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Sample records for cell lines mitochondria

  1. Peroxide effects of iodide excess on mitochondria in Fischer rat thyroid cell line in the early period

    Institute of Scientific and Technical Information of China (English)

    李敏

    2014-01-01

    Objective To investigate the peroxide effects of iodide excess on mitochondria in Fischer rat thyroid cell line(FRTL)in the early period.Methods After treatment with 0.0 mmol/L(control group)or 0.1 mmol/L potassium iodide(KI)for 2,4 and 24 h,respectively,changes of mitochondrial superoxide formation were assayed by flow cytometry and fluorescence microscopy using mitochondria-targeted hydroethidine(Mito SOX).

  2. 3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines

    International Nuclear Information System (INIS)

    Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells. (author)

  3. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    International Nuclear Information System (INIS)

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  4. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Akinori, E-mail: morita@tokushima-u.ac.jp [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8509 (Japan); Tanimoto, Keiji; Murakami, Tomoki; Morinaga, Takeshi [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Hosoi, Yoshio, E-mail: hosoi@med.tohoku.ac.jp [Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Department of Radiation Biology, Graduate School of Medicine, Tohoku University, Sendai 980-8575 (Japan)

    2014-01-24

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  5. Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

    Directory of Open Access Journals (Sweden)

    Yaíma L Lightfoot

    Full Text Available Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA depleted βLox5 cells, or βLox5 ρ(0 cells. βLox5 ρ(0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

  6. Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544

    International Nuclear Information System (INIS)

    Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21Waf1/Cip1, a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21Waf1/Cip1 paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21Waf1/Cip1 induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria

  7. Stem cell mitochondria during aging.

    Science.gov (United States)

    Min-Wen, Jason Chua; Jun-Hao, Elwin Tan; Shyh-Chang, Ng

    2016-04-01

    Mitochondria are the central hubs of cellular metabolism, equipped with their own mitochondrial DNA (mtDNA) blueprints to direct part of the programming of mitochondrial oxidative metabolism and thus reactive oxygen species (ROS) levels. In stem cells, many stem cell factors governing the intricate balance between self-renewal and differentiation have been found to directly regulate mitochondrial processes to control stem cell behaviors during tissue regeneration and aging. Moreover, numerous nutrient-sensitive signaling pathways controlling organismal longevity in an evolutionarily conserved fashion also influence stem cell-mediated tissue homeostasis during aging via regulation of stem cell mitochondria. At the genomic level, it has been demonstrated that heritable mtDNA mutations and variants affect mammalian stem cell homeostasis and influence the risk for human degenerative diseases during aging. Because such a multitude of stem cell factors and signaling pathways ultimately converge on the mitochondria as the primary mechanism to modulate cellular and organismal longevity, it would be most efficacious to develop technologies to therapeutically target and direct mitochondrial repair in stem cells, as a unified strategy to combat aging-related degenerative diseases in the future. PMID:26851627

  8. Mitochondria: pharmacological manipulation of cell death

    OpenAIRE

    Bouchier-Hayes, Lisa; Lartigue, Lydia; Newmeyer, Donald D.

    2005-01-01

    Cell death by apoptosis or necrosis is often important in the etiology and treatment of disease. Since mitochondria play important roles in cell death pathways, these organelles are potentially prime targets for therapeutic intervention. Here we discuss the mechanisms through which mitochondria participate in the cell death process and also survey some of the pharmacological approaches that target mitochondria in various ways.

  9. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

    Science.gov (United States)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. PMID:27163639

  10. Cell-to-cell movement of mitochondria in plants

    OpenAIRE

    Gurdon, Csanad; Svab, Zora; Feng, Yaping; Kumar, Dibyendu; Maliga, Pal

    2016-01-01

    We report cell-to-cell movement of mitochondria through a graft junction of two tobacco species, Nicotiana tabacum and Nicotiana sylvestris. The flowers of the N. tabacum line we used are male sterile due to a sterility-causing mitochondrial genome, whereas the N. sylvestris flowers are fertile. Grafting created an opportunity for organelle movement during the healing process when cell-to-cell connections at the graft junction were restored. We recognized N. sylvestris mitochondrial DNA trans...

  11. Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation.

    Directory of Open Access Journals (Sweden)

    Liliana Milani

    Full Text Available BACKGROUND: Doubly Uniparental Inheritance (DUI of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI. DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown

  12. Mitochondria, cholesterol and cancer cell metabolism.

    Science.gov (United States)

    Ribas, Vicent; García-Ruiz, Carmen; Fernández-Checa, José C

    2016-12-01

    Given the role of mitochondria in oxygen consumption, metabolism and cell death regulation, alterations in mitochondrial function or dysregulation of cell death pathways contribute to the genesis and progression of cancer. Cancer cells exhibit an array of metabolic transformations induced by mutations leading to gain-of-function of oncogenes and loss-of-function of tumor suppressor genes that include increased glucose consumption, reduced mitochondrial respiration, increased reactive oxygen species generation and cell death resistance, all of which ensure cancer progression. Cholesterol metabolism is disturbed in cancer cells and supports uncontrolled cell growth. In particular, the accumulation of cholesterol in mitochondria emerges as a molecular component that orchestrates some of these metabolic alterations in cancer cells by impairing mitochondrial function. As a consequence, mitochondrial cholesterol loading in cancer cells may contribute, in part, to the Warburg effect stimulating aerobic glycolysis to meet the energetic demand of proliferating cells, while protecting cancer cells against mitochondrial apoptosis due to changes in mitochondrial membrane dynamics. Further understanding the complexity in the metabolic alterations of cancer cells, mediated largely through alterations in mitochondrial function, may pave the way to identify more efficient strategies for cancer treatment involving the use of small molecules targeting mitochondria, cholesterol homeostasis/trafficking and specific metabolic pathways. PMID:27455839

  13. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

    OpenAIRE

    Tanya C. Burch; Rhim, Johng S.; Julius O Nyalwidhe

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mit...

  14. MITOCHONDRIA: INSIGHT TARGET OF DRUG DEVELOPMENT IN CANCER CELLS

    OpenAIRE

    Md. Ataur Rahman

    2012-01-01

    Mitochondria are involved in different physiological and pathological processes that are crucial for tumor cell physiology, growth and survival and its dysfunction leads to many human abnormalities, including cardiovascular diseases, neurodegenerative diseases, autoimmune disorders and cancer. The present review is focused on the different experimental and therapeutic cancer strategies addressed to either target mitochondria directly, or use mitochondria as mediators of apoptosis, although it...

