticulum (ER) and mitochondria. Among the detected zinc transporters, ER Zrt/Irt-like protein (ZIP)13 and mitochondrial ZIP8, which transport zinc from ER and mitochondria to cytosol, were significantly increased. Mitochondrial zinc transporter (ZnT) 4, which transports zinc from cytosol to mitochondria, was also increased. ER phosphorylated eukaryotic initiation factor 2α, activating transcription factor 4, and C/EBP homologous protein were significantly upregulated, and mitochondrial cytochrome c release and Bax insertion were detected in association with caspase-3 activation and apoptotic cell death. To define the role of zinc deficiency in ER and mitochondrial stress, H4IIEC3 cells were treated with 3 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine for 6 h with or without supplementation with zinc or N-acetylcysteine (NAC). The results demonstrated that zinc deprivation induced caspase-3 activation and apoptosis in association with ER and mitochondria dysfunction, which were inhibited by zinc as low as 10 μM but not by 2 mM NAC. These results suggest that chronic ethanol exposure induced in ER and mitochondrial zinc deficiency might activate intrinsic cell death signaling pathway, which could not be effectively rescued by antioxidant treatment. PMID:25767260

  5. Epidermal growth factor inhibits rat pancreatic cell proliferation, causes acinar cell hypertrophy, and prevents caerulein-induced desensitization of amylase release.

    PubMed

    Morisset, J; Larose, L; Korc, M

    1989-06-01

    The in vivo effects of epidermal growth factor (EGF) on pancreatic growth and digestive enzyme concentrations were compared with the actions of the pancreatic secretagogue caerulein in the adult rat. EGF (10 micrograms/kg BW) did not alter pancreatic weight or protein content. However, this concentration of EGF inhibited [3H]thymidine incorporation into DNA by 44%, decreased DNA content by 20%, and increased the concentrations of amylase, chymotrypsinogen, and protein by 106%, 232%, and 42%, respectively. Pancreatic acini prepared from EGF-treated rats exhibited a characteristic secretory response to caerulein that was superimposable to that obtained in acini from saline-treated rats. In both groups of acini half-maximal and maximal stimulation of amylase release occurred at approximately 5 pM and 50 pM caerulein, respectively. In contrast to EGF, caerulein (1 microgram/kg BW) increased pancreatic weight by 29% and protein content by 59%, and enhanced [3H]thymidine incorporation into DNA by 70%. Although caerulein increased the concentrations of pancreatic amylase and chymotrypsinogen by 38% and 297%, respectively, pancreatic acini prepared from caerulein-treated rats were less sensitive to the actions of caerulein in vitro when compared with acini from control rats. Indeed, the EC50 was shift from 4.8 pM to 9.8 pM after 4 days of treatment. EGF potentiated the actions of caerulein on pancreatic weight, protein content, and chymotrypsinogen concentration, and prevented the caerulein-induced alteration in the secretory responsiveness of the acinar cell. Conversely, caerulein reversed the inhibitory effect of EGF on thymidine incorporation. These findings suggest that EGF may modulate the trophic effects of certain gastrointestinal hormones, and may participate in the regulation of pancreatic exocrine function in vivo.

  6. Love is a battlefield: programmed cell death during fertilization.

    PubMed

    Heydlauff, Juliane; Groß-Hardt, Rita

    2014-03-01

    Plant development and growth is sustained by the constant generation of tremendous amounts of cells, which become integrated into various types of tissues and organs. What is all too often overlooked is that this thriving life also requires the targeted degeneration of selected cells, which undergo cell death according to genetically encoded programmes or environmental stimuli. The side-by-side existence of generation and demise is particularly evident in the haploid phase of the flowering plants cycle. Here, the lifespan of terminally differentiated accessory cells contrasts with that of germ cells, which by definition live on to form the next generation. In fact, with research in recent years it is becoming increasingly clear that the gametophytes of flowering plants constitute an attractive and powerful system for investigating the molecular mechanisms underlying selective cell death.

  7. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

    PubMed Central

    Escamez, Sacha; André, Domenique; Zhang, Bo; Bollhöner, Benjamin; Pesquet, Edouard; Tuominen, Hannele

    2016-01-01

    ABSTRACT We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells. PMID:26740571

  8. Live to die another way: modes of programmed cell death and the signals emanating from dying cells

    PubMed Central

    Fuchs, Yaron; Steller, Hermann

    2015-01-01

    Preface All life ends in death, but perhaps one of life’s grander ironies is that it also depends on death. Cell-intrinsic suicide pathways, termed programmed cell death (PCD), are crucial for animal development, tissue homeostasis and pathogenesis. Originally, PCD was virtually synonymous with apoptosis, but recently, alternative PCD mechanisms have been reported. Here, we provide an overview of several distinct PCD mechanisms, namely apoptosis, autophagy and necroptosis. In addition, we discuss the complex signals emanating from dying cells, which can either fuel regeneration or instruct additional killing. Further advances in understanding the physiological role of multiple cell death mechanisms and associated signals will be important to selectively manipulate PCD for therapeutic purposes. PMID:25991373

  9. Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

    PubMed Central

    2013-01-01

    Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation. PMID:23701745

  10. Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells.

    PubMed

    Niero, Evandro Luís de Oliveira; Machado-Santelli, Gláucia Maria

    2013-05-23

    Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.

  11. Neuroprotection by GH against excitotoxic-induced cell death in retinal ganglion cells.

    PubMed

    Martínez-Moreno, Carlos G; Ávila-Mendoza, José; Wu, Yilun; Arellanes-Licea, Elvira Del Carmen; Louie, Marcela; Luna, Maricela; Arámburo, Carlos; Harvey, Steve

    2016-08-01

    Retinal growth hormone (GH) has been shown to promote cell survival in retinal ganglion cells (RGCs) during developmental waves of apoptosis during chicken embryonic development. The possibility that it might also against excitotoxicity-induced cell death was therefore examined in the present study, which utilized quail-derived QNR/D cells as an in vitro RGC model. QNR/D cell death was induced by glutamate in the presence of BSO (buthionine sulfoxamide) (an enhancer of oxidative stress), but this was significantly reduced (P<0.01) in the presence of exogenous recombinant chicken GH (rcGH). Similarly, QNR/D cells that had been prior transfected with a GH plasmid to overexpress secreted and non-secreted GH. This treatment reduced the number of TUNEL-labeled cells and blocked their release of lactate dehydrogenase (LDH). In a further experiment with dissected neuroretinal explants from ED (embryonic day) 10 embryos, rcGH treatment of the explants also reduced (P<0.01) the number of glutamate-BSO-induced apoptotic cells and blocked the explant release of LDH. This neuroprotective action was likely mediated by increased STAT5 phosphorylation and increased bcl-2 production, as induced by exogenous rcGH treatment and the media from GH-overexpressing QNR/D cells. As rcGH treatment and GH-overexpression cells also increased the content of IGF-1 and IGF-1 mRNA this neuroprotective action of GH is likely to be mediated, at least partially, through an IGF-1 mechanism. This possibility is supported by the fact that the siRNA knockdown of GH or IGF-1 significantly reduced QNR/D cell viability, as did the immunoneutralization of IGF-1. GH is therefore neuroprotective against excitotoxicity-induced RGC cell death by anti-apoptotic actions involving IGF-1 stimulation. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Delineating the cell death mechanisms associated with skin electroporation.

    PubMed

    Schultheis, Katherine; Smith, Trevor R F; Kiosses, William B; Kraynyak, Kimberly A; Wong, Amelia; Oh, Janet; Broderick, Kate Elizabeth

    2018-06-28

    The immune responses elicited following delivery of DNA vaccines to the skin has previously been shown to be significantly enhanced by the addition of electroporation (EP) to the treatment protocol. Principally, EP increases the transfection of pDNA into the resident skin cells. In addition to increasing the levels of in vivo transfection, the physical insult induced by EP is associated with activation of innate pathways which are believed to mediate an adjuvant effect, further enhancing DNA vaccine responses. Here, we have investigated the possible mechanisms associated with this adjuvant effect, primarily focusing on the cell death pathways associated with the skin EP procedure independent of pDNA delivery. Using the minimally invasive CELLECTRA®-3P intradermal electroporation device that penetrates the epidermal and dermal layers of the skin, we have investigated apoptotic and necrotic cell death in relation to the vicinity of the electrode needles and electric field generated. Employing the well-established TUNEL assay, we detected apoptosis beginning as early as one hour after EP and peaking at the 4 hour time point. The majority of the apoptotic events were detected in the epidermal region directly adjacent to the electrode needle. Using a novel propidium iodide in vivo necrotic cell death assay, we detected necrotic events concentrated in the epidermal region adjacent to the electrode. Furthermore, we detected up-regulation of calreticulin expression on skin cells after EP, thus labeling these cells for uptake by dendritic cells and macrophages. These results allow us to delineate the cell death mechanisms occurring in the skin following intradermal EP independently of pDNA delivery. We believe these events contribute to the adjuvant effect observed following electroporation at the skin treatment site.

  13. Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice[W][OA

    PubMed Central

    Steffens, Bianka; Sauter, Margret

    2009-01-01

    In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H2O2). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-γ-S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Gα subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H2O2 was nearly completely abolished, indicating that signaling through Gα is essential. Ethylene and H2O2 were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Gα. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Gα activity is regulated posttranscriptionally. PMID:19656904

  14. Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

    PubMed Central

    Lang, Fangfang; Qin, Zhaoyang; Li, Fang; Zhang, Huilin; Fang, Zhenghui; Hao, Enkui

    2015-01-01

    Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells. PMID:26067645

  15. Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

    NASA Astrophysics Data System (ADS)

    Vasudevan, Srivathsan; Chen, George Chung Kit; Andika, Marta; Agarwal, Shuchi; Chen, Peng; Olivo, Malini

    2010-09-01

    Red blood cells (RBCs) have been found to undergo ``programmed cell death,'' or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

  16. Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level.

    PubMed

    Yoo, Jae-Myung; Lee, Youn-Sun; Choi, Heon-Kyo; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Oh, Seikwan; Yoo, Hwan-Soo

    2005-03-01

    Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

  17. Can dead bacterial cells be defined and are genes expressed after cell death?

    PubMed

    Trevors, J T

    2012-07-01

    There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

    PubMed Central

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Wong, Emily B; Suleman, Moosa; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-01

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

  19. Cyclic AMP regulates formation of mammary epithelial acini in vitro

    PubMed Central

    Nedvetsky, Pavel I.; Kwon, Sang-Ho; Debnath, Jayanta; Mostov, Keith E.

    2012-01-01

    Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus. PMID:22675028

  20. Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

    PubMed

    Lombardo, Tomás; Folgar, Martín Gil; Salaverry, Luciana; Rey-Roldán, Estela; Alvarez, Elida M; Carreras, María C; Kornblihtt, Laura; Blanco, Guillermo A

    2018-05-01

    Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 μM low-dose range (LDR) of CCCP. Within the 20-50 μM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage. © 2017

  1. ZBP1/DAI ubiquitination and sensing of influenza vRNPs activate programmed cell death

    PubMed Central

    Kuriakose, Teneema; Malireddi, R.K. Subbarao; Mishra, Ashutosh

    2017-01-01

    Innate sensing of influenza virus infection induces activation of programmed cell death pathways. We have recently identified Z-DNA–binding protein 1 (ZBP1) as an innate sensor of influenza A virus (IAV). ZBP1-mediated IAV sensing is critical for triggering programmed cell death in the infected lungs. Surprisingly, little is known about the mechanisms regulating ZBP1 activation to induce programmed cell death. Here, we report that the sensing of IAV RNA by retinoic acid inducible gene I (RIG-I) initiates ZBP1-mediated cell death via the RIG-I–MAVS–IFN-β signaling axis. IAV infection induces ubiquitination of ZBP1, suggesting potential regulation of ZBP1 function through posttranslational modifications. We further demonstrate that ZBP1 senses viral ribonucleoprotein (vRNP) complexes of IAV to trigger cell death. These findings collectively indicate that ZBP1 activation requires RIG-I signaling, ubiquitination, and vRNP sensing to trigger activation of programmed cell death pathways during IAV infection. The mechanism of ZBP1 activation described here may have broader implications in the context of virus-induced cell death. PMID:28634194

  2. Methylglyoxal-bis(guanylhydrazone), a polyamine analogue, sensitized γ-radiation-induced cell death in HL-60 leukemia cells Sensitizing effect of MGBG on γ-radiation-induced cell death.

    PubMed

    Kim, Jin Sik; Lee, Jin; Chung, Hai Won; Choi, Han; Paik, Sang Gi; Kim, In Gyu

    2006-09-01

    Methylglyoxal-bis(guanylhydrazone) (MGBG), a polyamine analogue, has been known to inhibit the biosynthesis of polyamines, which are important in cell proliferation. We showed that MGBG treatment significantly affected γ-radiation-induced cell cycle transition (G(1)/G(0)→S→G(2)/M) and thus γ-radiation-induced cell death. As determined by micronuclei and comet assay, we showed that it sensitized the cytotoxic effect induced by γ-radiation. One of the reasons is that polyamine depletion by MGBG treatment did not effectively protect against the chemical (OH) or physical damage to DNA caused by γ-radiation. Through in vitro experiment, we confirmed that DNA strand breaks induced by γ-radiation was prevented more effectively in the presence of polyamines (spermine and spermidine) than in the absence of polyamines. MGBG also blocks the cell cycle transition caused by γ-radiation (G(2) arrest), which helps protect cells by allowing time for DNA repair before entry into mitosis or apoptosis, via the down regulation of cyclin D1, which mediates the transition from G(1) to S phase of cell cycle, and ataxia telangiectasia mutated, which is involved in the DNA sensing, repair and cell cycle check point. Therefore, the abrogation of G(2) arrest sensitizes cells to the effect of γ-radiation. As a result, γ-radiation-induced cell death increased by about 2.5-3.0-fold in cells treated with MGBG. However, exogenous spermidine supplement partially relieved this γ-radiation-induced cytotoxicity and cell death. These findings suggest a potentially therapeutic strategy for increasing the cytotoxic efficacy of γ-radiation.

  3. Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells.

    PubMed

    Da Silva, Daniel; Lachaud, Christophe; Cotelle, Valérie; Brière, Christian; Grat, Sabine; Mazars, Christian; Thuleau, Patrice

    2011-05-01

    Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction. 

  4. Using stochastic cell division and death to probe minimal units of cellular replication

    NASA Astrophysics Data System (ADS)

    Chib, Savita; Das, Suman; Venkatesan, Soumya; Sai Narain Seshasayee, Aswin; Thattai, Mukund

    2018-03-01

    The invariant cell initiation mass measured in bacterial growth experiments has been interpreted as a minimal unit of cellular replication. Here we argue that the existence of such minimal units induces a coupling between the rates of stochastic cell division and death. To probe this coupling we tracked live and dead cells in Escherichia coli populations treated with a ribosome-targeting antibiotic. We find that the growth exponent from macroscopic cell growth or decay measurements can be represented as the difference of microscopic first-order cell division and death rates. The boundary between cell growth and decay, at which the number of live cells remains constant over time, occurs at the minimal inhibitory concentration (MIC) of the antibiotic. This state appears macroscopically static but is microscopically dynamic: division and death rates exactly cancel at MIC but each is remarkably high, reaching 60% of the antibiotic-free division rate. A stochastic model of cells as collections of minimal replicating units we term ‘widgets’ reproduces both steady-state and transient features of our experiments. Sub-cellular fluctuations of widget numbers stochastically drive each new daughter cell to one of two alternate fates, division or death. First-order division or death rates emerge as eigenvalues of a stationary Markov process, and can be expressed in terms of the widget’s molecular properties. High division and death rates at MIC arise due to low mean and high relative fluctuations of widget number. Isolating cells at the threshold of irreversible death might allow molecular characterization of this minimal replication unit.

  5. Histological and Finite Element Analysis of Cell Death due to Irreversible Electroporation

    PubMed Central

    Long, G.; Bakos, G.; Shires, P. K.; Gritter, L.; Crissman, J. W.; Harris, J. L.; Clymer, J. W.

    2014-01-01

    Irreversible electroporation (IRE) has been shown to be an effective method of killing cells locally. In contrast to radiofrequency ablation, the mechanism by which cells are thought to die via IRE is the creation of pores in cell membranes, without substantial increase in tissue temperature. To determine the degree to which cell death is non-thermal, we evaluated IRE in porcine hepatocytes in vivo. Using pulse widths of 10μs, bursts of 3 kV square-wave pulses were applied through a custom probe to the liver of an anesthetized pig. Affected tissue was evaluated histologically via stainings of hematoxylin & eosin (H&E), nitroblue tetrazolium (NBT) to monitor cell respiration and TUNEL to gauge apoptosis. Temperature was measured during the application of electroporation, and heat transfer was modeled via finite element analysis. Cell death was calculated via Arrhenius kinetics. Four distinct zones were observed within the ring return electrode; heat-fixed tissue, coagulation, necrotic, and viable. The Arrhenius damage integral estimated complete cell death only in the first zone, where the temperature exceeded 70°C, and partial or no cell death in the other zones, where maximum temperature was approximately 45°C. Except for a limited area near the electrode tip, cell death in IRE is predominantly due to a non-thermal mechanism. PMID:24000980

  6. Metabolic control of T-cell activation and death in SLE

    PubMed Central

    Fernandez, David; Perl, Andras

    2009-01-01

    Systemic lupus erythematosus (SLE) is characterized by abnormal T-cell activation and death, processes which are crucially dependent on the controlled production of reactive oxygen intermediates (ROI) and of ATP in mitochondria. The mitochondrial transmembrane potential (Δψm) has conclusively emerged as a critical checkpoint of ATP synthesis and cell death. Lupus T cells exhibit persistent elevation of Δψm or mitochondrial hyperpolarization (MHP) as well as depletion of ATP and glutathione which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. NO-induced mitochondrial biogenesis in normal T cells accelerates the rapid phase and reduces the plateau of Ca2+ influx upon CD3/CD28 co-stimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Treatment of SLE patients with rapamycin improves disease activity, normalizes CD3/CD28-induced Ca2+ fluxing but fails to affect MHP, suggesting that altered Ca2+ fluxing is downstream or independent of mitochondrial dysfunction. Understanding the molecular basis and consequences of MHP is essential for controlling T-cell activation and death signaling in SLE. Lupus T cells exhibit mitochondrial dysfunctionMitochondrial hyperpolarization (MHP) and ATP depletion predispose lupus T cells to death by necrosis which is pro-inflammatoryMHP is caused by depletion of glutathione and exposure to nitric oxide (NO)NO-induced mitochondrial biogenesis regenerates the Ca2+ signaling profile of lupus T cellsRapamycin treatment normalizes Ca2+ fluxing but not MHP, suggesting that the mammalian target of rapamycin, acts as a sensor and effector of MHP in SLE PMID:18722557

  7. Cell death in response to antimetabolites directed at thymidylate synthase.

    PubMed

    Barbour, Karen W; Berger, Franklin G

    2008-02-01

    Thymidylate synthase (TS) is an indispensable enzyme in the de novo biosynthesis of TMP during DNA replication and cell growth, and has, therefore, been an important target for several classes of antimetabolites used in cancer chemotherapy. While most investigations of the action of TS-directed agents have focused on apoptosis as the primary means of cell death, little is known regarding the role, if any, of non-apoptotic mechanisms. In the present study, we have examined the mode of cell death induced by several TS inhibitors. Apoptosis and necrosis in response to TS inhibitors was assessed. The roles of caspases and the transcriptional regulator nuclear factor kappa B (NFkappaB) in drug-induced cell death were analyzed. Finally, drug-mediated changes in expression of several proteins involved in regulation of apoptosis were analyzed. Though human colon tumor cells exposed to TS inhibitors undergo classical apoptosis, it is not the predominant mechanism of response; rather, a necrosis-like mechanism prevails. The apoptotic response to TS inhibitors is caspase-dependent, and is promoted by NFkappaB. In contrast, the necrosis-like response is independent of both caspases and NFkappaB. Exposure to TS inhibitors induces PARP cleavage, but does not alter expression of the pro or activated forms of caspases-3 or caspases-8, Fas, or FasL. Treatment with the death-inducing cytokine TNFalpha, like TS inhibitors, results in a limited extent of apoptosis that is both caspase- and NFkappaB-dependent; however, unlike TS inhibitors, the cytokine does not induce necrosis. Classical apoptosis occurs to a limited extent in human colon tumor cells exposed to TS inhibitors, with caspase-independent necrosis being the prinicipal mechanism of cell death. We suggest that the role of necrosis and necrosis-like mechanisms should be considered in future studies of the action of TS-directed antimetabolites, as well as other chemotherapeutic agents.

  8. Contact-independent cell death of human microglial cells due to pathogenic Naegleria fowleri trophozoites.

    PubMed

    Kim, Jong-Hyun; Kim, Daesik; Shin, Ho-Joon

    2008-12-01

    Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increase of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

  9. Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

    PubMed Central

    Kim, Jong-Hyun

    2008-01-01

    Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death. PMID:19127326

  10. Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons

    PubMed Central

    Wehner, Amanda B.; Abdesselem, Houari; Dickendesher, Travis L.; Imai, Fumiyasu; Yoshida, Yutaka; Giger, Roman J.; Pierchala, Brian A.

    2016-01-01

    ABSTRACT During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as ‘competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro. The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death. PMID:27143756

  11. Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death

    PubMed Central

    Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J.; Cheng, Qiang (Shawn); D’Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M.; Gonzalez Guzman, Michael J.; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K.; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G.; Ryan, Elizabeth P.; Colacci, Anna Maria; Hamid, Roslida A.; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K.; Woodrick, Jordan; Scovassi, A.Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H.; Lowe, Leroy; Park, Hyun Ho

    2015-01-01

    Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. PMID:26106145

  12. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    PubMed

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Ozone-Induced Cell Death in Tobacco Cultivar Bel W3 Plants. The Role of Programmed Cell Death in Lesion Formation

    PubMed Central

    Pasqualini, Stefania; Piccioni, Claudia; Reale, Lara; Ederli, Luisa; Della Torre, Guido; Ferranti, Francesco

    2003-01-01

    Treatment of the ozone-sensitive tobacco (Nicotiana tabacum L. cv Bel W3) with an ozone pulse (150 nL L–1 for 5 h) induced visible injury, which manifested 48 to 72 h from onset of ozone fumigation. The “classical” ozone symptoms in tobacco cv Bel W3 plants occur as sharply defined, dot-like lesions on the adaxial side of the leaf and result from the death of groups of palisade cells. We investigated whether this reaction had the features of a hypersensitive response like that which results from the incompatible plant-pathogen interaction. We detected an oxidative burst, the result of H2O2 accumulation at 12 h from the starting of fumigation. Ozone treatment induced deposition of autofluorescent compounds and callose 24 h from the start of treatment. Total phenolic content was also strongly stimulated at the 10th and 72nd h from starting fumigation, concomitant with an enhancement in phenylalanine ammonia-lyase a and phenylalanine ammonia-lyase b expression, as evaluated by reverse transcriptase-polymerase chain reaction. There was also a marked, but transient, increase in the mRNA level of pathogenesis-related-1a, a typical hypersensitive response marker. Overall, these results are evidence that ozone triggers a hypersensitive response in tobacco cv Bel W3 plants. We adopted four criteria for detecting programmed cell death in ozonated tobacco cv Bel W3 leaves: (a) early release of cytochrome c from mitochondria; (b) activation of protease; (c) DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA 3′-OH groups; and (d) ultrastructural changes characteristic of programmed cell death, including chromatin condensation and blebbing of plasma membrane. We, therefore, provide evidence that ozone-induced oxidative stress triggers a cell death program in tobacco cv Bel W3. PMID:14612586

  14. LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling

    PubMed Central

    2013-01-01

    Introduction Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression. Methods LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients. Results Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors. Conclusions These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression

  15. Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death.

    PubMed

    Koh, Eugene; Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

    2016-07-01

    Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. © 2016 American Society of Plant Biologists. All Rights Reserved.

  16. Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

    PubMed Central

    Koshiba, Taichi; Kobayashi, Masaru; Matoh, Toru

    2009-01-01

    Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD. PMID:19054807

  17. Cell death in Tetrahymena thermophila: new observations on culture conditions.

    PubMed

    Christensen, S T; Sørensen, H; Beyer, N H; Kristiansen, K; Rasmussen, L; Rasmussen, M I

    2001-01-01

    We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems. Copyright 2001 Academic Press.

  18. Megasporogenesis and programmed cell death in Tillandsia (Bromeliaceae).

    PubMed

    Papini, Alessio; Mosti, Stefano; Milocani, Eva; Tani, Gabriele; Di Falco, Pietro; Brighigna, Luigi

    2011-10-01

    The degeneration of three of four meiotic products is a very common process in the female gender of oogamous eukaryotes. In Tillandsia (and many other angiosperms), the surviving megaspore has a callose-free wall in chalazal position while the other three megaspores are completely embedded in callose. Therefore, nutrients and signals can reach more easily the functional megaspore from the nucellus through the chalazal pole with respect to the other megaspores. The abortion of three of four megaspores was already recognized as the result of a programmed cell death (PCD) process. We investigated the process to understand the modality of this specific type of PCD and its relationship to the asymmetric callose deposition around the tetrad. The decision on which of the four megaspores will be the supernumerary megaspores in angiosperms, and hence destined to undergo programmed cell death, appears to be linked to the callose layer deposition around the tetrad. During supernumerary megaspores degeneration, events leading to the deletion of the cells do not appear to belong to a single type of cell death. The first morphological signs are typical of autophagy, including the formation of autophagosomes. The TUNEL positivity and a change in morphology of mitochondria and chloroplasts indicate the passage to an apoptotic-like PCD phase, while the cellular remnants undergo a final process resembling at least partially (ER swelling) necrotic morphological syndromes, eventually leading to a mainly lipidic cell corpse still separated from the functional megaspore by a callose layer.

  19. Inhibitory effects of mouse bone marrow mesenchymal stem cell soup on staurospurine-induced cell death in MCF-7 and AGS.

    PubMed

    Zhaleh, M; Azadbakht, M; Bidmeshki Pour, A

    2017-01-01

    Staurospurine induces apoptosis in cell line. Bone Marrow Mesenchymal stem cells Soup is a promising tool for cell proliferation via a variety of secreted factors. In this study, we examined the effects of BMSCs Soup on Staurospurine induced-cell death in MCF-7 and AGS cells. There were three Groups: Group I: no incubation with BM Soup; Group II: incubated with 24 h BM Soup; Group III: incubation with 48 h BM Soup. There were two treatments in each group. The treatments were 1μM Staurospurine (Treatment 1) and 0.0 μM Staurospurine (Treatment 2). The cells were cultured in culture medium containing 0.2 % BSA. We obtained the cell viability, cell death and NO concentration. Our results showed that BM soup administration for 48 hours protectsed against 1μM staurosporine concentration induced cell death and reduced cell toxicity in MCF-7 and AGS cells. Cell viability and cell toxicity assay showed that BM soup in time dependent manner increased cell viability (p < 0.05) and cell death assay showed that cell death in time dependent manner was decreased(p < 0.05). Our data showed that BM soup with increasing NO concentration reduced staurospurine induced cell death and cell cytotoxicity (p < 0.05). It's concluded that BMSCs soup suppressed staurospurine-induced cytotoxicity activity process in MCF-7 and AGS cells (Fig. 9, Ref. 79).

  20. Tumour Vascular Shutdown and Cell Death Following Ultrasound-Microbubble Enhanced Radiation Therapy

    PubMed Central

    El Kaffas, Ahmed; Gangeh, Mehrdad J.; Farhat, Golnaz; Tran, William Tyler; Hashim, Amr; Giles, Anoja; Czarnota, Gregory J.

    2018-01-01

    High-dose radiotherapy effects are regulated by acute tumour endothelial cell death followed by rapid tumour cell death instead of canonical DNA break damage. Pre-treatment with ultrasound-stimulated microbubbles (USMB) has enabled higher-dose radiation effects with conventional radiation doses. This study aimed to confirm acute and longitudinal relationships between vascular shutdown and tumour cell death following radiation and USMB in a wild type murine fibrosarcoma model using in vivo imaging. Methods: Tumour xenografts were treated with single radiation doses of 2 or 8 Gy alone, or in combination with low-/high-concentration USMB. Vascular changes and tumour cell death were evaluated at 3, 24 and 72 h following therapy, using high-frequency 3D power Doppler and quantitative ultrasound spectroscopy (QUS) methods, respectively. Staining using in situ end labelling (ISEL) and cluster of differentiation 31 (CD31) of tumour sections were used to assess cell death and vascular distributions, respectively, as gold standard histological methods. Results: Results indicated a decrease in the power Doppler signal of up to 50%, and an increase of more than 5 dBr in cell-death linked QUS parameters at 24 h for tumours treated with combined USMB and radiotherapy. Power Doppler and quantitative ultrasound results were significantly correlated with CD31 and ISEL staining results (p < 0.05), respectively. Moreover, a relationship was found between ultrasound power Doppler and QUS results, as well as between micro-vascular densities (CD31) and the percentage of cell death (ISEL) (R2 0.5-0.9). Conclusions: This study demonstrated, for the first time, the link between acute vascular shutdown and acute tumour cell death using in vivo longitudinal imaging, contributing to the development of theoretical models that incorporate vascular effects in radiation therapy. Overall, this study paves the way for theranostic use of ultrasound in radiation oncology as a diagnostic modality to

  1. Does prostate acinar adenocarcinoma with Gleason Score 3+3=6 have the potential to metastasize?

    PubMed

    Montironi, Rodolfo; Scarpelli, Marina; Mazzucchelli, Roberta; Lopez-Beltran, Antonio; Santoni, Matteo; Briganti, Alberto; Montorsi, Francesco; Cheng, Liang

    2014-10-18

    There is a worldwide debate involving clinicians, uropathologists as well as patients and their families on whether Gleason score 6 adenocarcinoma should be labelled as cancer. We report a case of man diagnosed with biopsy Gleason score 6 acinar adenocarcinoma and classified as low risk (based on a PSA of 5 ng/mL and stage cT2a) whose radical prostatectomy specimen initially showed organ confined Gleason score 3+3=6, WHO nuclear grade 3, acinar adenocarcinoma with lymphovascular invasion and secondary deposit in a periprostatic lymph node. When deeper sections were cut to the point that almost all the slice present in the paraffin block was sectioned, a small tumor area (<5% of the whole tumor) of Gleason pattern 4 (poorly formed glands) was found in an extraprostatic position. The epilogue was that the additional finding changed the final Gleason score to 3+3=6 with tertiary pattern 4 and the stage to pT3a. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_190.

  2. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lyu, Qing; Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055; Tou, Fangfang

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary,more » our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.« less

  3. Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma.

    PubMed

    Serra, Pierre; Petat, Arthur; Maury, Jean-Michel; Thivolet-Bejui, Françoise; Chalabreysse, Lara; Barritault, Marc; Ebran, Nathalie; Milano, Gérard; Girard, Nicolas; Brevet, Marie

    2018-04-01

    The systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. This retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). Among 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). With the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Decoding cell death signals in liver inflammation.