  15. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

    Science.gov (United States)

    Schatten, H.; Lewis, M. L.; Chakrabarti, A.

    2001-01-01

    The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985. c 2001. Elsevier Science Ltd. All rights reserved.

  16. Mitochondria and Energetic Depression in Cell Pathophysiology

    Directory of Open Access Journals (Sweden)

    Stephan Zierz

    2009-05-01

    Full Text Available Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED, which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca2+ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS, mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD. However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis.

  17. Induction of apoptosis in human cancer cells by targeting mitochondria with gold nanoparticles.

    Science.gov (United States)

    Mkandawire, M M; Lakatos, M; Springer, A; Clemens, A; Appelhans, D; Krause-Buchholz, U; Pompe, W; Rödel, G; Mkandawire, M

    2015-06-28

    A major challenge in designing cancer therapies is the induction of cancer cell apoptosis, although activation of intrinsic apoptotic pathways by targeting gold nanoparticles to mitochondria is promising. We report an in vitro procedure targeting mitochondria with conjugated gold nanoparticles and investigating effects on apoptosis induction in the human breast cancer cell line Jimt-1. Gold nanoparticles were conjugated to a variant of turbo green fluorescent protein (mitoTGFP) harbouring an amino-terminal mitochondrial localization signal. Au nanoparticle conjugates were further complexed with cationic maltotriose-modified poly(propylene imine) third generation dendrimers. Fluorescence and transmission electron microscopy revealed that Au nanoparticle conjugates were directed to mitochondria upon transfection, causing partial rupture of the outer mitochondrial membrane, triggering cell death. The ability to target Au nanoparticles into mitochondria of breast cancer cells and induce apoptosis reveals an alternative application of Au nanoparticles in photothermal therapy of cancer. PMID:26022234

  18. Cell-to-cell movement of mitochondria in plants.

    Science.gov (United States)

    Gurdon, Csanad; Svab, Zora; Feng, Yaping; Kumar, Dibyendu; Maliga, Pal

    2016-03-22

    We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer fromNicotiana sylvestrisintoNicotiana tabacumcells. The alloplasmicN. tabacumline we used carriesNicotiana undulatacytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer fromN. sylvestristoN. tabacumcould be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility toorf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation. PMID:26951647

  19. Mitochondria in human pluripotent stem cell apoptosis.

    Science.gov (United States)

    TeSlaa, Tara; Setoguchi, Kiyoko; Teitell, Michael A

    2016-04-01

    Human pluripotent stem cells (hPSCs) have great potential in regenerative medicine because they can differentiate into any cell type in the body. Genome integrity is vital for human development and for high fidelity passage of genetic information across generations through the germ line. To ensure genome stability, hPSCs maintain a lower rate of mutation than somatic cells and undergo rapid apoptosis in response to DNA damage and additional cell stresses. Furthermore, cellular metabolism and the cell cycle are also differentially regulated between cells in pluripotent and differentiated states and can aid in protecting hPSCs against DNA damage and damaged cell propagation. Despite these safeguards, clinical use of hPSC derivatives could be compromised by tumorigenic potential and possible malignant transformation from failed to differentiate cells. Since hPSCs and mature cells differentially respond to cell stress, it may be possible to specifically target undifferentiated cells for rapid apoptosis in mixed cell populations to enable safer use of hPSC-differentiated cells in patients. PMID:26828436

  20. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes.

    Science.gov (United States)

    Burch, Tanya C; Rhim, Johng S; Nyalwidhe, Julius O

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT(2) PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  1. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

    Directory of Open Access Journals (Sweden)

    Tanya C. Burch

    2016-01-01

    Full Text Available Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells and RC77T/E (malignant cells were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions.

  2. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    Science.gov (United States)

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  3. Morphological and functional changes of mitochondria in apoptotic esophageal carcinoma cells induced by arsenic trioxide

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Jian Shen; Qiao-Shan Li; Cai-Yun Chen; Jiong-Yu Chen; Yi Zeng

    2002-01-01

    AIM: To demonstrate that mitochondrial morphological andfunctional changes are an important intermediate link in thecourse of apoptosis in esophageal carcinoma cells inducedby As2O3.METHODS: The esophageal carcinoma cell line SHEEC1,established in our laboratory, was cultured in 199 growthmedium, supplemented with 100mL@ L-1 calf serum and3 mol@L-1 As2O3( the same below). After 2, 4, 6, 12, 24 hof drug adding, the SHEEC1 cells were collected for light-and electron-microscopic examination. The mitochondriawere labeled by Rhodamine fluorescence probe and thefluorescence intensity of the mitochondria was measured byflow cytometer and cytofluorimetric analysis. Further, themitochondrial transmembrane potential ( MTP, ΔΨm )change was also calculated.RESULTS: The mitochondrial morphological change afteradding As2O3 could be divided into three stages. In theearly-stage (2-6h) after adding As2O3, an adaptiveproliferation of mitochondria appeared; in the mid-stage (6-12 h ) e degenerative change was observed; and in the late-stage (12-24 h ) the mitochondria swelled with outermembrana broken down and then calls death with apoptoticchanges of nucleus. The functional change of themitochondria indicated by fluorescent intensity, whichreflected the MTP status of mitochondria, was in accordancewith morphological change of the mitochondria. Thefluorescent intensity increased at early-stage, declined inmid-stage and decreased to the lowest in the late@ stage. 24h after As2O3 adding, the cell nucleus showed typicalapoptotic changes.CONCLUSION: Under the inducement of As2O3, the earlyapoptotic changes of SHEEC1 cells were the apparentmorphological and functional changes of mitochondria,afterwards the nucleus changes followed. lt is consideredthat changes of mitochondria are an important intermediatelink in the course of apoptosis of esophageal carcinomacalls induced by As2O3.

  4. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    International Nuclear Information System (INIS)

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies

  5. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell.