    PubMed

    Brenner, Catherine; Galluzzi, Lorenzo; Kepp, Oliver; Kroemer, Guido

    2013-09-01

    Inflammation can be either beneficial or detrimental to the liver, depending on multiple factors. Mild (i.e., limited in intensity and destined to resolve) inflammatory responses have indeed been shown to exert consistent hepatoprotective effects, contributing to tissue repair and promoting the re-establishment of homeostasis. Conversely, excessive (i.e., disproportionate in intensity and permanent) inflammation may induce a massive loss of hepatocytes and hence exacerbate the severity of various hepatic conditions, including ischemia-reperfusion injury, systemic metabolic alterations (e.g., obesity, diabetes, non-alcoholic fatty liver disorders), alcoholic hepatitis, intoxication by xenobiotics and infection, de facto being associated with irreversible liver damage, fibrosis, and carcinogenesis. Both liver-resident cells (e.g., Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells) and cells that are recruited in response to injury (e.g., monocytes, macrophages, dendritic cells, natural killer cells) emit pro-inflammatory signals including - but not limited to - cytokines, chemokines, lipid messengers, and reactive oxygen species that contribute to the apoptotic or necrotic demise of hepatocytes. In turn, dying hepatocytes release damage-associated molecular patterns that-upon binding to evolutionary conserved pattern recognition receptors-activate cells of the innate immune system to further stimulate inflammatory responses, hence establishing a highly hepatotoxic feedforward cycle of inflammation and cell death. In this review, we discuss the cellular and molecular mechanisms that account for the most deleterious effect of hepatic inflammation at the cellular level, that is, the initiation of a massive cell death response among hepatocytes. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  5. CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

    PubMed

    Leclair, Pascal; Liu, Chi-Chao; Monajemi, Mahdis; Reid, Gregor S; Sly, Laura M; Lim, Chinten James

    2018-05-10

    CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

  6. Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

    PubMed

    Christopher, Karen L; Pedler, Michelle G; Shieh, Biehuoy; Ammar, David A; Petrash, J Mark; Mueller, Niklaus H

    2014-02-01

    In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Fas/Fas ligand regulation mediates cell death in human Ewing's sarcoma cells treated with melatonin

    PubMed Central

    García-Santos, G; Martin, V; Rodríguez-Blanco, J; Herrera, F; Casado-Zapico, S; Sánchez-Sánchez, A M; Antolín, I; Rodríguez, C

    2012-01-01

    Background: Despite recent advances in cancer therapy, the 5-year survival rate for Ewing's sarcoma is still very low, and new therapeutic approaches are necessary. It was found previously that melatonin induces cell death in the Ewing's sarcoma cell line, SK-N-MC, by activating the extrinsic apoptotic pathway. Methods: Melatonin actions were analysed by metabolic viability/survival cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein activation/expression and electrophoretic mobility shift assay for transcription factor activation. Results: Melatonin increases the expression of Fas and its ligand Fas L, this increase being responsible for cell death induced by the indolamine. Melatonin also produces a transient increase in intracellular oxidants and activation of the redox-regulated transcription factor Nuclear factor-kappaB. Inhibition of such activation prevents cell death and Fas/Fas L upregulation. Cytotoxic effect and Fas/Fas L regulation occur in all Ewing's cell lines studied, and do not occur in the other tumour cell lines studied where melatonin does not induce cell death. Conclusion: Our data offers new insights in the study of alternative therapeutic strategies in the treatment of Ewing's sarcoma. Further attention deserves to be given to the differences in the cellular biology of sensitive tumours that could explain the cytotoxic effect of melatonin and the increase in the level of free radicals caused by this molecule, in particular cancer types. PMID:22382690

  8. Induction of non-apoptotic cell death by morphinone in human promyelocytic leukemia HL-60 cells.

    PubMed

    Takeuchi, Risa; Hoshijima, Hiroshi; Nagasaka, Hiroshi; Chowdhury, Shahead Ali; Kikuchi, Hirotaka; Kanda, Yumiko; Kunii, Shiro; Kawase, Masami; Sakagami, Hiroshi

    2006-01-01

    As previously suggested, codeinone (oxidation product of codeine) induces non-apoptotic cell death, characterized by marginal caspase activation and the lack of DNA fragmentation in HL-60 human promyelocytic leukemia cells, which was inhibited by N-acetyl-L-cysteine. Whether, morphinone, an oxidative metabolite of morphine, also induced a similar type of cell death in HL-60 cells was investigated. Morphinone showed slightly higher cytotoxic activity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, NA, Ca9-22, promyelocytic leukemia HL-60, cervical carcinoma HeLa) than against normal oral human cells (gingival fibroblast HGF, pulp cells HPC, periodontal ligament fibroblast HPLF). Morphinone also induced an almost undetectable level of internucleosomal DNA fragmentation in the HL-60 cells. Morphinone did not activate caspase-8 or -9 in these cells. Morphinone dose-dependently activated caspase-3 in both HL-60 and HSC-2 cell lines, but to a much lesser extent than actinomycin D. Electron microscopy demonstrated that morphinone induced mitochondrial shrinkage, vacuolization and production of autophagosome and the loss of cell surface microvilli, without destruction of cell surface and nuclear membranes in the HL-60 cells. The autophagy inhibitor 3-methyladenine (0.3-10 mM) slightly inhibited the morphinone-induced cytotoxicity, when corrected for its own cytotoxicity. These data suggest that morphinone induces non-apoptotic cell death in HL-60 cells.

  9. Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hojka-Osinska, Anna, E-mail: hojka@immuno.iitd.pan.wroc.pl; Ziolo, Ewa, E-mail: ziolo@immuno.iitd.pan.wroc.pl; Rapak, Andrzej, E-mail: rapak@immuno.iitd.pan.wroc.pl

    Highlights: Black-Right-Pointing-Pointer The combination of fenretinide and indomethacin induces a high level of cell death. Black-Right-Pointing-Pointer Apoptotic pathway is caspase-independent. Black-Right-Pointing-Pointer Jurkat cells undergo AIF-mediated cell death. -- Abstract: Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergisticmore » effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug-indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.« less

  10. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship.

    PubMed

    Strasser, A; Harris, A W; Cory, S

    1991-11-29

    Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.

  11. Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

    PubMed Central

    Nishikawa, Yukihiro; Okuzaki, Daisuke; Fukushima, Kohshiro; Mukai, Satomi; Ohno, Shouichi; Ozaki, Yuki; Yabuta, Norikazu; Nojima, Hiroshi

    2015-01-01

    Withaferin A (WA), a major bioactive component of the Indian herb Withania somnifera, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death. PMID:26230090

  12. Regulation of programmed cell death or apoptosis in atherosclerosis.

    PubMed

    Geng, Y J

    1997-01-01

    Intimal thickening caused by accumulation of cells, lipids, and connective tissue characterizes atherosclerosis, an arterial disease that leads to cardiac and cerebral infarction. Apoptosis, or genetically programmed cell death, is important for the development and morphogenesis of organs and tissues. As in other tissues, cells of cardiovascular tissues can undergo apoptosis. Increased apoptosis has been found in both human and animal atherosclerotic lesions, mediating tissue turnover and lesion development. In addition to vascular cells, many activated immune cells, mainly macrophages and T cells, are present in atherosclerotic lesions, where these cells produce biologically active substances such as the proinflammatory cytokines tumor necrosis factor, interleukin-1 (IL-1), and interferon-gamma. Simultaneous exposure to these cytokines may trigger apoptosis of vascular smooth muscle cells. The products of death-regulating genes including Fas/Fas ligand, members of IL-1 beta cysteinyl protease (caspase) family, the tumor suppressive gene p53, and the protooncogene c-myc have been found in vascular cells and may participate in the regulation of vascular apoptosis during the development of atherosclerosis. Abnormal occurrence of apoptosis may take place in atherosclerotic lesions, including attenuation or acceleration of the apoptotic death process. The former may cause an increase in the cellularity of the lesions, and the latter can reduce cellular components important for maintaining the integrity and stability of the plaques. Clarification of the molecular mechanism that regulates apoptosis may help design a new strategy for treatment of patients with atherosclerosis and its major complications, heart attack and stroke.

  13. Apoptosis inducing factor gene depletion inhibits zearalenone-induced cell death in a goat Leydig cell line.

    PubMed

    Yang, Diqi; Jiang, Tingting; Lin, Pengfei; Chen, Huatao; Wang, Lei; Wang, Nan; Zhao, Fan; Tang, Keqiong; Zhou, Dong; Wang, Aihua; Jin, Yaping

    2017-01-01

    Zearalenone (ZEA) is a contaminant of human food and animal feedstuffs that causes health hazards. However, the signal pathways underlying ZEA toxicity remain elusive. The aims of this study were to determine which pathways are involved in ZEA-induced cell death and investigate the effect of apoptosis inducing factor (AIF) on cell death during ZEA treatment in the immortalized goat Leydig cell line hTERT-GLC. This study showed that ZEA-induced cell death in hTERT-GLCs works via endoplasmic reticulum (ER) stress, the caspase-dependent pathway, the caspase-independent pathway and autophagy. Recombinant lentiviral vectors were constructed to silence AIF expression in hTERT-GLCs. Flow cytometry results showed that knockdown of AIF diminished ZEA-induced cell apoptosis in hTERT-GLCs. Furthermore, we found AIF depletion down-regulated phosphoIRE1α, GRP78, CHOP and promoted the switch of LC3-I to LC3-II. Therefore, ZEA induces cytotoxicity in hTERT-GLCs via different pathways, while AIF-mediated signaling plays a critical role in ZEA-induced cell death in hTERT-GLCs. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Silicon does not mitigate cell death in cultured tobacco BY-2 cells subjected to salinity without ethylene emission.

    PubMed

    Liang, Xiaolei; Wang, Huahua; Hu, Yanfeng; Mao, Lina; Sun, Lili; Dong, Tian; Nan, Wenbin; Bi, Yurong

    2015-02-01

    Silicon induces cell death when ethylene is suppressed in cultured tobacco BY-2 cells. There is a crosstalk between Si and ethylene signaling. Silicon (Si) is beneficial for plant growth. It alleviates both biotic and abiotic stresses in plants. How Si works in plants is still mysterious. This study investigates the mechanism of Si-induced cell death in tobacco BY-2 cell cultures when ethylene is suppressed. Results showed that K2SiO3 alleviated the damage of NaCl stress. Si treatment rapidly increased ethylene emission and the expression of ethylene biosynthesis genes. Treatments with Si + Ag and Si + aminooxyacetic acid (AOA, ethylene biosynthesis inhibitor) reduced the cell growth and increased cell damage. The treatment with Si + Ag induced hydrogen peroxide (H2O2) generation and ultimately cell death. Some nucleus of BY-2 cells treated with Si + Ag appeared TUNEL positive. The inhibition of H2O2 and nitric oxide (NO) production reduced the cell death rate induced by Si + Ag treatment. Si eliminated the up-regulation of alternative pathway by Ag. These data suggest that ethylene plays an important role in Si function in plants. Without ethylene, Si not only failed to enhance plant resistance, but also elevated H2O2 generation and further induced cell death in tobacco BY-2 cells.

  15. D-galactose induces necroptotic cell death in neuroblastoma cell lines.

    PubMed

    Li, Na; He, Yangyan; Wang, Ling; Mo, Chunfen; Zhang, Jie; Zhang, Wei; Li, Junhong; Liao, Zhiyong; Tang, Xiaoqiang; Xiao, Hengyi

    2011-12-01

    D-Galactose (D-gal) can induce oxidative stress in non-cancer cells and result in cell damage by disturbing glucose metabolism. However, the effect of D-gal on cancer cells is yet to be explored. In this study, we investigated the toxicity of D-gal to malignant cells specifically neuroblastoma cells. As the results, high concentrations of D-gal had significant toxicity to cancer cells, whereas the same concentrations of glucose had no; the viability loss via D-gal treatment was prominent to malignant cells (Neuro2a, SH-SY5Y, PC-3, and HepG2) comparing to non-malignant cells (NIH3T3 and LO(2)). Differing from the apoptosis induced by H(2) O(2), D-gal damaged cells showed the characters of necrotic cell death, such as trypan blue-tangible and early phase LDH leakage. Further experiments displayed that the toxic effect of D-gal can be alleviated by necroptosis inhibitor Necrostatin (Nec-1) and autophagy inhibitor 3-methyladenine (3-MA) but not by caspase inhibitor z-VAD-fmk. D-Gal treatment can transcriptionally up-regulate the genes relevant to necroptosis (Bmf, Bnip3) and autophagy (Atg5, TIGAR) but not the genes related to apoptosis (Caspase3, Bax, and p53). D-Gal did not activate Caspase-3, but prompted puncta-like GFP-LC3 distribution, an indicator for activated autophagy. The involvement of aldose reductase (AR)-mediated polyol pathway was proved because the inhibitor of AR can attenuate the toxicity of D-gal and D-gal treatment elevates the expression of AR. This study demonstrates for the first time that D-gal can induce non-apoptotic but necroptotic cell death in neuroblastoma cells and provides a new clue for developing the strategy against apoptosis-resistant cancers. Copyright © 2011 Wiley Periodicals, Inc.

  16. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kito, Hiroaki; Yamazaki, Daiju; Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cellmore » turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.« less

  17. Mixed acinar-neuroendocrine-ductal carcinoma of the pancreas: a tale of three lineages.

    PubMed

    Anderson, Mark J; Kwong, Christina A; Atieh, Mohammed; Pappas, Sam G

    2016-06-02

    Most pancreatic cancers arise from a single cell type, although mixed pancreatic carcinomas represent a rare exception. The rarity of these aggressive malignancies and the limitations of fine-needle aspiration (FNA) pose significant barriers to diagnosis and appropriate management. We report a case of a 54-year-old man presenting with abdominal pain, jaundice and a hypodense lesion within the uncinate process on CT. FNA suggested poorly differentiated adenocarcinoma, which was subsequently resected via pancreaticoduodenectomy. Pathological analysis yielded diagnosis of invasive mixed acinar-neuroendocrine-ductal pancreatic carcinoma. Given the rare and deadly nature of these tumours, clinicians must be aware of their pathophysiology and do practice with a high degree of clinical suspicion, when appropriate. Surgical resection and thorough pathological analysis with immunohistochemical staining and electron microscopy remain the standards of care for mixed pancreatic tumours without gross evidence of metastasis. Diligent characterisation of the presentation and histological findings associated with these neoplasms should continue in order to promote optimal diagnostic and therapeutic strategies. 2016 BMJ Publishing Group Ltd.

  18. bis-Dehydroxy-Curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy.

    PubMed

    Basile, Valentina; Belluti, Silvia; Ferrari, Erika; Gozzoli, Chiara; Ganassi, Sonia; Quaglino, Daniela; Saladini, Monica; Imbriano, Carol

    2013-01-01

    The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways. In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death. Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

  19. Tales of cannibalism, suicide, and murder: Programmed cell death in C. elegans.

    PubMed

    Kinchen, Jason M; Hengartner, Michael O

    2005-01-01

    "Life is pleasant. Death is peaceful. It's the transition that's troublesome," said Isaac Asimov. Indeed, much scientific work over the last hundred years centered around attempts either to stave off or to induce the onset of death, at both the organismal and the cellular levels. In this quest, the nematode C. elegans has proven an invaluable tool, first, in the articulation of the genetic pathway by which programmed cell death proceeds, and also as a continuing source of inspiration. It is our purpose in this Chapter to familiarize the reader with the topic of programmed cell death in C. elegans and its relevance to current research in the fields of apoptosis and cell corpse clearance.

  20. Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

    PubMed

    Brimson, James M; Tencomnao, Tewin

    2011-07-26

    Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H₂DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  1. Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity.

    PubMed

    Stelmashook, E V; Lozier, E R; Goryacheva, E S; Mergenthaler, P; Novikova, S V; Zorov, D B; Isaev, N K

    2010-09-27

    The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca(2+)](i)) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca(2+)](i)). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca(2+)](i) increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca(2+)](i). Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca(2+)](i), demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca(2+)](i) and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca(2+)](i) and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  2. Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

    PubMed

    Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

    2017-08-01

    Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

    PubMed

    Blank, Norbert; Burger, Renate; Duerr, Birgit; Bakker, Frank; Wohlfarth, Anika; Dumitriu, Ingrid; Kalden, Joachim R; Herrmann, Martin

    2002-08-01

    Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered. Copyright 2002 Wiley-Liss, Inc.

  4. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in severalmore » cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.« less

  5. Persistent random walk of cells involving anomalous effects and random death

    NASA Astrophysics Data System (ADS)

    Fedotov, Sergei; Tan, Abby; Zubarev, Andrey

    2015-04-01

    The purpose of this paper is to implement a random death process into a persistent random walk model which produces sub-ballistic superdiffusion (Lévy walk). We develop a stochastic two-velocity jump model of cell motility for which the switching rate depends upon the time which the cell has spent moving in one direction. It is assumed that the switching rate is a decreasing function of residence (running) time. This assumption leads to the power law for the velocity switching time distribution. This describes the anomalous persistence of cell motility: the longer the cell moves in one direction, the smaller the switching probability to another direction becomes. We derive master equations for the cell densities with the generalized switching terms involving the tempered fractional material derivatives. We show that the random death of cells has an important implication for the transport process through tempering of the superdiffusive process. In the long-time limit we write stationary master equations in terms of exponentially truncated fractional derivatives in which the rate of death plays the role of tempering of a Lévy jump distribution. We find the upper and lower bounds for the stationary profiles corresponding to the ballistic transport and diffusion with the death-rate-dependent diffusion coefficient. Monte Carlo simulations confirm these bounds.

  6. Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baba, Miyako; Inoue, Masahiro; Itoh, Kazuyuki

    2008-09-12

    CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited themore » association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells.« less

  7. Inhibition of paraquat-induced autophagy accelerates the apoptotic cell death in neuroblastoma SH-SY5Y cells.

    PubMed

    González-Polo, Rosa A; Niso-Santano, Mireia; Ortíz-Ortíz, Miguel A; Gómez-Martín, Ana; Morán, José M; García-Rubio, Lourdes; Francisco-Morcillo, Javier; Zaragoza, Concepción; Soler, Germán; Fuentes, José M

    2007-06-01

    Autophagy is a degradative mechanism involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. This phenomenon of autophagy has been observed in neurons from patients with Parkinson's disease (PD), suggesting a functional role for autophagy in neuronal cell death. On the other hand, it has been demonstrated that exposure to pesticides can be a risk factor in the incidence of PD. In this sense, paraquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium dichloride), a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant MPP(+) (1-methyl-4-phenyl-pyridine), has been suggested as a potential etiologic factor for the development of PD. The current study shows, for the first time, that low concentrations of PQ induce several characteristics of autophagy in human neuroblastoma SH-SY5Y cells. In this way, PQ induced the accumulation of autophagic vacuoles (AVs) in the cytoplasm and the recruitment of a LC3-GFP fusion protein to AVs. Furthermore, the cells treated with PQ showed an increase of the long-lived protein degradation which is blocked in the presence of the autophagy inhibitor 3-methyladenine and regulated by the mammalian target of rapamycin (mTOR) signaling. Finally, the cells succumbed to cell death with hallmarks of apoptosis such as phosphatidylserine exposure, caspase activation, and chromatin condensation. While caspase inhibition retarded cell death, autophagy inhibition accelerated the apoptotic cell death induced by PQ. Altogether, these findings show the relationship between autophagy and apoptotic cell death in human neuroblastoma cells treated with PQ.

  8. Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

    PubMed Central

    McPhee, C K; Balgley, B M; Nelson, C; Hill, J H; Batlevi, Y; Fang, X; Lee, C S; Baehrecke, E H

    2013-01-01

    Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. PMID:22935612

  9. Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

    PubMed

    Klein, Johanna C; Wild, Clarissa A; Lang, Stephan; Brandau, Sven

    2016-06-01

    Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy.

  10. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung

    Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis inmore » T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.« less

  11. Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

    PubMed

    Narayanan, Kannan Badri; Ali, Manaf; Barclay, Barry J; Cheng, Qiang Shawn; D'Abronzo, Leandro; Dornetshuber-Fleiss, Rita; Ghosh, Paramita M; Gonzalez Guzman, Michael J; Lee, Tae-Jin; Leung, Po Sing; Li, Lin; Luanpitpong, Suidjit; Ratovitski, Edward; Rojanasakul, Yon; Romano, Maria Fiammetta; Romano, Simona; Sinha, Ranjeet K; Yedjou, Clement; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Amedei, Amedeo; Brown, Dustin G; Ryan, Elizabeth P; Colacci, Annamaria; Hamid, Roslida A; Mondello, Chiara; Raju, Jayadev; Salem, Hosni K; Woodrick, Jordan; Scovassi, A Ivana; Singh, Neetu; Vaccari, Monica; Roy, Rabindra; Forte, Stefano; Memeo, Lorenzo; Kim, Seo Yun; Bisson, William H; Lowe, Leroy; Park, Hyun Ho

    2015-06-01

    Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

    PubMed

    Boyan, George; Graf, Philip; Ehrhardt, Erica

    2018-03-01

    We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

  13. Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

    PubMed Central

    Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function. PMID:26029925

  14. DRAM Triggers Lysosomal Membrane Permeabilization and Cell Death in CD4+ T Cells Infected with HIV

    PubMed Central

    Laforge, Mireille; Limou, Sophie; Harper, Francis; Casartelli, Nicoletta; Rodrigues, Vasco; Silvestre, Ricardo; Haloui, Houda; Zagury, Jean-Francois; Senik, Anna; Estaquier, Jerome

    2013-01-01

    Productive HIV infection of CD4+ T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. PMID:23658518

  15. Cell Death Control by Matrix Metalloproteinases1[OPEN

    PubMed Central

    Zimmermann, Dirk; Sieferer, Elke; Pfannstiel, Jens

    2016-01-01

    In contrast to mammalian matrix metalloproteinases (MMPs) that play important roles in the remodeling of the extracellular matrix in animals, the proteases responsible for dynamic modifications of the plant cell wall are largely unknown. A possible involvement of MMPs was addressed by cloning and functional characterization of Sl2-MMP and Sl3-MMP from tomato (Solanum lycopersicum). The two tomato MMPs were found to resemble mammalian homologs with respect to gelatinolytic activity, substrate preference for hydrophobic amino acids on both sides of the scissile bond, and catalytic properties. In transgenic tomato seedlings silenced for Sl2/3-MMP expression, necrotic lesions were observed at the base of the hypocotyl. Cell death initiated in the epidermis and proceeded to include outer cortical cell layers. In later developmental stages, necrosis spread, covering the entire stem and extending into the leaves of MMP-silenced plants. The subtilisin-like protease P69B was identified as a substrate of Sl2- and Sl3-MMP. P69B was shown to colocalize with Sl-MMPs in the apoplast of the tomato hypocotyl, it exhibited increased stability in transgenic plants silenced for Sl-MMP activity, and it was cleaved and inactivated by Sl-MMPs in vitro. The induction of cell death in Sl2/3-MMP-silenced plants depended on P69B, indicating that Sl2- and Sl3-MMP act upstream of P69B in an extracellular proteolytic cascade that contributes to the regulation of cell death in tomato. PMID:27208293

  16. Antioxidant gene therapy against neuronal cell death

    PubMed Central

    Navarro-Yepes, Juliana; Zavala-Flores, Laura; Annadurai, Anandhan; Wang, Fang; Skotak, Maciej; Chandra, Namas; Li, Ming; Pappa, Aglaia; Martinez-Fong, Daniel; Razo, Luz Maria Del; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2014-01-01

    Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy. PMID:24333264

  17. Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

    Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

  18. Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells.

    PubMed

    Woo, Seon Min; Seo, Seung Un; Min, Kyoung-Jin; Im, Seung-Soon; Nam, Ju-Ock; Chang, Jong-Soo; Kim, Shin; Park, Jong-Wook; Kwon, Taeg Kyu

    2018-04-27

    Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants ( N -acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

  19. The inducers of immunogenic cell death for tumor immunotherapy.

    PubMed

    Li, Xiuying

    2018-01-01

    Immunotherapy is a promising treatment modality that acts by selectively harnessing the host immune defenses against cancer. An effective immune response is often needed to eliminate tumors following treatment which can trigger the immunogenicity of dying tumor cells. Some treatment modalities (such as photodynamic therapy, high hydrostatic pressure or radiotherapy) and agents (some chemotherapeutic agents, oncolytic viruses) have been used to endow tumor cells with immunogenicity and/or increase their immunogenicity. These treatments and agents can boost the antitumor capacity by inducing immune responses against tumor neoantigens. Immunogenic cell death is a manner of cell death that can induce the emission of immunogenic damage-associated molecular patterns (DAMPs). DAMPs are sufficient for immunocompetent hosts to trigger the immune system. This review focuses on the latest developments in the treatment modalities and agents that can induce and/or enhance the immunogenicity of cancer cells.

  20. Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

    NASA Astrophysics Data System (ADS)

    Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

    2008-02-01

    Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

  1. Augmented trophoblast cell death in preeclampsia can proceed via ceramide-mediated necroptosis

    PubMed Central

    Bailey, Liane Jennifer; Alahari, Sruthi; Tagliaferro, Andrea; Post, Martin; Caniggia, Isabella

    2017-01-01

    Preeclampsia, a serious hypertensive disorder of pregnancy, is characterized by elevated ceramide (CER) content that is responsible for heightened trophoblast cell death rates via apoptosis and autophagy. Whether trophoblast cells undergo necroptosis, a newly characterized form of regulated necrosis, and the potential role of CER in this process remain to be established. Herein, we report that exposure of both JEG3 cells and primary isolated cytotrophoblasts to C16:0 CER in conjunction with a caspase-8 inhibitor (Q-VD-OPh) promoted necroptotic cell death, as evidenced by increased expression and association of receptor-interacting protein kinases RIP1 and RIP3, as well as phosphorylation of mixed lineage kinase domain-like (MLKL) protein. MLKL activation and oligomerization could be abrogated by pretreatment with the necroptosis inhibitor necrostatin-1 (Nec-1). CER+Q-VD-OPH-treated primary trophoblasts displayed striking necrotic morphology along with disrupted fusion processes as evidenced by maintenance of E-cadherin-stained membrane boundaries and reduced glial cell missing-1 expression, but these events were effectively reversed using Nec-1. Of clinical relevance, we established an increased susceptibility to necroptotic cell death in preeclamptic placentae relative to normotensive controls. In preeclampsia, increased necrosome (RIP1/RIP3) protein levels, as well as MLKL activation and oligomerization associated with necrotic cytotrophoblast morphology. In addition, caspase-8 activity was reduced in severe early-onset preeclampsia cases. This study is the first to report that trophoblast cells undergo CER-induced necroptotic cell death, thereby contributing to the increased placental dysfunction and cell death found in preeclampsia. PMID:28151467

  2. Crocetin shifts autophagic cell survival to death of breast cancer cells in chemotherapy.

    PubMed

    Zhang, Ailian; Li, Jincheng

    2017-03-01

    The chemotherapy with fluorouracil is not always effective, in which some breast cancer cells may survive the fluorouracil treatment through enhanced autophagy. Crocetin is the major constituent of saffron, a Chinese traditional herb, which has recently found to have multiple pharmacological effects, including anticancer. However, the effects of Crocetin on the outcome of fluorouracil therapy for breast cancer have not been studied. Here, we showed that fluorouracil treatment inhibited the growth of breast cancer cells, in either a Cell Counting Kit-8 assay or an MTT assay. Inhibition of autophagy further suppressed breast cancer cell growth, suggesting that the breast cancer cells increased autophagic cell survival during fluorouracil treatment. However, Crocetin significantly increased the suppressive effects of fluorouracil on breast cancer cell growth, without affecting either cell apoptosis or autophagy. Inhibition of autophagy at the presence of Crocetin partially abolished the suppressive effects on breast cancer cell growth, suggesting that Crocetin may increase autophagic cell death in fluorouracil-treated breast cancer cells. Furthermore, Crocetin decreased Beclin-1 levels but increased ATG1 levels in fluorouracil-treated breast cancer cells. Together, these data suggest that Crocetin may shift autophagic cell survival to autophagic cell death in fluorouracil-treated breast cancer cells, possibly through modulation of the expression of ATG1 and Beclin-1.

  3. Vacuolar processing enzyme in plant programmed cell death

    PubMed Central

    Hatsugai, Noriyuki; Yamada, Kenji; Goto-Yamada, Shino; Hara-Nishimura, Ikuko

    2015-01-01

    Vacuolar processing enzyme (VPE) is a cysteine proteinase originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants, and it is known to be an ortholog of animal asparaginyl endopeptidase (AEP/VPE/legumain). VPE has been shown to exhibit enzymatic properties similar to that of caspase 1, which is a cysteine protease that mediates the programmed cell death (PCD) pathway in animals. Although there is limited sequence identity between VPE and caspase 1, their predicted three-dimensional structures revealed that the essential amino-acid residues for these enzymes form similar pockets for the substrate peptide YVAD. In contrast to the cytosolic localization of caspases, VPE is localized in vacuoles. VPE provokes vacuolar rupture, initiating the proteolytic cascade leading to PCD in the plant immune response. It has become apparent that the VPE-dependent PCD pathway is involved not only in the immune response, but also in the responses to a variety of stress inducers and in the development of various tissues. This review summarizes the current knowledge on the contribution of VPE to plant PCD and its role in vacuole-mediated cell death, and it also compares VPE with the animal cell death executor caspase 1. PMID:25914711

  4. Cell birth, cell death, cell diversity and DNA breaks: how do they all fit together?

    NASA Technical Reports Server (NTRS)

    Gilmore, E. C.; Nowakowski, R. S.; Caviness, V. S. Jr; Herrup, K.

    2000-01-01

    Substantial death of migrating and differentiating neurons occurs within the developing CNS of mice that are deficient in genes required for repair of double-stranded DNA breaks. These findings suggest that large-scale, yet previously unrecognized, double-stranded DNA breaks occur normally in early postmitotic and differentiating neurons. Moreover, they imply that cell death occurs if the breaks are not repaired. The cause and natural function of such breaks remains a mystery; however, their occurrence has significant implications. They might be detected by histological methods that are sensitive to DNA fragmentation and mistakenly interpreted to indicate cell death when no relationship exists. In a broader context, there is now renewed speculation that DNA recombination might be occurring during neuronal development, similar to DNA recombination in developing lymphocytes. If this is true, the target gene(s) of recombination and their significance remain to be determined.

  5. Mangiferin induces cell death against rhabdomyosarcoma through sustained oxidative stress.

    PubMed

    Padma, Vishwanadha Vijaya; Kalaiselvi, Palanisamy; Yuvaraj, Rangasamy; Rabeeth, M

    2015-06-01

    Embryonic rhabdomyosarcoma (RD) is the most prevalent type of cancer among children. The present study aimed to investigate cell death induced by mangiferin in RD cells. The Inhibitory concentration (IC 50 ) value of mangiferin was determined by an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Cell death induced by mangiferin against RD cells was determined through lactate dehydrogenase and nitric oxide release, intracellular calcium levels, reactive oxygen species generation, antioxidant status, mitochondrial calcium level, and mitochondrial membrane potential. Furthermore, acridine orange/ethidium bromide staining was performed to determine early/late apoptotic event. Mangiferin induced cell death in RD cells with an IC 50 value of 70 μM. The cytotoxic effect was reflected in a dose-dependent increase in lactate dehydrogenase leakage and nitric oxide release during mangiferin treatment. Mangiferin caused dose dependent increase in reactive oxygen species generation, intracellular calcium levels with subsequent decrease in antioxidant status (catalase, superoxide dismutase, glutathione-S-transferase, and glutathione) and loss of mitochondrial membrane potential in RD cells. Further data from fluorescence microscopy suggest that mangiferin caused cell shrinkage and nuclear condensation along with the occurrence of a late event of apoptosis. Results of the present study shows that mangiferin can act as a promising chemopreventive agent against RD by inducing sustained oxidative stress.