    Science.gov (United States)

    Jianping, Du

    2010-01-01

    Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compared to chromosome genomes, the mitochondria genomes were not restricted by introns so they were mutated(fall back) easy. The result is that mitochondria lose function and internal environment of somatic cell become acid and evoked chromosome genomes to mutate, in the end somatic cells become cancer cells. It is the trigger of somatic cell transforming to cancer cell that mitochondria genome happen mutation and lose function. PMID:20181100

  6. Mitochondria: An intriguing target for killing tumour-initiating cells

    Czech Academy of Sciences Publication Activity Database

    Yan, B.; Dong, L.; Neužil, Jiří

    2016-01-01

    Roč. 26, JAN 2016 (2016), s. 86-93. ISSN 1567-7249 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : Tumour-initiating cells * ALPHA-TOCOPHERYL SUCCINATE * Therapeutic resistance * Mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.249, year: 2014

  7. Bax translocation into mitochondria during dihydroartemisinin(DHA)-induced apoptosis in human lung adenocarcinoma cells

    Science.gov (United States)

    Lu, Ying-ying; Chen, Tong-sheng; Qu, Jun-Le

    2009-02-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. This study was investigated in human lung adenocarconoma ASTC-a-1 cell line and aimed to determine whether the apoptotic process was mediated by Bax activation and translocation during DHA-induced apoptosis. In this study, DHA induced a time-dependent apoptotic cell death, which was assayed by Cell Counting Kit (CCK-8) and Hoechst 33258 staining. Detection of Bax aggregation and translocation to mitochondria was observed in living cells which were co-transfected with GFP-Bax and Dsred-mito plasmid using confocal fluorescence microscope technique. Overall, these results demonstrated that Bax activation and translocation to mitochondria occurred during DHA-induced apoptosis.

  8. Mutation of mitochondria genome: trigger of somatic cell transforming to cancer cell

    OpenAIRE

    Jianping Du

    2010-01-01

    Abstract Nearly 80 years ago, scientist Otto Warburg originated a hypothesis that the cause of cancer is primarily a defect in energy metabolism. Following studies showed that mitochondria impact carcinogenesis to remodel somatic cells to cancer cells through modifying the genome, through maintenance the tumorigenic phenotype, and through apoptosis. And the Endosymbiotic Theory explains the origin of mitochondria and eukaryotes, on the other hands, the mitochondria also can fall back. Compare...

  9. MITOCHONDRIA: INSIGHT TARGET OF DRUG DEVELOPMENT IN CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Md. Ataur Rahman

    2012-09-01

    Full Text Available Mitochondria are involved in different physiological and pathological processes that are crucial for tumor cell physiology, growth and survival and its dysfunction leads to many human abnormalities, including cardiovascular diseases, neurodegenerative diseases, autoimmune disorders and cancer. The present review is focused on the different experimental and therapeutic cancer strategies addressed to either target mitochondria directly, or use mitochondria as mediators of apoptosis, although its total molecular mechanism has not been elucidated. Therefore, the role of mitochondria in the etiology and progression of several function and explore potential therapeutic benefits of targeting mitochondria in the disease processes. Newly evolving advances in disease diagnostics and therapy will further facilitate future growth in the field of mitochondrian biology, where there is a dire need for sensitive and more affordable diagnostic tools and an urgency to develop effective therapies and identify reliable drug to predict accurately the response to a cancer therapy. These approaches to treat mitochondrial dysfunction rationally could lead to selective protection of cells in different tissues and various disease states. To avoid mitochondrial liabilities, routine screens need to be positioned within the drug-development process as targets of drug-induced cytotoxicity or cancer promotion, as regulators of apoptosis, as sources of cell signalling through reactive oxygen species, and mitochondrial control of specific nuclear responses. However, several novel mitochondrial targets are now emerging, including the potential to manipulate the mitochondrial pool to maintain function via biogenesis and mitophagy. Forthcoming insights into the fine regulation of mitochondrial apoptosis will likely open future perspectives for cancer drug development.

  10. Well-functioning cell mitochondria promote good health

    OpenAIRE

    Chowanadisai, Winyoo; Shenoy, Sonia F; Sharman, Edward; Carl L. Keen; Liu, Jiankang; Rucker, Robert B

    2011-01-01

    Mitochondriol function can be directly linked to protection from certain chronic diseases and conditions, such as heart disease, diabetes, metabolic syndrome and chronic inflammation, as well as the aging processes. Mitochondria are central to normal glucose, amino acid and fatty acid metabolism, in addition to antioxidant modulation and virtually all aspects of cell turnover and maintenance. Nutrition plays an essential role in optimizing such functions. We describe strategies for the regula...

  11. Ion transport by mitochondria-rich cells in toad skin

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Ussing, H H; Spring, K R

    1987-01-01

    The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution......) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions...

  12. Role of mitochondria on muscle cell death and meat tenderization.

    Science.gov (United States)

    Sierra, Verónica; Oliván, Mamen

    2013-05-01

    The possibility that mitochondria are involved in cellular dysfunction is particularly high in situations associated with increases in free radical activity, like hypoxia or ischemia; therefore its potential role in the muscle post-mortem metabolism is reviewed. In the dying muscle, different routes of cell death catabolism (apoptosis, autophagy) may occur having great influence on the process of conversion of muscle into meat. Mitochondria are the first and also one of the main organelles affected by post-mortem changes; therefore they are decisive in the subsequent cellular responses influencing the pathway to cell demise and thus, the final meat quality. Depending on the cell death programme followed by muscle cells after exsanguination, diverse proteases would be activated to a different extent, which is also reviewed in order to understand how they affect meat tenderization. This review also summarizes recent patents relating cell death processes and meat tenderness. Further research is encouraged as there is still a need of knowledge on cell death post-mortem processes to increase our understanding of the conversion of muscle into meat. PMID:23432120

  13. Clozapine-induced mitochondria alterations and inflammation in brain and insulin-responsive cells.

    Directory of Open Access Journals (Sweden)

    Verόnica Contreras-Shannon

    Full Text Available BACKGROUND: Metabolic syndrome (MetS is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined. METHODOLOGY/PRINCIPAL FINDINGS: Cultured mouse myoblasts (C2C12, adipocytes (3T3-L1, hepatocytes (FL-83B, and monocytes (RAW 264.7 were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line. CONCLUSIONS/SIGNIFICANCE: Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated

  14. Mitochondria as therapeutic targets for cancer stem cells

    Institute of Scientific and Technical Information of China (English)

    In Sung Song; Jeong Yu Jeong; Seung Hun Jeong; Hyoung Kyu Kim; Kyung Soo Ko; Byoung Doo Rhee; Nari Kim; Jin Han

    2015-01-01

    Cancer stem cells (CSCs) are maintained by theirsomatic stem cells and are responsible for tumorinitiation, chemoresistance, and metastasis. Evidencefor the CSCs existence has been reported for a numberof human cancers. The CSC mitochondria have beenshown recently to be an important target for cancertreatment, but clinical significance of CSCs and theirmitochondria properties remain unclear. Mitochondriatargetedagents are considerably more effectivecompared to other agents in triggering apoptosis ofCSCs, as well as general cancer cells, via mitochondrialdysfunction. Mitochondrial metabolism is altered incancer cells because of their reliance on glycolyticintermediates, which are normally destined for oxidativephosphorylation. Therefore, inhibiting cancer-specificmodifications in mitochondrial metabolism, increasingreactive oxygen species production, or stimulatingmitochondrial permeabilization transition could bepromising new therapeutic strategies to activate celldeath in CSCs as well, as in general cancer cells. Thisreview analyzed mitochondrial function and its potentialas a therapeutic target to induce cell death in CSCs.Furthermore, combined treatment with mitochondriatargeteddrugs will be a promising strategy for thetreatment of relapsed and refractory cancer.