  6. Cell block eleven, looking from the "Death Row" exercise yard, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Cell block eleven, looking from the "Death Row" exercise yard, facing north (note cell block fifteen to the right and cell block fourteen in the distance_ - Eastern State Penitentiary, 2125 Fairmount Avenue, Philadelphia, Philadelphia County, PA

  7. Stress and death of cnidarian host cells play a role in cnidarian bleaching.

    PubMed

    Paxton, Camille W; Davy, Simon K; Weis, Virginia M

    2013-08-01

    Coral bleaching occurs when there is a breakdown of the symbiosis between cnidarian hosts and resident Symbiodinium spp. Multiple mechanisms for the bleaching process have been identified, including apoptosis and autophagy, and most previous work has focused on the Symbiodinium cell as the initiator of the bleaching cascade. In this work we show that it is possible for host cells to initiate apoptosis that can contribute to death of the Symbiodinium cell. First we found that colchicine, which results in apoptosis in other animals, causes cell death in the model anemone Aiptasia sp. but not in cultured Symbiodinium CCMP-830 cells or in cells freshly isolated from host Aiptasia (at least within the time frame of our study). In contrast, when symbiotic Aiptasia were incubated in colchicine, cell death in the resident Symbiodinium cells was observed, suggesting a host effect on symbiont mortality. Using live-cell confocal imaging of macerated symbiotic host cell isolates, we identified a pattern where the initiation of host cell death was followed by mortality of the resident Symbiodinium cells. This same pattern was observed in symbiotic host cells that were subjected to temperature stress. This research suggests that mortality of symbionts during temperature-induced bleaching can be initiated in part by host cell apoptosis.

  8. Glutathione peroxidase 4 overexpression inhibits ROS-induced cell death in diffuse large B-cell lymphoma.

    PubMed

    Kinowaki, Yuko; Kurata, Morito; Ishibashi, Sachiko; Ikeda, Masumi; Tatsuzawa, Anna; Yamamoto, Masahide; Miura, Osamu; Kitagawa, Masanobu; Yamamoto, Kouhei

    2018-02-20

    Regulation of oxidative stress and redox systems has important roles in carcinogenesis and cancer progression, and for this reason has attracted much attention as a new area of cancer therapeutic targets. Glutathione peroxidase 4 (GPX4), an antioxidant enzyme, has biological important functions such as signaling cell death by suppressing peroxidation of membrane phospholipids. However, few studies exist on the expression and clinical relevance of GPX4 in malignant lymphomas such as diffuse large B-cell lymphoma. In this study, we assessed the expression of GPX4 immunohistochemically. GPX4 was expressed in 35.5% (33/93) cases of diffuse large B-cell lymphoma. The GPX4-positive group had poor overall survival (P = 0.0032) and progression-free survival (P = 0.0004) compared with those of the GPX4-negative group. In a combined analysis of GPX4 and 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, there was a negative correlation between GPX4 and 8-hydroxydeoxyguanosine (P = 0.0009). The GPX4-positive and 8-hydroxydeoxyguanosine-negative groups had a significantly worse prognosis than the other groups in both overall survival (P = 0.0170) and progression-free survival (P = 0.0005). These results suggest that the overexpression of GPX4 is an independent prognostic predictor in diffuse large B-cell lymphoma. Furthermore, in vitro analysis demonstrated that GPX4-overexpressing cells were resistant to reactive oxygen species-induced cell death (P = 0.0360). Conversely, GPX4-knockdown cells were sensitive to reactive oxygen species-induced cell death (P = 0.0111). From these data, we conclude that GPX4 regulates reactive oxygen species-induced cell death. Our results suggest a novel therapeutic strategy using the mechanism of ferroptosis, as well as a novel prognostic predictor of diffuse large B-cell lymphoma.

  9. Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells

    PubMed Central

    Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

    2017-01-01

    Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity. PMID:28139717

  10. Deferasirox-induced iron depletion promotes BclxL downregulation and death of proximal tubular cells.

    PubMed

    Martin-Sanchez, Diego; Gallegos-Villalobos, Angel; Fontecha-Barriuso, Miguel; Carrasco, Susana; Sanchez-Niño, Maria Dolores; Lopez-Hernandez, Francisco J; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto; Sanz, Ana Belén

    2017-01-31

    Iron deficiency has been associated with kidney injury. Deferasirox is an oral iron chelator used to treat blood transfusion-related iron overload. Nephrotoxicity is the most serious and common adverse effect of deferasirox and may present as an acute or chronic kidney disease. However, scarce data are available on the molecular mechanisms of nephrotoxicity. We explored the therapeutic modulation of deferasirox-induced proximal tubular cell death in culture. Deferasirox induced dose-dependent tubular cell death and AnexxinV/7AAD staining showed features of apoptosis and necrosis. However, despite inhibiting caspase-3 activation, the pan-caspase inhibitor zVAD-fmk failed to prevent deferasirox-induced cell death. Moreover, zVAD increased deferasirox-induced cell death, a feature sometimes found in necroptosis. Electron microscopy identified mitochondrial injury and features of necrosis. However, neither necrostatin-1 nor RIP3 knockdown prevented deferasirox-induced cell death. Deferasirox caused BclxL depletion and BclxL overexpression was protective. Preventing iron depletion protected from BclxL downregulation and deferasirox cytotoxicity. In conclusion, deferasirox promoted iron depletion-dependent cell death characterized by BclxL downregulation. BclxL overexpression was protective, suggesting a role for BclxL downregulation in iron depletion-induced cell death. This information may be used to develop novel nephroprotective strategies. Furthermore, it supports the concept that monitoring kidney tissue iron depletion may decrease the risk of deferasirox nephrotoxicity.

  11. Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

    PubMed

    Kitahara, Yusuke; Kawane, Kohki; Nagata, Shigekazu

    2010-09-01

    The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

  12. Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

    PubMed

    Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

    2018-01-01

    Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. How well can morphology assess cell death modality? A proteomics study

    PubMed Central

    Chernobrovkin, Alexey L; Zubarev, Roman A

    2016-01-01

    While the focus of attempts to classify cell death programs has finally shifted in 2010s from microscopy-based morphological characteristics to biochemical assays, more recent discoveries have put the underlying assumptions of many such assays under severe stress, mostly because of the limited specificity of the assays. On the other hand, proteomics can quantitatively measure the abundances of thousands of proteins in a single experiment. Thus proteomics could develop a modern alternative to both semiquantitative morphology assessment as well as single-molecule biochemical assays. Here we tested this hypothesis by analyzing the proteomes of cells dying after been treated with various chemical agents. The most striking finding is that, for a multivariate model based on the proteome changes in three cells lines, the regulation patterns of the 200–500 most abundant proteins typically attributed to household type more accurately reflect that of the proteins directly interacting with the drug than any other protein subset grouped by common function or biological process, including cell death. This is in broad agreement with the 'rigid cell death mechanics' model where drug action mechanism and morphological changes caused by it are bijectively linked. This finding, if confirmed, will open way for a broad use of proteomics in death modality assessment. PMID:27752363

  14. Role of leptin in modulation of Porphyromonas gingivalis lipopolysaccharide-induced up-regulation of endothelin-1 in salivary gland acinar cells.

    PubMed

    Slomiany, Bronislaw L; Slomiany, Amalia

    2005-08-01

    Leptin, a pleiotropic cytokine that regulates food intake and metabolic and endocrine responses, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection. In this study, we report that in sublingual salivary gland acinar cells leptin plays a role in the suppression of up-regulation in endothelin-1 (ET-1), induced by the LPS of a periodontopathic bacterium P. gingivalis. We show that P. gingivalisLPS detrimental effect on salivary mucin synthesis, associated with up-regulation (3.9-fold) in ET-1 generation and the enhancement (3.2-fold) in endothelin-converting enzyme-1 (ECE-1) activity, was subject to a dose-dependent suppression by leptin. The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, as well as by ERK inhibitor, PD98059. However, while the blockade of ERK led also to amplification in the impedance by leptin of the LPS-induced expression of ECE-1 and ET-1, the effect was not observed in the presence of wortmannin. The findings are the first to demonstrate that leptin counters the pathological consequences of P. gingivalisinfection on the synthesis of salivary mucin through the involvement in signaling events of PI3K and ERK pathways. We also show that the ERK cascade represents a critical signaling target for leptin in the LPS-induced up-regulation in ET-1.

  15. Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress

    PubMed Central

    Lin, Abraham; Truong, Billy; Patel, Sohil; Kaushik, Nagendra; Choi, Eun Ha; Fridman, Gregory; Fridman, Alexander; Miller, Vandana

    2017-01-01

    A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy. PMID:28467380

  16. 'Hints' in the killer protein gasdermin D: unveiling the secrets of gasdermins driving cell death.

    PubMed

    Qiu, Shiqiao; Liu, Jing; Xing, Feiyue

    2017-04-01

    Pyroptosis is a lytic form of cell death distinguished from apoptosis, ferroptosis, necrosis, necroptosis, NETosis, oncosis, pyronecrosis and autophagy. Proinflammatory caspases cleave a gasdermin D (GSDMD) protein to generate a 31 kDa N-terminal domain. The cleavage relieves the intramolecular inhibition on the gasdermin-N domain, which then moves to the plasma membrane to exhibit pore-forming activity. Thus, GSDMD acts as the final and direct executor of pyroptotic cell death. Owing to the selective targeting of the inner leaflet of the plasma membrane with the pore-forming that determines pyroptotic cell death, GSDMD could be a potential target to control cell death or extracellular bacterial infections. Intriguingly, other gasdermin family members also share similar N-terminal domains, but they present different cell death programs. Herein, we summarize features and functions of the novel player proteins in cell death, including GSDMD triggering pyroptosis, Gsdma3/GSDMA initiating autophagy/apoptosis and DFNA5 inducing apoptosis/secondary necrosis. The gasdermin N terminus appears to be a novel pore-forming protein. This provides novel insight into the underlying roles and mechanisms of lytic or nonlytic forms of programmed cell death, as well as their potential applications in inflammation-associated diseases.

  17. Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish.

    PubMed

    Viringipurampeer, I A; Shan, X; Gregory-Evans, K; Zhang, J P; Mohammadi, Z; Gregory-Evans, C Y

    2014-05-01

    Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

  18. Cystine uptake through the cystine/glutamate antiporter xCT triggers glioblastoma cell death under glucose deprivation.

    PubMed

    Goji, Takeo; Takahara, Kazuhiko; Negishi, Manabu; Katoh, Hironori

    2017-12-01

    Oncogenic signaling in cancer cells alters glucose uptake and utilization to supply sufficient energy and biosynthetic intermediates for survival and sustained proliferation. Oncogenic signaling also prevents oxidative stress and cell death caused by increased production of reactive oxygen species. However, elevated glucose metabolism in cancer cells, especially in glioblastoma, results in the cells becoming sensitive to glucose deprivation ( i.e. in high glucose dependence), which rapidly induces cell death. However, the precise mechanism of this type of cell death remains unknown. Here, we report that glucose deprivation alone does not trigger glioblastoma cell death. We found that, for cell death to occur in glucose-deprived glioblastoma cells, cystine and glutamine also need to be present in culture media. We observed that cystine uptake through the cystine/glutamate antiporter xCT under glucose deprivation rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death. We conclude that although cystine uptake is crucial for production of antioxidant glutathione in cancer cells its transport through xCT also induces oxidative stress and cell death in glucose-deprived glioblastoma cells. Combining inhibitors targeting cancer-specific glucose metabolism with cystine and glutamine treatment may offer a therapeutic approach for glioblastoma tumors exhibiting high xCT expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells.

    PubMed

    Anderson, Chastain; Majeste, Andrew; Hanus, Jakub; Wang, Shusheng

    2016-12-01

    Cigarette smoking remains one of the leading causes of preventable death worldwide. Vascular cell death and dysfunction is a central or exacerbating component in the majority of cigarette smoking related pathologies. The recent development of the electronic nicotine delivery systems known as e-cigarettes provides an alternative to conventional cigarette smoking; however, the potential vascular health risks of e-cigarette use remain unclear. This study evaluates the effects of e-cigarette aerosol extract (EAE) and conventional cigarette smoke extract (CSE) on human umbilical vein endothelial cells (HUVECs). A laboratory apparatus was designed to produce extracts from e-cigarettes and conventional cigarettes according to established protocols for cigarette smoking. EAE or conventional CSE was applied to human vascular endothelial cells for 4-72 h, dependent on the assay. Treated cells were assayed for reactive oxygen species, DNA damage, cell viability, and markers of programmed cell death pathways. Additionally, the anti-oxidants α-tocopherol and n-acetyl-l-cysteine were used to attempt to rescue e-cigarette induced cell death. Our results indicate that e-cigarette aerosol is capable of inducing reactive oxygen species, causing DNA damage, and significantly reducing cell viability in a concentration dependent fashion. Immunofluorescent and flow cytometry analysis indicate that both the apoptosis and programmed necrosis pathways are triggered by e-cigarette aerosol treatment. Additionally, anti-oxidant treatment provides a partial rescue of the induced cell death, indicating that reactive oxygen species play a causal role in e-cigarette induced cytotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Ursodeoxycholic acid effectively kills drug-resistant gastric cancer cells through induction of autophagic death.

    PubMed

    Lim, Sung-Chul; Han, Song Iy

    2015-09-01

    Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance

  1. The cell on the edge of life and death: Crosstalk between autophagy and apoptosis.

    PubMed

    Kasprowska-Liśkiewicz, Daniela

    2017-09-21

    Recently, the crosstalk between autophagy and apoptosis has attracted broader attention. Basal autophagy serves to maintain cell homeostasis, while the upregulation of this process is an element of stress response that enables the cell to survive under adverse conditions. Autophagy may also determine the fate of the cell through its interactions with cell death pathways. The protein networks that control the initiation and the execution phase of these two processes are highly interconnected. Several scenarios for the crosstalk between autophagy and apoptosis exist. In most cases, the activation of autophagy represents an attempt of the cell to cope with stress, and protects the cell from apoptosis or delays its initiation. Generally, the simultaneous activation of pro-survival and pro-death pathways is prevented by the mutual inhibitory crosstalk between autophagy and apoptosis. But in some circumstances, autophagy or the proteins of the core autophagic machinery may promote cellular demise through excessive self-digestion (so-called "autophagic cell death") or by stimulating the activation of other cell death pathways. It is controversial whether cells actually die via autophagy, which is why the term "autophagic cell death" has been under intense debate lately. This review summarizes the recent findings on the multilevel crosstalk between autophagy and apoptosis in aspects of common regulators, mutual inhibition of these processes, the stimulation of apoptosis by autophagy or autophagic proteins and finally the role of autophagy as a death-execution mechanism.

  2. Cell death during Drosophila melanogaster early oogenesis is mediated through autophagy.

    PubMed

    Nezis, Ioannis P; Lamark, Trond; Velentzas, Athanassios D; Rusten, Tor Erik; Bjørkøy, Geir; Johansen, Terje; Papassideri, Issidora S; Stravopodis, Dimitrios J; Margaritis, Lukas H; Stenmark, Harald; Brech, Andreas

    2009-04-01

    Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.