  15. Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells.

    Directory of Open Access Journals (Sweden)

    Britta Muster

    Full Text Available BACKGROUND: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2-3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10-24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. CONCLUSION/SIGNIFICANCE: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.

  16. Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Benjamin Wiench

    2012-01-01

    Full Text Available Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

  17. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function.

    Science.gov (United States)

    Katrangi, Eyad; D'Souza, Gerard; Boddapati, Sarathi V; Kulawiec, Mariola; Singh, Keshav K; Bigger, Brian; Weissig, Volkmar

    2007-12-01

    Mitochondrial DNA mutations are the direct cause of several physiological disorders and are also associated with the aging process. The modest progress made over the past two decades towards manipulating the mitochondrial genome and understanding its function within living mammalian cells means that cures for mitochondrial DNA mutations are still elusive. Here, we report that transformed mammalian cells internalize exogenous isolated mitochondria upon simple co-incubation. We first demonstrate the physical presence of internalized mitochondria within recipient cells using fluorescence microscopy. Second, we show that xenogenic transfer of murine mitochondria into human cells lacking functional mitochondria can functionally restore respiration in cells lacking mtDNA. Third, utilizing the natural competence of isolated mitochondria to take up linear DNA molecules, we demonstrate the feasibility of using cellular internalization of isolated exogenous mitochondria as a potential tool for studying mitochondrial genetics in living mammalian cells. PMID:18069915

  18. Correlation between chloride flux via the mitochondria-rich cells and transepithelial water movement in isolated frog skin (Rana esculenta)

    DEFF Research Database (Denmark)

    Nielsen, Robert

    Antidiuretic hormone; chloride transport; electroosmosis; Frog skin; Intercalated cells; Local osmosis; Mitochondria-rich cells.......Antidiuretic hormone; chloride transport; electroosmosis; Frog skin; Intercalated cells; Local osmosis; Mitochondria-rich cells....

  19. Mitochondria, cellular stress resistance, somatic cell depletion and lifespan.

    Science.gov (United States)

    Robb, Ellen L; Page, Melissa M; Stuart, Jeffrey A

    2009-03-01

    The causes of aging and determinants of maximum lifespan in animal species are multifaceted and complex. However, a wealth of experimental data suggests that mitochondria are involved both in the aging process and in regulating lifespan. Here we outline a somatic cell depletion (SCD) model to account for correlations between: (1) mitochondrial reactive oxygen species and lifespan; (2) mitochondrial antioxidant enzymes and lifespan; (3) mitochondrial DNA mutation and lifespan and (4) cellular stress resistance and lifespan. We examine the available data from within the framework of the SCD model, in which mitochondrial dysfunction leading to cell death and gradual loss of essential somatic cells eventually contributes to the decline in physiological performance that limits lifespan. This model is useful in explaining many of the mitochondrial manipulations that alter maximum lifespan in a variety of animal species; however, there are a number of caveats and critical experiments outstanding, and these are outlined in this review. PMID:20021396

  20. Mitochondria and Mitophagy: The Yin and Yang of Cell Death Control

    OpenAIRE

    Kubli, Dieter A.; Gustafsson, Åsa B.

    2012-01-01

    Mitochondria are primarily responsible for providing the contracting cardiac myocyte with a continuous supply of ATP. However, mitochondria can rapidly change into death-promoting organelles. In response to changes in the intracellular environment, mitochondria become producers of excessive reactive oxygen species and release pro-death proteins, resulting in disrupted ATP synthesis and activation of cell death pathways. Interestingly, cells have developed a defense mechanism against aberrant ...

  1. Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts.

    Science.gov (United States)

    Griffin, Carly; Karnik, Aditya; McNulty, James; Pandey, Siyaram

    2011-01-01

    The naturally occurring Amaryllidaceae alkaloid pancratistatin exhibits potent apoptotic activity against a large panel of cancer cells lines and has an insignificant effect on noncancerous cell lines, although with an elusive cellular target. Many current chemotherapeutics induce apoptosis via genotoxic mechanisms and thus have low selectivity. The observed selectivity of pancratistatin for cancer cells promoted us to consider the hypothesis that this alkaloid targets cancer cell mitochondria rather than DNA or its replicative machinery. In this study, we report that pancratistatin decreased mitochondrial membrane potential and induced apoptotic nuclear morphology in p53-mutant (HT-29) and wild-type p53 (HCT116) colorectal carcinoma cell lines, but not in noncancerous colon fibroblast (CCD-18Co) cells. Interestingly, pancratistatin was found to be ineffective against mtDNA-depleted (ρ(0)) cancer cells. Moreover, pancratistatin induced cell death in a manner independent of Bax and caspase activation, and did not alter β-tubulin polymerization rate nor cause double-stranded DNA breaks. For the first time we report the efficacy of pancratistatin in vivo against human colorectal adenocarcinoma xenografts. Intratumor administration of pancratistatin (3 mg/kg) caused significant reduction in the growth of subcutaneous HT-29 tumors in Nu/Nu mice (n = 6), with no apparent toxicity to the liver or kidneys as indicated by histopathologic analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Altogether, this work suggests that pancratistatin may be a novel mitochondria-targeting compound that selectively induces apoptosis in cancer cells and significantly reduces tumor growth. PMID:21220492

  2. Nuclear genes involved in mitochondria-to-nucleus communication in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Gabrielson Edward

    2002-11-01

    Full Text Available Abstract Background The interaction of nuclear and mitochondrial genes is an essential feature in maintenance of normal cellular function. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, mitochondrial DNA (mtDNA encodes 13 subunits and rest of the subunits are encoded by nuclear DNA. Mutations in mitochondrial genes encoding the 13 subunits have been reported in a variety of cancers. However, little is known about the nuclear response to impairment of mitochondrial function in human cells. Results We isolated a Rho0 (devoid of mtDNA derivative of a breast cancer cell line. Our study suggests that depletion of mtDNA results in oxidative stress, causing increased lipid peroxidation in breast cancer cells. Using a cDNA microarray we compared differences in the nuclear gene expression profile between a breast cancer cell line (parental Rho+ and its Rho0 derivative impaired in mitochondrial function. Expression of several nuclear genes involved in cell signaling, cell architecture, energy metabolism, cell growth, apoptosis including general transcription factor TFIIH, v-maf, AML1, was induced in Rho0 cells. Expression of several genes was also down regulated. These include phospholipase C, agouti related protein, PKC gamma, protein tyrosine phosphatase C, phosphodiestarase 1A (cell signaling, PIBF1, cytochrome p450, (metabolism and cyclin dependent kinase inhibitor p19, and GAP43 (cell growth and differentiation. Conclusions Mitochondrial impairment in breast cancer cells results in altered expression of nuclear genes involved in signaling, cellular architecture, metabolism, cell growth and differentiation, and apoptosis. These genes may mediate the cross talk between mitochondria and the nucleus.

  3. Methods for Mitochondria and Mitophagy Flux Analyses in Stem Cells of Resting and Regenerating Skeletal Muscle.

    Science.gov (United States)

    García-Prat, Laura; Martínez-Vicente, Marta; Muñoz-Cánoves, Pura

    2016-01-01

    Mitochondria generate most of the cell's supply of ATP as a source of energy. They are also implicated in the control of cell's growth and death. Because of these critical functions, mitochondrial fitness is key for cellular homeostasis. Often, however, mitochondria become defective following damage or stress. To prevent accumulation of damaged mitochondria, the cells clear them through mitophagy, which is defined as the selective degradation of mitochondria by autophagy (the process for degradation of long-lived proteins and damaged organelles in lysosomes). Recently, constitutive mitophagic activity has been reported in quiescent muscle stem cells (satellite cells), which sustain regeneration of skeletal muscle. In response to muscle damage, these cells activate, expand, and differentiate to repair damaged myofibers. Mitophagy was shown to be required for maintenance of satellite cells in their healthy quiescent state. Conversely, damaged mitochondria accumulated in satellite cells with aging and this was attributed to defective mitophagy. This caused increased levels of reactive oxygen species (ROS) and loss of muscle stem cell regenerative capacity at old age. In this chapter, we describe different experimental strategies to evaluate mitochondria status and mitophagy in muscle stem cells from mice. They should improve our ability to study muscle stem homeostasis in adult life, and their loss of function in aging and disease. PMID:27492176

  4. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

    Science.gov (United States)

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-01-01

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

  5. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott;

    2014-01-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial...... rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional...... DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly...

  6. Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells

    International Nuclear Information System (INIS)

    Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSoxTM Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation. (author)

  7. The Salmonella Translocated Effector SopA Is Targeted to the Mitochondria of Infected Cells

    OpenAIRE

    Layton, Abigail N.; Brown, Philip J; Galyov, Edouard E.

    2005-01-01

    This study investigates the Salmonella effector protein SopA. We show that in Salmonella enterica serovar Dublin-infected cells, SopA1-347 fused to two carboxy-terminal hemagglutinin tags partially colocalized with mitochondria. Transfection of eukaryotic cells with a panel of constructs encoding truncated versions of SopA identified that amino acids 100 to 347 were sufficient to target SopA to the mitochondria.

  8. The antineoplastic agent α-bisabolol promotes cell death by inducing pores in mitochondria and lysosomes.

    Science.gov (United States)

    Rigo, Antonella; Vinante, Fabrizio

    2016-08-01

    The sesquiterpene α-bisabolol (α-BSB) has been shown to be an effective cytotoxic agent for a variety of human cancer cells in culture and animal models. However, much of its intracellular action remains elusive. We evaluated the cytotoxic action of α-BSB against CML-T1, Jurkat and HeLa cell lines, as preclinical models for myeloid, lymphoid and epithelial neoplasias. The approach included single cell analysis (flow cytometry, immunocytology) combined with cytotoxicity and proliferation assays to characterize organelle damage, autophagy, cytostatic effect, and apoptosis. The study focuses on the relevant steps in the cytotoxic cascade triggered by α-BSB: (1) the lipid rafts through which α-BSB enters the cells, (2) the opening of pores in the mitochondria and lysosomes, (3) the activation of both caspase-dependent and caspase-independent cell death pathways, (4) the induction of autophagy and (5) apoptosis. The effectiveness of α-BSB as an agent against tumor cells is grounded on its capability to act on different layers of cell regulation to elicit different concurrent death signals, thereby neutralizing a variety of aberrant survival mechanisms leading to treatment resistance in neoplastic cell. PMID:27278818

  9. Dithiothreitol abrogates the effect of arsenic trioxide on normal rat liver mitochondria and human hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Arsenic trioxide (ATO) is a known environmental toxicant and a potent chemotherapeutic agent. Significant correlation has been reported between consumption of arsenic-contaminated water and occurrence of liver cancer; moreover, ATO-treated leukemia patients also suffers from liver toxicity. Hence, modulation of ATO action may help to prevent populations suffering from arsenic toxicity as well as help reduce the drug-related side effects. Dithiothreitol (DTT) is a well-known dithiol agent reported to modulate the action of ATO. Controversial reports exist regarding the effect of DTT on ATO-induced apoptosis in leukemia cells. To the best of our knowledge, no report illustrates the modulatory effect of DTT on ATO-induced liver toxicity, the prime target for arsenic. Mitochondria serve as the doorway to apoptosis and have been implicated in ATO-induced cell death. Hence, we attempted to study the modulatory effect of DTT on ATO-induced dysfunction of mammalian liver mitochondria and human hepatocellular carcinoma cell line (Hep3B). We, for the first time, report that ATO produces complex I-mediated electron transfer inhibition, reactive oxygen species (ROS) generation, respiration inhibition, and ATO-induced ROS-mediated mitochondrial permeability transition (MPT) opening. DTT at low concentration (100 μM and less) prevents the effect of ATO-induced complex I-malfunctions. DTT protects mitochondria from ATO-mediated opening of MPT and membrane potential depolarization. DTT also prevented ATO-induced Hep3B cell death. Thus, at low concentrations DTT abrogates the effect of ATO on rat liver mitochondria and Hep3B cell line. Therefore, the present result suggests, that use of low concentration of dithiols as food supplement may prevent arsenic toxicity in affected population

  10. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection

  11. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  12. The role of mitochondria in defence mechanisms of human endothelial cells

    OpenAIRE

    Wilkinson, J R

    2010-01-01

    Introduction: Mitochondria are considered to be the powerhouse of the cell being the primary generators of ATP, they also have numerous other important functions including; being the main generator of reactive oxygen species (ROS) and a central role in apoptosis. As the main intracellular source of ROS, many people believe that mitochondria play a significant role in ageing. Senescence is associated with ageing and has been associated with atherosclerotic vascular disease. The concept of huma...