  3. Rational development of a cytotoxic peptide to trigger cell death.

    PubMed

    Boohaker, Rebecca J; Zhang, Ge; Lee, Michael W; Nemec, Kathleen N; Santra, Santimukul; Perez, J Manuel; Khaled, Annette R

    2012-07-02

    Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

  4. MLKL-PITPα signaling-mediated necroptosis contributes to cisplatin-triggered cell death in lung cancer A549 cells.

    PubMed

    Jing, Lin; Song, Fei; Liu, Zhenyu; Li, Jianghua; Wu, Bo; Fu, Zhiguang; Jiang, Jianli; Chen, Zhinan

    2018-02-01

    Necroptosis has been reported to be involved in cisplatin-induced cell death, but the mechanisms underlying the occurrence of necroptosis are not fully elucidated. In this study, we show that apart from apoptosis, cisplatin induces necroptosis in A549 cells. The alleviation of cell death by two necroptosis inhibitors-necrostatin-1 (Nec-1) and necrosulfonamide (NSA), and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) at serine 358, suggest the involvement of receptor-interacting protein kinase 1 (RIPK1)-RIPK3-MLKL signaling in cisplatin-treated A549 cells. Additionally, the initiation of cisplatin-induced necroptosis relies on autocrine tumor necrosis factor alpha (TNF-α). Furthermore, we present the first evidence that phosphatidylinositol transfer protein alpha (PITPα) is involved in MLKL-mediated necroptosis by interacting with the N terminal MLKL on its sixth helix and the preceding loop, which facilitates MLKL oligomerization and plasma membrane translocation in necroptosis. Silencing of PITPα expression interferes with MLKL function and reduces cell death. Our data elucidate that cisplatin-treated lung cancer cells undergo a new type of programmed cell death called necroptosis and shed new light on how MLKL translocates to the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death

    PubMed Central

    Cao, Chike; Liu, Yanling; Lehmann, Michael

    2007-01-01

    Cell death during Drosophila melanogaster metamorphosis is controlled by the steroid hormone 20-hydroxyecdysone (20E). Elements of the signaling pathway that triggers death are known, but it is not known why some tissues, and not others, die in response to a particular hormone pulse. We found that loss of the tissue-specific transcription factor Fork head (Fkh) is both required and sufficient to specify a death response to 20E in the larval salivary glands. Loss of fkh itself is a steroid-controlled event that is mediated by the 20E-induced BR-C gene, and that renders the key death regulators hid and reaper hormone responsive. These results implicate the D. melanogaster FOXA orthologue Fkh with a novel function as a competence factor for steroid-controlled cell death. They explain how a specific tissue is singled out for death, and why this tissue survives earlier hormone pulses. More generally, they suggest that cell identity factors like Fkh play a pivotal role in the normal control of developmental cell death. PMID:17339378

  6. Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

    PubMed

    Shin, Yoo Seob; Cha, Hyun Young; Lee, Bok-Soon; Kang, Sung Un; Hwang, Hye Sook; Kwon, Hak Cheol; Kim, Chul-Ho; Choi, Eun Chang

    2016-04-01

    The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

  7. Tumor necrosis factor (TNF) biology and cell death.

    PubMed

    Bertazza, Loris; Mocellin, Simone

    2008-01-01

    Tumor necrosis factor (TNF) was the first cytokine to be used in humans for cancer therapy. However, its role in the treatment of cancer patients is debated. Most uncertainties in this field stem from the knowledge that the pathways directly activated or indirectly affected upon TNF engagement with its receptors can ultimately lead to very different outcomes in terms of cell survival. In this article, we summarize the fundamental molecular biology aspects of this cytokine. Such a basis is a prerequisite to critically approach the sometimes conflicting preclinical and clinical findings regarding the relationship between TNF, tumor biology and anticancer therapy. Although the last decade has witnessed remarkable advances in this field, we still do not know in detail how cells choose between life and death after TNF stimulation. Understanding this mechanism will not only shed new light on the physiological significance of TNF-driven programmed cell death but also help investigators maximize the anticancer potential of this cytokine.

  8. Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hemendinger, Richelle A., E-mail: richelle.hemendinger@carolinashealthcare.org; Armstrong, Edward J.; Brooks, Benjamin Rix

    Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC{sub 50} (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolatemore » (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC{sub 50} (concentration at which 50% of maximal cell death is inhibited) of 0.6 {mu}M and 0.4 {mu}M, respectively. In contrast, MTHF (up to 10 {mu}M) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS.« less

  9. Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

    PubMed Central

    Wang, Guohao; Liu, Xudong; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

    2016-01-01

    The Huntington’s disease (HD) protein, huntingtin (HTT), is essential for early development. Because suppressing the expression of mutant HTT is an important approach to treat the disease, we must first understand the normal function of Htt in adults versus younger animals. Using inducible Htt knockout mice, we found that Htt depletion does not lead to adult neurodegeneration or animal death at >4 mo of age, which was also verified by selectively depleting Htt in neurons. On the other hand, young Htt KO mice die at 2 mo of age of acute pancreatitis due to the degeneration of pancreatic acinar cells. Importantly, Htt interacts with the trypsin inhibitor, serine protease inhibitor Kazal-type 3 (Spink3), to inhibit activation of digestive enzymes in acinar cells in young mice, and transgenic HTT can rescue the early death of Htt KO mice. These findings point out age- and cell type-dependent vital functions of Htt and the safety of knocking down neuronal Htt expression in adult brains as a treatment. PMID:26951659

  10. Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

    PubMed

    Kang, Jongkyun; Yeom, Eunbyul; Lim, Janghoo; Choi, Kwang-Wook

    2014-01-01

    The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM) pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp) posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

  11. [Novel Anticancer Strategy Targeting Switch Mechanisms in Two Types of Cell Death: Necrosis and Apoptosis].

    PubMed

    Sato, Akira

    2017-01-01

     Two types of cell death, necrosis and apoptosis, are defined in terms of cell death morphological features. We have been studying the mechanisms by which cell death processes are switched during the treatment of mouse tumor FM3A with anticancer, 5-fluoro-2'-deoxyuridine (FUdR): it induces original clone F28-7 to necrosis, but its sub-clone F28-7-A to apoptosis. We identified several such switch regulators of cell death: heat shock protein 90 (HSP90), lamin-B1, cytokeratin-19, and activating transcription factor 3 (ATF3), by using transcriptomic, proteomic analyses and siRNA screening. For example, the inhibition of HSP90 by its inhibitor geldanamycin in F28-7 caused a shift from necrosis to apoptosis. We also observed that the knockdown of lamin-B1, cytokeratin-19, or ATF3 expression in F28-7 resulted in a shift from necrosis to apoptosis. Recently, we used microRNA (miRNA, miR) microarray analyses to investigate the miRNA expression profiles in these sister cells. The miR-351 and miR-743a were expressed at higher levels in F28-7-A than in F28-7. Higher expression of miR-351 or miR-743a in F28-7, induced by transfecting the miR mimics, resulted in a switch of cell death mode: necrosis to apoptosis. Furthermore, transfection of an miR-351 inhibitor into F28-7-A resulted in morphological changes, and mode of cell death from apoptosis to necrosis. These findings suggest that the identified cell death regulators may have key roles in switching cell death mode. Possible mechanisms involving cell death regulators in the switch of necrosis or apoptosis are discussed. We propose a novel anticancer strategy targeting the switch regulators of necrosis or apoptosis.

  12. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

    PubMed Central

    Galluzzi, L; Bravo-San Pedro, J M; Vitale, I; Aaronson, S A; Abrams, J M; Adam, D; Alnemri, E S; Altucci, L; Andrews, D; Annicchiarico-Petruzzelli, M; Baehrecke, E H; Bazan, N G; Bertrand, M J; Bianchi, K; Blagosklonny, M V; Blomgren, K; Borner, C; Bredesen, D E; Brenner, C; Campanella, M; Candi, E; Cecconi, F; Chan, F K; Chandel, N S; Cheng, E H; Chipuk, J E; Cidlowski, J A; Ciechanover, A; Dawson, T M; Dawson, V L; De Laurenzi, V; De Maria, R; Debatin, K-M; Di Daniele, N; Dixit, V M; Dynlacht, B D; El-Deiry, W S; Fimia, G M; Flavell, R A; Fulda, S; Garrido, C; Gougeon, M-L; Green, D R; Gronemeyer, H; Hajnoczky, G; Hardwick, J M; Hengartner, M O; Ichijo, H; Joseph, B; Jost, P J; Kaufmann, T; Kepp, O; Klionsky, D J; Knight, R A; Kumar, S; Lemasters, J J; Levine, B; Linkermann, A; Lipton, S A; Lockshin, R A; López-Otín, C; Lugli, E; Madeo, F; Malorni, W; Marine, J-C; Martin, S J; Martinou, J-C; Medema, J P; Meier, P; Melino, S; Mizushima, N; Moll, U; Muñoz-Pinedo, C; Nuñez, G; Oberst, A; Panaretakis, T; Penninger, J M; Peter, M E; Piacentini, M; Pinton, P; Prehn, J H; Puthalakath, H; Rabinovich, G A; Ravichandran, K S; Rizzuto, R; Rodrigues, C M; Rubinsztein, D C; Rudel, T; Shi, Y; Simon, H-U; Stockwell, B R; Szabadkai, G; Tait, S W; Tang, H L; Tavernarakis, N; Tsujimoto, Y; Vanden Berghe, T; Vandenabeele, P; Villunger, A; Wagner, E F; Walczak, H; White, E; Wood, W G; Yuan, J; Zakeri, Z; Zhivotovsky, B; Melino, G; Kroemer, G

    2015-01-01

    Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death. PMID:25236395

  13. Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

    PubMed

    Galluzzi, L; Bravo-San Pedro, J M; Vitale, I; Aaronson, S A; Abrams, J M; Adam, D; Alnemri, E S; Altucci, L; Andrews, D; Annicchiarico-Petruzzelli, M; Baehrecke, E H; Bazan, N G; Bertrand, M J; Bianchi, K; Blagosklonny, M V; Blomgren, K; Borner, C; Bredesen, D E; Brenner, C; Campanella, M; Candi, E; Cecconi, F; Chan, F K; Chandel, N S; Cheng, E H; Chipuk, J E; Cidlowski, J A; Ciechanover, A; Dawson, T M; Dawson, V L; De Laurenzi, V; De Maria, R; Debatin, K-M; Di Daniele, N; Dixit, V M; Dynlacht, B D; El-Deiry, W S; Fimia, G M; Flavell, R A; Fulda, S; Garrido, C; Gougeon, M-L; Green, D R; Gronemeyer, H; Hajnoczky, G; Hardwick, J M; Hengartner, M O; Ichijo, H; Joseph, B; Jost, P J; Kaufmann, T; Kepp, O; Klionsky, D J; Knight, R A; Kumar, S; Lemasters, J J; Levine, B; Linkermann, A; Lipton, S A; Lockshin, R A; López-Otín, C; Lugli, E; Madeo, F; Malorni, W; Marine, J-C; Martin, S J; Martinou, J-C; Medema, J P; Meier, P; Melino, S; Mizushima, N; Moll, U; Muñoz-Pinedo, C; Nuñez, G; Oberst, A; Panaretakis, T; Penninger, J M; Peter, M E; Piacentini, M; Pinton, P; Prehn, J H; Puthalakath, H; Rabinovich, G A; Ravichandran, K S; Rizzuto, R; Rodrigues, C M; Rubinsztein, D C; Rudel, T; Shi, Y; Simon, H-U; Stockwell, B R; Szabadkai, G; Tait, S W; Tang, H L; Tavernarakis, N; Tsujimoto, Y; Vanden Berghe, T; Vandenabeele, P; Villunger, A; Wagner, E F; Walczak, H; White, E; Wood, W G; Yuan, J; Zakeri, Z; Zhivotovsky, B; Melino, G; Kroemer, G

    2015-01-01

    Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

  14. 24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis.

    PubMed

    Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

    2011-07-15

    24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.

  15. 24(S)-Hydroxycholesterol Induces Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis*

    PubMed Central

    Yamanaka, Kazunori; Saito, Yoshiro; Yamamori, Tohru; Urano, Yasuomi; Noguchi, Noriko

    2011-01-01

    24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis. PMID:21613228

  16. Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe.

    PubMed

    Hara, Yusuke; Sudo, Tatsuya; Togane, Yu; Akagawa, Hiromi; Tsujimura, Hidenobu

    2018-04-01

    Programmed cell death is a conserved strategy for neural development both in vertebrates and invertebrates and is recognized at various developmental stages in the brain from neurogenesis to adulthood. To understand the development of the central nervous system, it is essential to reveal not only molecular mechanisms but also the role of neural cell death (Pinto-Teixeira et al., 2016). To understand the role of cell death in neural development, we investigated the effect of inhibition of cell death on optic lobe development. Our data demonstrate that, in the optic lobe of Drosophila, cell death occurs in neural precursor cells and neurons before neurite formation and functions to prevent various developmental abnormalities. When neuronal cell death was inhibited by an effector caspase inhibitor, p35, multiple abnormal neuropil structures arose during optic lobe development-e.g., enlarged or fused neuropils, misrouted neurons and abnormal neurite lumps. Inhibition of cell death also induced morphogenetic defects in the lamina and medulla development-e.g., failures in the separation of the lamina and medulla cortices and the medulla rotation. These defects were reproduced in the mutant of an initiator caspase, dronc. If cell death was a mechanism for removing the abnormal neuropil structures, we would also expect to observe them in mutants defective for corpse clearance. However, they were not observed in these mutants. When dead cell-membranes were visualized with Apoliner, they were observed only in cortices and not in neuropils. These results suggest that the cell death occurs before mature neurite formation. Moreover, we found that inhibition of cell death induced ectopic neuroepithelial cells, neuroblasts and ganglion mother cells in late pupal stages, at sites where the outer and inner proliferation centers were located at earlier developmental stages. Caspase-3 activation was observed in the neuroepithelial cells and neuroblasts in the proliferation centers

  17. Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice.

    PubMed

    He, Ping; Yang, Jong Won; Yang, Vincent W; Bialkowska, Agnieszka B

    2018-04-01

    Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. We performed studies in adult mice with conditional disruption of Klf5 (Klf5 fl/fl ) and/or expression of Kras G12D (LSL-Kras G12D ) via Cre ERTM recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of Kras G12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-Kras G12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-Cre ERTM ;LSL-Kras G12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-Cre ERTM ;LSL-Kras G12D ;Klf5 fl

  18. Cell-cycle control in the face of damage--a matter of life or death.

    PubMed

    Clarke, Paul R; Allan, Lindsey A

    2009-03-01

    Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

  19. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

    PubMed

    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  20. Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo.

    PubMed

    Voyatzis, Sylvie; Muzerelle, Aude; Gaspar, Patricia; Nicol, Xavier

    2012-01-01

    Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.

  1. Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

    PubMed

    Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

    2009-03-31

    Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

  2. Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration.

    PubMed

    Kaarniranta, Kai; Tokarz, Paulina; Koskela, Ali; Paterno, Jussi; Blasiak, Janusz

    2017-04-01

    Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.

  3. The Phytoalexin Resveratrol Regulates the Initiation of Hypersensitive Cell Death in Vitis Cell

    PubMed Central

    Chang, Xiaoli; Heene, Ernst; Qiao, Fei; Nick, Peter

    2011-01-01

    Resveratrol is a major phytoalexin produced by plants in response to various stresses and promotes disease resistance. The resistance of North American grapevine Vitis rupestris is correlated with a hypersensitive reaction (HR), while susceptible European Vitis vinifera cv. ‘Pinot Noir’ does not exhibit HR, but expresses basal defence. We have shown previously that in cell lines derived from the two Vitis species, the bacterial effector Harpin induced a rapid and sensitive accumulation of stilbene synthase (StSy) transcripts, followed by massive cell death in V. rupestris. In the present work, we analysed the function of the phytoalexin resveratrol, the product of StSy. We found that cv. ‘Pinot Noir’ accumulated low resveratrol and its glycoside trans-piceid, whereas V. rupestris produced massive trans-resveratrol and the toxic oxidative δ-viniferin, indicating that the preferred metabolitism of resveratrol plays role in Vitis resistance. Cellular responses to resveratrol included rapid alkalinisation, accumulation of pathogenesis-related protein 5 (PR5) transcripts, oxidative burst, actin bundling, and cell death. Microtubule disruption and induction of StSy were triggered by Harpin, but not by resveratrol. Whereas most responses proceeded with different amplitude for the two cell lines, the accumulation of resveratrol, and the competence for resveratrol-induced oxidative burst differed in quality. The data lead to a model, where resveratrol, in addition to its classical role as antimicrobial phytoalexin, represents an important regulator for initiation of HR-related cell death. PMID:22053190

  4. A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death.

    PubMed

    Ma, Hongming; Dang, Ying; Wu, Yonggan; Jia, Gengxiang; Anaya, Edgar; Zhang, Junli; Abraham, Sojan; Choi, Jang-Gi; Shi, Guojun; Qi, Ling; Manjunath, N; Wu, Haoquan

    2015-07-28

    West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the ER-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Mechanism of neem limonoids-induced cell death in cancer: role of oxidative phosphorylation

    PubMed Central

    Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph; O’Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay; Yadava, Nagendra; Chandra, Dhyan

    2016-01-01

    We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. PMID:26627937

  6. Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

    PubMed

    Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

    2016-01-01

    We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Eosinophilic Otitis Media: the Aftermath of Eosinophil Extracellular Trap Cell Death.