  13. PKCα translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    WU; Qiao(吴乔); LIU; Su(刘苏); DING; Liang(丁亮); YE; Xiaofeng(叶晓峰); SU; Wenjin(苏文金)

    2002-01-01

    PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKC? was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCα from both mitochondria and cytosol to nucleus as clearly shown by laser-scanning-confocal microscopy, while the protein level of PKCα was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCα was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCα from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCα translocation.

  14. Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

    Institute of Scientific and Technical Information of China (English)

    Young Geol Yoon; Michael Duane Koob

    2011-01-01

    Due to technical difficulties, the genetic transformation of mitochondria in mammalian cells is still a challenge. In this report, we described our attempts to transform mammalian mitochondria with an engineered mitochondrial genome based on selection using a drug resistance gene. Because the standard drug-resistant neomycin phosphotransferase confers resistance to high concentrations of G418 when targeted to the mitochondria, we generated a recoded neomycin resistance gene that uses the mammalian mitochondrial genetic code to direct the synthesis of this protein in the mitochondria, but not in the nucleus (mitochondrial version). We also generated a universal version of the recoded neomycin resistance gene that allows synthesis of the drug-resistant proteins both in the mitochondria and nucleus. When we transfected these recoded neomycin resistance genes that were incorporated into the mouse mitochondrial genome clones into mouse tissue culture cells by electroporation, no DNA constructs were delivered into the mitochondria. We found that the universal version of the recoded neomycin resistance gene was expressed in the nucleus and thus conferred drug resistance to G418 selection, while the synthetic mitochondrial version of the gene produced no background drug-resistant cells from nuclear transformation. These recoded synthetic drug-resistant genes could be a useful tool for selecting mitochondrial genetic transformants as a precise technology for mitochondrial transformation is developed.

  15. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

    Science.gov (United States)

    Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

    2016-04-01

    Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms

  16. Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

    Science.gov (United States)

    Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

    2006-02-01

    We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

  17. Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

    International Nuclear Information System (INIS)

    Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

  18. Study on the role of mitochondria in sodium butyrate-induced apoptosis of ovarian carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Liu Wei; Tang Chunsheng; Rong Fengnian

    2005-01-01

    Objective:To investigate the role of mitochondria in sodium butyrate-induced apoptosis of ovarian carcinoma cells in vitro.Methods:Human ovarian epithelial cancer 3AO cells were cultured in vitro and treated with sodium butyrate of different concentration for different time. The characters of apoptosis were assessed through light microscopy and DNA ladder analysis. The morphological changes of mitochondria were detected through electron and epifluorescence microscopy. The functional changes of mitochondria and the expression of Bcl-2/Bax protein were analyzed by flow cytometry.Results:As the concentration of sodium butyrate rose to 4mmol/L, the morphologic characters of apoptosis were found by light microscopy, DNA ladder was observed. Under epifluorescence microscope the fluorescence of the control group was stronger than that of the experimental group. Under electron microscope swelled mitochondria was detected. Flow cytometry analysis showed mitochondria transmembrane potentials decreased and there were down-regulate of Bcl-2 protein and up-regulate of the Bax protein(P<0.05).Conclusion:Sodium butyrate can induce apoptosis of 3AO cells in a time-dose dependent manner. Mitochondrion may play a key role in the procedure of apoptosis of ovarian cancer cells.

  19. Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    DEFF Research Database (Denmark)

    Havelund, Jesper; Thelen, Jay J.; Møller, Ian Max

    2013-01-01

    functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of...

  20. Human mitochondrial transcription factor A functions in both nuclei and mitochondria and regulates cancer cell growth

    International Nuclear Information System (INIS)

    Highlights: → Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. → mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. → Overexpression of mtTFA enhances the growth of cancer cells, whereas downregulation of mtTFA inhibits their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). → Knockdown of mtTFA expression induces p21-dependent G1 cell cycle arrest. -- Abstract: Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

  1. Biochemistry, proteomics and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    Directory of Open Access Journals (Sweden)

    Jesper Foged Havelund

    2013-03-01

    Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

  2. Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-Bromopyruvate

    OpenAIRE

    Chen, Zhao; Zhang, Hui; Lu, Weiqin; Huang, Peng

    2009-01-01

    It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study...

  3. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  4. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway.

    Science.gov (United States)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-01

    Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection. PMID:25499817

  5. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    Science.gov (United States)

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain. PMID:24880782

  6. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    International Nuclear Information System (INIS)

    Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  7. 叶酰聚谷氨酸合成酶基因在甲氨蝶呤对映体获得性耐药A549细胞株中的表达差异%Differential gene expression of folylpolyglutamate synthetase in cytoplasm and mitochondria in acquired methotrexate enantiomers resistant to lung cancer A549 cell lines

    Institute of Scientific and Technical Information of China (English)

    周红艳; 何晓东; 孙余婕; 凡任芝; 孙利; 沈佐君

    2011-01-01

    index of MTX resistance cell lines were detected by MTT.The gene expressions of FPGS in cytoplasm and mitochondria were detected by real-time quantitative polymerase chain reaction (PCR).Results The resistance indice of D-( - )-MTX resistant cell lines were higher than those of L-( + )-MTX resistant cells (32.7±9.3 vs 11.5 ±2.9,P <0.05).The resistant indice of L-( + )-MTX /A549 were from 5 to 15,which mean the middle resistance.The resistant indice of D-( - )-MTX/A549 were more than 15,which mean the severe resistance.The expression of mFPGS had difference between resistant cell lines of L-( + )and D-( - )-MTX except at 15 μmol/L MTX (at 25 μmol/L,1.3 ±0.7 vs.2.3 ±0.9;at 35 μmol/L,1.1 ±0.9 vs.2.6±0.3;at 45 μmol/L,1.0±1.0 vs.1.4±0.8;at 55 μmol/L,0.2±0.1 vs.1.0±0.2;all P<0.05).The expressions of cFPGS had no difference between resistant cell lines of L-( + ) and D-( - )-MTX at 15 μmol/L MTX,while at 25 -55 μmol/L,the cFPGS levels and resistance indice of D-( - )-MTX/A549 resistant cell lines showed a highly negative correlation ( r = - 0.95,P<0.05 ).Conclusion There may be a different mechanism between the first time treatment with 15 μmol/L dosage and the continual treatment with more than 25 μmol/L dosage in A549 cell lines.There had higher resistant index in D-( - )-MTX/A549 cell line than in L-( + )-MTX/A549 cell line.The results indicated that the difference in chirality should be considered in clinical treatment with MTX.

  8. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (DMID) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  9. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    International Nuclear Information System (INIS)

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD

  10. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of); Shin, Ki Soon [Department of Biology, Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kang, Shin Jung, E-mail: sjkang@sejong.ac.kr [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of)

    2013-08-09

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

  11. Are maternal mitochondria the selfish entities that are masters of the cells of eukaryotic multicellular organisms?

    OpenAIRE

    Agnati, Luigi F.; Barlow, Peter W; Baldelli, E.; Baluška, Frantisek

    2009-01-01

    The Energide concept, as well as the endosymbiotic theory of eukaryotic cell organization and evolution, proposes that present-day cells of eukaryotic organisms are mosaics of specialized and cooperating units, or organelles. Some of these units were originally free-living prokaryotes, which were engulfed during evolutionary time. Mitochondria represent one of these types of previously independent organisms, the Energide, is another type. This new perspective on the organization of the cell h...

  12. Role of Mitochondria-rich Cells for Passive Chloride Transport, discussion of Ussing's Contribution to Our Understanding of Shunt Pathways in Epithelia

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Kristensen, Poul; Nedergaard, Signe Nielsen;

    2001-01-01

    Toad skin, Mitochondria-rich cells, Chloride channels, Epithelial shunt pathways, Leaky epithelia, Recirculation theory of isotonic transport......Toad skin, Mitochondria-rich cells, Chloride channels, Epithelial shunt pathways, Leaky epithelia, Recirculation theory of isotonic transport...

  13. Bezielle selectively targets mitochondria of cancer cells to inhibit glycolysis and OXPHOS.

    Directory of Open Access Journals (Sweden)

    Vivian Chen

    Full Text Available Bezielle (BZL101 is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells.

  14. Dracocephalum: Novel Anticancer Plant Acting on Liver Cancer Cell Mitochondria

    Directory of Open Access Journals (Sweden)

    Mojtaba Talari

    2014-01-01

    Full Text Available Dracocephalum kotschyi Boiss. (Labiatae is a native Iranian medicinal plant which has been used in combination with Peganum harmala L. as a remedy for many forms of human cancer especially leukemia and gastrointestinal malignancies. Hepatocellular carcinoma (HCC is the third leading cause of cancer-related death worldwide. In this investigation HCC was induced by a single intraperitoneal injection of diethylnitrosamine (DEN in corn oil at 200 mg/kg body weight to rats. Two weeks after DEN administration, cancer development was promoted with dietary 2-acetylaminofluorene (2-AAF (0.02%, w/w for 2 weeks. Serum alpha-fetoprotein (AFP concentration, serum alanine transaminase (ALT, aspartate transaminase (AST, and alkaline phosphatase (ALP activities were also determined for confirmation of hepatocellular carcinoma induction. Then rat hepatocytes were isolated with collagen perfusion technique and tumoral hepatocytes were sorted by flow cytometry. Finally isolated mitochondria obtained from both tumoral and nontumoral hepatocytes were used for any probable toxic effect of Dracocephalum kotschyi ethanolic extract. Our results showed that D. kotschyi extract (250 µg/mL induced reactive oxygen species (ROS formation, mitochondrial membrane permeabilization (MMP, and mitochondrial swelling and cytochrome c release only in tumoral but not nontumoral hepatocyte. These findings propose Dracocephalum kotschyi as a promising candidate for future anticancer research.

  15. Biology of SNU Cell Lines

    OpenAIRE

    Ku, Ja-Lok; Park, Jae-Gahb

    2005-01-01

    SNU (Seoul National University) cell lines have been established from Korean cancer patients since 1982. Of these 109 cell lines have been characterized and reported, i.e., 17 colorectal carcinoma, 12 hepatocellular carcinoma, 11 gastric carcinoma, 12 uterine cervical carcinoma, 17 B-lymphoblastoid cell lines derived from cancer patients, 5 ovarian carcinoma, 3 malignant mixed Mllerian tumor, 6 laryngeal squamous cell carcinoma, 7 renal cell carcinoma, 9 brain tumor, 6 biliary tract, and 4 pa...

  16. Mitochondria, calcium and pro-apoptotic proteins as mediators in cell death signaling

    Directory of Open Access Journals (Sweden)

    S.S. Smaili

    2003-02-01

    Full Text Available Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca2+-dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca2+ content. Mitochondria are endowed with multiple Ca2+ transport mechanisms by which they take up and release Ca2+ across their inner membrane. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (Dym. The collapse of Dym along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway.

  17. Are maternal mitochondria the selfish entities that are masters of the cells of eukaryotic multicellular organisms?

    Science.gov (United States)

    Agnati, Luigi F; Barlow, Peter W; Baldelli, E; Baluska, Frantisek

    2009-01-01

    The Energide concept, as well as the endosymbiotic theory of eukaryotic cell organization and evolution, proposes that present-day cells of eukaryotic organisms are mosaics of specialized and cooperating units, or organelles. Some of these units were originally free-living prokaryotes, which were engulfed during evolutionary time. Mitochondria represent one of these types of previously independent organisms, the Energide, is another type. This new perspective on the organization of the cell has been further expanded to reveal the concept of a public milieu, the cytosol, in which Energides and mitochondria live, each with their own private internal milieu. The present paper discusses how the endosymbiotic theory implicates a new hypothesis about the hierarchical and communicational organization of the integrated prokaryotic components of the eukaryotic cell and provides a new angle from which to consider the theory of evolution and its bearing upon cellular complexity. Thus, it is proposed that the "selfish gene" hypothesis of Dawkins1 is not the only possible perspective for comprehending genomic and cellular evolution. Our proposal is that maternal mitochondria are the selfish "master" entities of the eukaryotic cell with respect not only to their propagation from cell-to-cell and from generation-to-generation but also to their regulation of all other cellular functions. However, it should be recognized that the concept of "master" and "servant" cell components is a metaphor; in present-day living organisms their organellar components are considered to be interdependent and inseparable. PMID:19513277

  18. Timeline: Mitochondria.

    Science.gov (United States)

    Evans, Allyson

    2016-03-01

    Mitochondria perform diverse yet interconnected cellular functions and are dynamically regulated by complex signaling pathways. Interest in this fascinating organelle has recently undergone a renaissance due to a series of discoveries revealing that mitochondrial function goes beyond the generation of molecular fuel. This Timeline highlights some of the groundbreaking events over the last 30 years in our understanding of how mitochondria are made and turned over, and how they integrate and signal within the cell and organism. To view this Timeline, open or download the PDF. PMID:26942681

  19. Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells.