    PubMed

    Ueki, Shigeharu; Ohta, Nobuo; Takeda, Masahide; Konno, Yasunori; Hirokawa, Makoto

    2017-05-01

    Eosinophilic otitis media (EOM) is a refractory disease characterized by the accumulation of eosinophils in middle ear effusion and mucosa. We summarize current knowledge regarding the clinical characteristics and management of EOM. Although eosinophil activation in inflamed foci is involved in the pathogenesis of EOM, little is known about the fate of the eosinophils and aftermath of their cell death. We discuss the possibility that eosinophils undergo non-apoptotic cell death that worsens tissue damage and increases effusion viscosity. Unlike chronic otitis media, EOM is strongly associated with an allergic background. Corticosteroids are currently the only effective pharmacological treatment, and surgical intervention is often required. Mucosal eosinophils infiltrate extensively into the middle ear cavity where they are stimulated by locally produced activators including interleukin-5 and eotaxin. The eosinophils undergo cytolysis in the effusion, which represents a major fate of activated eosinophils in vivo. Recent data revealed cytolysis could be renamed as extracellular trap cell death (ETosis). ETosis represents suicidal cell death involving total cell degranulation and development of sticky chromatin structures (extracellular traps (ETs)). The characteristics of eosinophil- and neutrophil-derived ET polymers might contribute to the difference in viscosity of secretions between EOM and common chronic otitis media. The extracellular products remaining after eosinophil ETosis are an important aspect of EOM pathology. The concept of ETosis also has novel implications for potential therapeutic modalities in various eosinophilic disorders.

  8. The MST/Hippo Pathway and Cell Death: A Non-Canonical Affair

    PubMed Central

    Fallahi, Emma; O’Driscoll, Niamh A.; Matallanas, David

    2016-01-01

    The MST/Hippo signalling pathway was first described over a decade ago in Drosophila melanogaster and the core of the pathway is evolutionary conserved in mammals. The mammalian MST/Hippo pathway regulates organ size, cell proliferation and cell death. In addition, it has been shown to play a central role in the regulation of cellular homeostasis and it is commonly deregulated in human tumours. The delineation of the canonical pathway resembles the behaviour of the Hippo pathway in the fly where the activation of the core kinases of the pathway prevents the proliferative signal mediated by the key effector of the pathway YAP. Nevertheless, several lines of evidence support the idea that the mammalian MST/Hippo pathway has acquired new features during evolution, including different regulators and effectors, crosstalk with other essential signalling pathways involved in cellular homeostasis and the ability to actively trigger cell death. Here we describe the current knowledge of the mechanisms that mediate MST/Hippo dependent cell death, especially apoptosis. We include evidence for the existence of complex signalling networks where the core proteins of the pathway play a central role in controlling the balance between survival and cell death. Finally, we discuss the possible involvement of these signalling networks in several human diseases such as cancer, diabetes and neurodegenerative disorders. PMID:27322327

  9. Oxidative stress activates the TRPM2-Ca2+-CaMKII-ROS signaling loop to induce cell death in cancer cells.

    PubMed

    Wang, Qian; Huang, Lihong; Yue, Jianbo

    2017-06-01

    High intracellular levels of reactive oxygen species (ROS) cause oxidative stress that results in numerous pathologies, including cell death. Transient potential receptor melastatin-2 (TRPM2), a Ca 2+ -permeable cation channel, is mainly activated by intracellular adenosine diphosphate ribose (ADPR) in response to oxidative stress. Here we studied the role and mechanisms of TRPM2-mediated Ca 2+ influx on oxidative stress-induced cell death in cancer cells. We found that oxidative stress activated the TRPM2-Ca 2+ -CaMKII cascade to inhibit early autophagy induction, which ultimately led to cell death in TRPM2 expressing cancer cells. On the other hand, TRPM2 knockdown switched cells from cell death to autophagy for survival in response to oxidative stress. Moreover, we found that oxidative stress activated the TRPM2-CaMKII cascade to further induce intracellular ROS production, which led to mitochondria fragmentation and loss of mitochondrial membrane potential. In summary, our data demonstrated that oxidative stress activates the TRPM2-Ca 2+ -CaMKII-ROS signal loop to inhibit autophagy and induce cell death. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Changes in metabolic markers in insulin-producing β-cells during hypoxia-induced cell death as studied by NMR metabolomics.

    PubMed

    Tian, Lianji; Kim, Hoe Suk; Kim, Heyonjin; Jin, Xing; Jung, Hye Seung; Park, Kyong Soo; Cho, Kyoung Won; Park, Sunghyouk; Moon, Woo Kyung

    2013-08-02

    This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic β-cell line INS-1 to identify potential early and late biomarkers for predicting hypoxia-induced cell death. INS-1 cells were incubated under normoxic conditions (95% air, 5% CO₂) or hypoxic conditions (1% O₂, 5% CO₂, 95% N₂) for 2, 4, 6, 12, or 24 h. The biological changes indicating the process of cell death were analyzed using the MTT assay, flow cytometry, Western blotting, and immunostaining. Changes in the metabolic profiles from cell lysates were identified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy, and the spectra were analyzed by the multivariate model Orthogonal Projections to Latent Structure-Discriminant Analysis. Cell viability decreased approximately 40% after 12-24 h of hypoxia, coincident with a high level of cleaved caspase-3. A high level of HIF-1α was detected in the 12-24 h hypoxic conditions. The metabolite profiles were altered according to the degree of exposure to hypoxia. A spectral analysis showed significant differences in creatine-containing compounds at the early stage (2-6 h) and taurine-containing compounds at the late stage (12-24 h), with the detection of HIF-1α and cleaved caspase-3 in cells exposed to hypoxia compared to normoxia. Glycerophosphocholine decreased during the early stage hypoxia. The change in taurine- and creatine-containing compounds and choline species could be involved in the β-cell death process as inhibitors or activators of cell death. Our results imply that assessment by ¹H NMR spectroscopy would be a useful tool to predict the cell death process and to identify molecules regulating hypoxia-induced cell death mechanisms.

  11. Induction of cytosine arabinoside-resistant human myeloid leukemia cell death through autophagy regulation by hydroxychloroquine.

    PubMed

    Kim, Yundeok; Eom, Ju-In; Jeung, Hoi-Kyung; Jang, Ji Eun; Kim, Jin Seok; Cheong, June-Won; Kim, Young Sam; Min, Yoo Hong

    2015-07-01

    We investigated the effects of the autophagy inhibitor hydroxychloroquine (HCQ) on cell death of cytosine arabinoside (Ara-C)-resistant human acute myeloid leukemia (AML) cells. Ara-C-sensitive (U937, AML-2) and Ara-C-resistant (U937/AR, AML-2/AR) human AML cell lines were used to evaluate HCQ-regulated cytotoxicity, autophagy, and apoptosis as well as effects on cell death-related signaling pathways. We found that HCQ-induced dose- and time-dependent cell death in Ara-C-resistant cells compared to Ara-C-sensitive cell lines. The extent of cell death and features of HCQ-induced autophagic markers including increase in microtubule-associated protein light chain 3 (LC3) I conversion to LC3-II, beclin-1, ATG5, as well as green fluorescent protein-LC3 positive puncta and autophagosome were remarkably greater in U937/AR cells. Also, p62/SQSTM1 was increased in response to HCQ. p62/SQSTM1 protein interacts with both LC3-II and ubiquitin protein and is degraded in autophagosomes. Therefore, a reduction of p62/SQSTM1 indicates increased autophagic degradation, whereas an increase of p62/SQSTM1 by HCQ indicates inhibited autophagic degradation. Knock down of p62/SQSTM1 using siRNA were prevented the HCQ-induced LC3-II protein level as well as significantly reduced the HCQ-induced cell death in U937/AR cells. Also, apoptotic cell death and caspase activation in U937/AR cells were increased by HCQ, provided evidence that HCQ-induced autophagy blockade. Taken together, our data show that HCQ-induced apoptotic cell death in Ara-C-resistant AML cells through autophagy regulation. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  12. Regulation of cell death and cell survival gene expression during ovarian follicular development and atresia.

    PubMed

    Jiang, Jin-Yi; Cheung, Carmen K M; Wang, Yifang; Tsang, Benjamin K

    2003-01-01

    Mammalian ovarian follicular development and atresia is closely regulated by the cross talk of cell death and cell survival signals, which include endocrine hormones (gonadotropins) and intra-ovarian regulators (gonadal steroids, cytokines and growth factors). The fate of the follicle is dependent on a delicate balance in the expression and actions of factors promoting follicular cell proliferation, growth and differentiation and of those inducing programmed cell death (apoptosis). As an important endocrine hormone, FSH binds to its granulosa cell receptors and promotes ovarian follicle survival and growth not only by stimulating proliferation and estradiol secretion of these cells, but also inhibiting the apoptosis by up-regulating the expression of intracellular anti-apoptotic proteins, such as XIAP and FLIP. In addition, intra-ovarian regulators, such as TGF-alpha and TNF-alpha, also play an important role in the control of follicular development and atresia. In response to FSH, Estradiol-17 beta synthesized from the granulosa cells stimulates thecal expression of TGF-alpha, which in turn increases granulosa cell XIAP expression and proliferation. The death receptor and ligand, Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal, culminating in caspase-mediated apoptosis and follicular atresia. In contrast, TNF-alpha has both survival and pro-apoptotic function in the follicle, depending on the receptor subtype activated, but has been shown to promote granulosa cell survival by increasing XIAP and FLIP expression via the IkappaB-NFkappaB pathway. The pro-apoptotic action of TNF-alpha is mediated through the activation of caspases, via its receptor- (i.e. Caspases-8 and -3) and mitochrondria- (i.e. Caspase-9 and -3) death pathways. In the present manuscript, we have reviewed the actions and interactions of gonadotropins and intra-ovarian regulators in the control of granulosa cell fate and ultimately follicular destiny. We have

  13. Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

    PubMed

    Sakao, Masayuki; Sakaguchi, Yutaku; Suzuki, Ryo; Takahashi, Yu; Kishimoto, Masanobu; Fukui, Toshiro; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

    The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

  14. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells.

    PubMed

    Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

    2017-01-01

    Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3'-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson's disease.

  15. MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells

    PubMed Central

    Ito, Keisuke; Eguchi, Yutaka; Imagawa, Yusuke; Akai, Shuji; Mochizuki, Hideki; Tsujimoto, Yoshihide

    2017-01-01

    Regulation of cell death is potentially a powerful treatment modality for intractable diseases such as neurodegenerative diseases. Although there have been many reports about the possible involvement of various types of cell death in neurodegenerative diseases, it is still unclear exactly how neurons die in patients with these diseases, thus treatment strategies based on cell death regulation have not been established yet. To obtain some insight into the mechanisms of cell death involved in neurodegenerative diseases, we studied the effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line SH-SY5Y (a widely used model of Parkinson’s disease). We found that MPP+ predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a necroptosis inhibitor, and by an indole-containing compound (3,3′-diindolylmethane: DIM). However, it occurred independently of receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating that this form of cell death was not necroptosis. MPP+-induced cell death was also inhibited by several inhibitors of ferroptosis, including ferrostatin-1 (Fer-1). Although MPP+-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death seemed to be distinct from ferroptosis because MPP+-induced death (but not ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied by ATP depletion and mitochondrial swelling. Further investigation of MPP+-induced non-apoptotic cell death may be useful for understanding the mechanisms of neuronal loss and for treatment of neurodegenerative diseases such as Parkinson’s disease. PMID:28250973

  16. Bcl-2 proteins and autophagy regulate mitochondrial dynamics during programmed cell death in the Drosophila ovary.

    PubMed

    Tanner, Elizabeth A; Blute, Todd A; Brachmann, Carrie Baker; McCall, Kimberly

    2011-01-01

    The Bcl-2 family has been shown to regulate mitochondrial dynamics during cell death in mammals and C. elegans, but evidence for this in Drosophila has been elusive. Here, we investigate the regulation of mitochondrial dynamics during germline cell death in the Drosophila melanogaster ovary. We find that mitochondria undergo a series of events during the progression of cell death, with remodeling, cluster formation and uptake of clusters by somatic follicle cells. These mitochondrial dynamics are dependent on caspases, the Bcl-2 family, the mitochondrial fission and fusion machinery, and the autophagy machinery. Furthermore, Bcl-2 family mutants show a striking defect in cell death in the ovary. These data indicate that a mitochondrial pathway is a major mechanism for activation of cell death in Drosophila oogenesis.

  17. Effect of vitamin E on 24(S)-hydroxycholesterol-induced necroptosis-like cell death and apoptosis.

    PubMed

    Nakazawa, Takaya; Miyanoki, Yuta; Urano, Yasuomi; Uehara, Madoka; Saito, Yoshiro; Noguchi, Noriko

    2017-05-01

    24(S)-Hydroxycholesterol (24S-OHC) has diverse physiological and pathological functions. In particular, cytotoxic effects of 24S-OHC in neuronal cells are important in development of neurodegenerative diseases. 24S-OHC induces necroptosis-like cell death in SH-SY5Y cells expressing little caspase-8. In the present study, 24S-OHC was found to induce apoptosis as determined by caspase-3 activation in all-trans-retinoic acid (atRA)-treated SH-SY5Y cells in which expression of caspase-8 was induced. 24S-OHC-induced cell death was inhibited by α-tocopherol (α-Toc) but not by α-tocotrienol (α-Toc3) in SH-SY5Y cells regardless of whether cells were treated with atRA. In contrast, cumene hydroperoxide (CumOOH)-induced cell death was significantly inhibited by α-Toc and α-Toc3. In atRA-treated SH-SY5Y cells, generation of reactive oxygen species (ROS) was induced by stimulation with CumOOH but was not induced by stimulation with 24S-OHC. These results suggest that inhibition of 24S-OHC-induced cell death by α-Toc cannot be explained by its radical scavenging antioxidant activity. Esterification of 24S-OHC followed by lipid droplet (LD) formation due to acyl-CoA:cholesterol acyltransferase 1 (ACAT1) are key events in 24S-OHC-induced cell death in atRA-treated SH-SY5Y cells as demonstrated by inhibition of cell death by ACAT1 inhibitor. LD number was not changed by treatment with either α-Toc or α-Toc3. The different physical properties of α-Toc and α-Toc3 may account for their different inhibitory effects on 24S-OHC-induced cell death. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Cell Death Pathways in Mutant Rhodopsin Rat Models Identifies Genotype-Specific Targets Controlling Retinal Degeneration.

    PubMed

    Viringipurampeer, Ishaq A; Gregory-Evans, Cheryl Y; Metcalfe, Andrew L; Bashar, Emran; Moritz, Orson L; Gregory-Evans, Kevin

    2018-06-18

    Retinitis pigmentosa (RP) is a group of inherited neurological disorders characterized by rod photoreceptor cell death, followed by secondary cone cell death leading to progressive blindness. Currently, there are no viable treatment options for RP. Due to incomplete knowledge of the molecular signaling pathways associated with RP pathogenesis, designing therapeutic strategies remains a challenge. In particular, preventing secondary cone photoreceptor cell loss is a key goal in designing potential therapies. In this study, we identified the main drivers of rod cell death and secondary cone loss in the transgenic S334ter rhodopsin rat model, tested the efficacy of specific cell death inhibitors on retinal function, and compared the effect of combining drugs to target multiple pathways in the S334ter and P23H rhodopsin rat models. The primary driver of early rod cell death in the S334ter model was a caspase-dependent process, whereas cone cell death occurred though RIP3-dependent necroptosis. In comparison, rod cell death in the P23H model was via necroptotic signaling, whereas cone cell loss occurred through inflammasome activation. Combination therapy of four drugs worked better than the individual drugs in the P23H model but not in the S334ter model. These differences imply that treatment modalities need to be tailored for each genotype. Taken together, our data demonstrate that rationally designed genotype-specific drug combinations will be an important requisite to effectively target primary rod cell loss and more importantly secondary cone survival.

  19. Improving Accuracy in Arrhenius Models of Cell Death: Adding a Temperature-Dependent Time Delay.

    PubMed

    Pearce, John A

    2015-12-01

    The Arrhenius formulation for single-step irreversible unimolecular reactions has been used for many decades to describe the thermal damage and cell death processes. Arrhenius predictions are acceptably accurate for structural proteins, for some cell death assays, and for cell death at higher temperatures in most cell lines, above about 55 °C. However, in many cases--and particularly at hyperthermic temperatures, between about 43 and 55 °C--the particular intrinsic cell death or damage process under study exhibits a significant "shoulder" region that constant-rate Arrhenius models are unable to represent with acceptable accuracy. The primary limitation is that Arrhenius calculations always overestimate the cell death fraction, which leads to severely overoptimistic predictions of heating effectiveness in tumor treatment. Several more sophisticated mathematical model approaches have been suggested and show much-improved performance. But simpler models that have adequate accuracy would provide useful and practical alternatives to intricate biochemical analyses. Typical transient intrinsic cell death processes at hyperthermic temperatures consist of a slowly developing shoulder region followed by an essentially constant-rate region. The shoulder regions have been demonstrated to arise chiefly from complex functional protein signaling cascades that generate delays in the onset of the constant-rate region, but may involve heat shock protein activity as well. This paper shows that acceptably accurate and much-improved predictions in the simpler Arrhenius models can be obtained by adding a temperature-dependent time delay. Kinetic coefficients and the appropriate time delay are obtained from the constant-rate regions of the measured survival curves. The resulting predictions are seen to provide acceptably accurate results while not overestimating cell death. The method can be relatively easily incorporated into numerical models. Additionally, evidence is presented

  20. Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

    PubMed Central

    Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

    2016-01-01

    Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

  1. Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: A protective role

    PubMed Central

    Cheng, Yu-Chi; Chang, Jer-Ming; Chen, Chien-An

    2015-01-01

    Endoplasmic reticulum stress occurs in a variety of patho-physiological mechanisms and there has been great interest in managing this pathway for the treatment of clinical diseases. Autophagy is closely interconnected with endoplasmic reticulum stress to counteract the possible injurious effects related with the impairment of protein folding. Studies have shown that glomerular podocytes exhibit high rate of autophagy to maintain as terminally differentiated cells. In this study, podocytes were exposed to tunicamycin and thapsigargin to induce endoplasmic reticulum stress. Thapsigargin/tunicamycin treatment induced a significant increase in endoplasmic reticulum stress and of cell death, represented by higher GADD153 and GRP78 expression and propidium iodide flow cytometry, respectively. However, thapsigargin/tunicamycin stimulation also enhanced autophagy development, demonstrated by monodansylcadaverine assay and LC3 conversion. To evaluate the regulatory effects of autophagy on endoplasmic reticulum stress-induced cell death, rapamycin (Rap) or 3-methyladenine (3-MA) was added to enhance or inhibit autophagosome formation. Endoplasmic reticulum stress-induced cell death was decreased at 6 h, but was not reduced at 24 h after Rap+TG or Rap+TM treatment. In contrast, endoplasmic reticulum stress-induced cell death increased at 6 and 24 h after 3-MA+TG or 3-MA+TM treatment. Our study demonstrated that thapsigargin/tunicamycin treatment induced endoplasmic reticulum stress which resulted in podocytes death. Autophagy, which counteracted the induced endoplasmic reticulum stress, was simultaneously enhanced. The salvational role of autophagy was supported by adding Rap/3-MA to mechanistically regulate the expression of autophagy and autophagosome formation. In summary, autophagy helps the podocytes from cell death and may contribute to sustain the longevity as a highly differentiated cell lineage. PMID:25322957

  2. Yeast Genetics for Delineating Bax/Bcl Pathway of Cell Death Regulation.

    DTIC Science & Technology

    1998-07-01

    differences in tosol. The cytosol also became electron dense ("cyto- the copy number of the episomal plasmid from which solic condensation"), similar to...Cell Death & Differ . 3, 229-236. (1993). The C. eheans cell death gene ccd-3 encodes a protein similar ¶Xhitc. K., Tahaoglu, E., and Steller, H. (1996...components may be used in different functional contexts. Similar modules might exist in metazoan apoptotic pathways. Even though yeast does not contain

  3. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    PubMed

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  4. Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

    PubMed Central

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

  5. Effect of blue light radiation on curcumin-induced cell death of breast cancer cells

    NASA Astrophysics Data System (ADS)

    Zeng, X. B.; Leung, A. W. N.; Xia, X. S.; Yu, H. P.; Bai, D. Q.; Xiang, J. Y.; Jiang, Y.; Xu, C. S.

    2010-06-01

    In the present study, we have successfully set up a novel blue light source with the power density of 9 mW/cm2 and the wavelength of 435.8 nm and then the novel light source was used to investigate the effect of light radiation on curcumin-induced cell death. The cytotoxicity was investigated 24 h after the treatment of curcumin and blue light radiation together using MTT reduction assay. Nuclear chromatin was observed using a fluorescent microscopy with Hoechst33258 staining. The results showed blue light radiation could significantly enhance the cytotoxicity of curcumin on the MCF-7 cells and apoptosis induction. These findings demonstrated that blue light radiation could enhance curcumin-induced cell death of breast cancer cells, suggesting light radiation may be an efficient enhancer of curcumin in the management of breast cancer.

  6. Fluoxetine Prevents Oligodendrocyte Cell Death by Inhibiting Microglia Activation after Spinal Cord Injury

    PubMed Central

    Lee, Jee Y.; Kang, So R.

    2015-01-01

    Abstract Oligodendrocyte cell death and axon demyelination after spinal cord injury (SCI) are known to be important secondary injuries contributing to permanent neurological disability. Thus, blocking oligodendrocyte cell death should be considered for therapeutic intervention after SCI. Here, we demonstrated that fluoxetine, an antidepressant drug, alleviates oligodendrocyte cell death by inhibiting microglia activation after SCI. After injury at the T9 level with a Precision Systems and Instrumentation (Lexington, KY) device, fluoxetine (10 mg/kg, intraperitoneal) was administered once a day for the indicated time points. Immunostaining with CD11b (OX-42) antibody and quantification analysis showed that microglia activation was significantly inhibited by fluoxetine at 5 days after injury. Fluoxetine also significantly inhibited activation of p38 mitogen-activated protein kinase (p38-MAPK) and expression of pro-nerve growth factor (pro-NGF), which is known to mediate oligodendrocyte cell death through the p75 neurotrophin receptor after SCI. In addition, fluoxetine attenuated activation of Ras homolog gene family member A and decreased the level of phosphorylated c-Jun and, ultimately, alleviated caspase-3 activation and significantly reduced cell death of oligodendrocytes at 5 days after SCI. Further, the decrease of myelin basic protein, myelin loss, and axon loss in white matter was also significantly blocked by fluoxetine, as compared to vehicle control. These results suggest that fluoxetine inhibits oligodendrocyte cell death by inhibiting microglia activation and p38-MAPK activation, followed by pro-NGF production after SCI, and provide a potential usage of fluoxetine for a therapeutic agent after acute SCI in humans. PMID:25366938

  7. HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53.

    PubMed

    Hallgren, O; Gustafsson, L; Irjala, H; Selivanova, G; Orrenius, S; Svanborg, C

    2006-02-01

    HAMLET (Human alpha-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.

  8. Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

    PubMed

    de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

    2010-12-21

    The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

  9. Vitamin K3 triggers human leukemia cell death through hydrogen peroxide generation and histone hyperacetylation.

    PubMed

    Lin, Changjun; Kang, Jiuhong; Zheng, Rongliang

    2005-10-01

    Vitamin K3 (VK3) is a well-known anticancer agent, but its mechanism remains elusive. In the present study, VK3 was found to simultaneously induce cell death, reactive oxygen species (ROS) generation, including superoxide anion (O2*-) and hydrogen peroxide (H2O2) generation, and histone hyperacetylation in human leukemia HL-60 cells in a concentration- and time-dependent manner. Catalase (CAT), an antioxidant enzyme that specifically scavenges H2O2, could significantly diminish both histone acetylation increase and cell death caused by VK3, whereas superoxide dismutase (SOD), an enzyme that specifically eliminates O2*-, showed no effect on both of these, leading to the conclusion that H2O2 generation, but not O2*- generation, contributes to VK3-induced histone hyperacetylation and cell death. This conclusion was confirmed by the finding that enhancement of VK3-induced H2O2 generation by vitamin C (VC) could significantly promote both the histone hyperacetylation and cell death. Further studies suggested that histone hyperacetylation played an important role in VK3-induced cell death, since sodium butyrate, a histone deacetylase (HDAC) inhibitor, showed no effect on ROS generation, but obviously potentiated VK3-induced histone hyperacetylation and cell death. Collectively, these results demonstrate a novel mechanism for the anticancer activity of VK3, i.e., VK3 induced tumor cell death through H2O2 generation, which then further induced histone hyperacetylation.

  10. An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death.

    PubMed

    Garcia-Belinchón, Mercè; Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Casanelles, Elisenda; Ribas, Judit; Yuste, Victor J

    2015-08-21

    Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death. © 2015 by The American Society for

  11. An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death*

    PubMed Central

    Garcia-Belinchón, Mercè; Sánchez-Osuna, María; Martínez-Escardó, Laura; Granados-Colomina, Carla; Pascual-Guiral, Sònia; Iglesias-Guimarais, Victoria; Casanelles, Elisenda; Ribas, Judit; Yuste, Victor J.

    2015-01-01

    Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as “apoptosis-necrosis continuum.” To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death. PMID:26124276

  12. Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

    PubMed Central

    Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

    2009-01-01

    Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development. PMID:19279211

  13. Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

    PubMed Central

    de Koning, Pieter J. A.; Kummer, J. Alain; de Poot, Stefanie A. H.; Quadir, Razi; Broekhuizen, Roel; McGettrick, Anne F.; Higgins, Wayne J.; Devreese, Bart; Worrall, D. Margaret; Bovenschen, Niels

    2011-01-01

    Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death. PMID:21857942

  14. Ultrastructural aspects of autoschizis: a new cancer cell death induced by the synergistic action of ascorbate/menadione on human bladder carcinoma cells.

    PubMed

    Gilloteaux, J; Jamison, J M; Arnold, D; Taper, H S; Summers, J L

    2001-01-01

    Scanning and transmission electron microscopy were employed to further characterize the cytotoxic effects of a ascorbic acid/menadione (or vitamin C/vitamin K3) combination on a human bladder carcinoma T24 cell line. Following 1-h treatment T24 cells display membrane and mitochondrial defects as well as excision of cytoplasmic fragments that contain no organelles. These continuous self-excisions reduce the cell size. Concomitant, nuclear changes, chromatin disassembly, nucleolar condensation and fragmentation, and decreased nuclear volume lead to cell death via a process similar to karyorrhexis and karyolysis. Because this cell death is achieved through a progressive loss of cytoplasm due to self-morsellation, the authors named this mode of cell death autoschizis (from the Greek autos, self, and schizein, to split, as defined in Scanning. 1998; 20: 564-575). This morphological characterization of autoschizic cell death confirms and extends the authors previous reports and demonstrates that this cell death is distinct from apoptosis.

  15. Hop/STI1 modulates retinal proliferation and cell death independent of PrP{sup C}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Arruda-Carvalho, Maithe; Njaine, Brian; Silveira, Mariana S.

    Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP{sup C}). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP{sup C} dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 ({alpha}-STI1) blocked both ganglion cell and NBL cell deathmore » independent of PrP{sup C}. cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while {alpha}-STI1 increased proliferation in the developing retina, both independent of PrP{sup C}. We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP{sup C}.« less

  16. Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures.

    PubMed

    Koehl, Julia; Djulic, Alma; Kirner, Veronika; Nguyen, Tach Thao; Heiser, Ingrid

    2007-12-01

    The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture. At higher quercinin concentrations (2 nM), H(2)O(2) formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H(2)O(2) production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as alpha-amino-oxy-acetic acid (AOA) and CoCl(2) also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.

  17. Role of Hsp-70 in triptolide-mediated cell death of neuroblastoma.

    PubMed

    Antonoff, Mara B; Chugh, Rohit; Skube, Steven J; Dudeja, Vikas; Borja-Cacho, Daniel; Clawson, Kimberly A; Vickers, Selwyn M; Saluja, Ashok K

    2010-09-01

    Our recent work demonstrated that treatment of neuroblastoma with triptolide causes apoptotic cell death in vitro and decreases tumor size in vivo. Triptolide therapy has been associated with reduced expression of Hsp-70, suggesting a mechanism of cell killing involving Hsp-70 inhibition. The principal objective of this study was to investigate the role of Hsp-70 in triptolide-mediated cell death in neuroblastoma. Neuroblastoma cells were transfected with Hsp-70-specific siRNA. Viability, caspase activity, and phosphatidylserine externalization were subsequently measured. An orthotopic, syngeneic murine tumor model was developed, and randomized mice received daily injections of triptolide or vehicle. At 21 d, mice were sacrificed. Immunohistochemisty was used to characterize Hsp-70 levels in residual tumors, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to identify cells undergoing apoptosis. Targeted silencing of Hsp-70 with siRNA significantly decreased cellular viability, augmented caspase-3 activity, and resulted in increased annexin-V staining. These effects parallel those findings obtained following treatment with triptolide. Residual tumors from triptolide-treated mice showed minimal staining with Hsp-70 immunohistochemistry, while control tumors stained prominently. Tumors from treated mice demonstrated marked staining with the TUNEL assay, while control tumors showed no evidence of apoptosis. Use of siRNA to suppress Hsp-70 expression in neuroblastoma resulted in apoptotic cell death, similar to the effects of triptolide. Residual tumors from triptolide-treated mice expressed decreased levels of Hsp-70 and demonstrated significant apoptosis. These findings support the hypothesis that Hsp-70 inhibition plays a significant role in triptolide-mediated neuroblastoma cell death. Copyright 2010 Elsevier Inc. All rights reserved.

  18. E4F1 deficiency results in oxidative stress–mediated cell death of leukemic cells

    PubMed Central

    Hatchi, Elodie; Rodier, Genevieve; Lacroix, Matthieu; Caramel, Julie; Kirsh, Olivier; Jacquet, Chantal; Schrepfer, Emilie; Lagarrigue, Sylviane; Linares, Laetitia Karine; Lledo, Gwendaline; Tondeur, Sylvie; Dubus, Pierre

    2011-01-01

    The multifunctional E4F1 protein was originally discovered as a target of the E1A viral oncoprotein. Growing evidence indicates that E4F1 is involved in key signaling pathways commonly deregulated during cell transformation. In this study, we investigate the influence of E4F1 on tumorigenesis. Wild-type mice injected with fetal liver cells from mice lacking CDKN2A, the gene encoding Ink4a/Arf, developed histiocytic sarcomas (HSs), a tumor originating from the monocytic/macrophagic lineage. Cre-mediated deletion of E4F1 resulted in the death of HS cells and tumor regression in vivo and extended the lifespan of recipient animals. In murine and human HS cell lines, E4F1 inactivation resulted in mitochondrial defects and increased production of reactive oxygen species (ROS) that triggered massive cell death. Notably, these defects of E4F1 depletion were observed in HS cells but not healthy primary macrophages. Short hairpin RNA–mediated depletion of E4F1 induced mitochondrial defects and ROS-mediated death in several human myeloid leukemia cell lines. E4F1 protein is overexpressed in a large subset of human acute myeloid leukemia samples. Together, these data reveal a role for E4F1 in the survival of myeloid leukemic cells and support the notion that targeting E4F1 activities might have therapeutic interest. PMID:21708927

  19. Sulfated lentinan induced mitochondrial dysfunction leads to programmed cell death of tobacco BY-2 cells.

    PubMed

    Wang, Jie; Wang, Yaofeng; Shen, Lili; Qian, Yumei; Yang, Jinguang; Wang, Fenglong

    2017-04-01

    Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H 2 O 2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H 2 DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD. Copyright © 2016. Published by Elsevier Inc.

  20. Hedgehog inhibition promotes a switch from Type II to Type I cell death receptor signaling in cancer cells.

    PubMed

    Kurita, Satoshi; Mott, Justin L; Cazanave, Sophie C; Fingas, Christian D; Guicciardi, Maria E; Bronk, Steve F; Roberts, Lewis R; Fernandez-Zapico, Martin E; Gores, Gregory J

    2011-03-31

    TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.