    Science.gov (United States)

    Shahzidi, Susan; Cunderlíková, Beata; Więdłocha, Antoni; Zhen, Yan; Vasovič, Vlada; Nesland, Jahn M; Peng, Qian

    2011-11-01

    Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways. PMID:21881674

  20. Mitochondria-Targeted Analogues of Metformin Exhibit Enhanced Antiproliferative and Radiosensitizing Effects in Pancreatic Cancer Cells.

    Science.gov (United States)

    Cheng, Gang; Zielonka, Jacek; Ouari, Olivier; Lopez, Marcos; McAllister, Donna; Boyle, Kathleen; Barrios, Christy S; Weber, James J; Johnson, Bryon D; Hardy, Micael; Dwinell, Michael B; Kalyanaraman, Balaraman

    2016-07-01

    Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP(+)). In particular, the analogue Mito-Met10, synthesized by attaching TPP(+) to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904-15. ©2016 AACR. PMID:27216187

  1. Control of Cell Differentiation by Mitochondria, Typically Evidenced in Dictyostelium Development

    Directory of Open Access Journals (Sweden)

    Yasuo Maeda

    2013-11-01

    Full Text Available In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, cell movement, and pattern formation. This has been most strikingly realized in development of the cellular slime mold Dictyostelium. For example, the expression of the mitochondrial ribosomal protein S4 (mt-rps4 gene is required for the initial differentiation. The Dictyostelium homologue (Dd-TRAP1 of TRAP-1 (tumor necrosis receptor-associated protein 1, a mitochondrial molecular chaperone belonging to the Hsp90 family, allows the prompt transition of cells from growth to differentiation through a novel prestarvation factor (PSF-3 in growth medium. Moreover, a cell-type-specific organelle named a prespore-specific vacuole (PSV is constructed by mitochondrial transformation with the help of the Golgi complex. Mitochondria are also closely involved in a variety of cellular activities including CN-resistant respiration and apoptosis. These mitochondrial functions are reviewed in this article, with special emphasis on the regulation of Dictyostelium development.

  2. Control of cell differentiation by mitochondria, typically evidenced in dictyostelium development.

    Science.gov (United States)

    Maeda, Yasuo; Chida, Junji

    2013-01-01

    In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration. Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, cell movement, and pattern formation. This has been most strikingly realized in development of the cellular slime mold Dictyostelium. For example, the expression of the mitochondrial ribosomal protein S4 (mt-rps4) gene is required for the initial differentiation. The Dictyostelium homologue (Dd-TRAP1) of TRAP-1 (tumor necrosis receptor-associated protein 1), a mitochondrial molecular chaperone belonging to the Hsp90 family, allows the prompt transition of cells from growth to differentiation through a novel prestarvation factor (PSF-3) in growth medium. Moreover, a cell-type-specific organelle named a prespore-specific vacuole (PSV) is constructed by mitochondrial transformation with the help of the Golgi complex. Mitochondria are also closely involved in a variety of cellular activities including CN-resistant respiration and apoptosis. These mitochondrial functions are reviewed in this article, with special emphasis on the regulation of Dictyostelium development. PMID:24970198

  3. Tumor cell phenotype is sustained by selective MAPK oxidation in mitochondria.

    Directory of Open Access Journals (Sweden)

    Soledad Galli

    Full Text Available Mitochondria are major cellular sources of hydrogen peroxide (H(2O(2, the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2O(2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2O(2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2O(2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2O(2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2O(2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei, the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2O(2 level. Like this, high H(2O(2 or directed mutation of redox-sensitive ERK2 Cys(214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to

  4. A novel class of mitochondria-targeted soft electrophiles modifies mitochondrial proteins and inhibits mitochondrial metabolism in breast cancer cells through redox mechanisms.

    Directory of Open Access Journals (Sweden)

    Praveen K Vayalil

    Full Text Available Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration in vitro. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.

  5. Structural modifications of mitochondria-targeted chlorambucil alter cell death mechanism but preserve MDR evasion.

    Science.gov (United States)

    Jean, Sae Rin; Pereira, Mark P; Kelley, Shana O

    2014-08-01

    Multidrug resistance (MDR) remains one of the major obstacles in chemotherapy, potentially rendering a multitude of drugs ineffective. Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common resistance factors that are present in the nucleus or cytosol. In particular, mitochondria-targeted chlorambucil (mt-Cbl) has increased potency and activity against resistant cancer cells compared to the parent compound chlorambucil (Cbl). However, it was found that, due to its high reactivity, mt-Cbl induces a necrotic type of cell death via rapid nonspecific alkylation of mitochondrial proteins. Here, we demonstrate that by tuning the alkylating activity of mt-Cbl via chemical modification, the rate of generation of protein adducts can be reduced, resulting in a shift of the cell death mechanism from necrosis to a more controlled apoptotic pathway. Moreover, we demonstrate that all of the modified mt-Cbl compounds effectively evade MDR resulting from cytosolic GST-μ upregulation by rapidly accumulating in mitochondria, inducing cell death directly from within. In this study, we systematically elucidated the advantages and limitations of targeting alkylating agents with varying reactivity to mitochondria. PMID:24922525

  6. Pluripotent stem cell lines

    OpenAIRE

    Yu, Junying; Thomson, James A.

    2008-01-01

    The derivation of human embryonic stem cells 10 years ago ignited an explosion of public interest in stem cells, yet this achievement depended on prior decades of research on mouse embryonic carcinoma cells and embryonic stem cells. In turn, the recent derivation of mouse and human induced pluripotent stem cells depended on the prior studies on mouse and human embryonic stem cells. Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in vitro while ma...

  7. Isolation of mitochondria from CNS

    OpenAIRE

    Kristian, Tibor

    2010-01-01

    This unit contains a protocol describing the isolation of brain mitochondria by using discontinuous Percoll gradient centrifugation. The Percoll density gradient centrifugation separates synaptosomes, myelin, and free non-synaptic mitochondria released from cells during tissue homogenization into individual fractions. Mitochondria entrapped in synaptosomes (synaptic mitochondria) can be liberated using nitrogen cavitation and then further purified by Percoll gradient centrifugation. These met...

  8. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    Science.gov (United States)

    Villalobo, A; Lehninger, A L

    1980-03-25

    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered. PMID:6766937

  9. Edaravone protects PC12 cells from ischemic-like injury via attenuating the damage to mitochondria

    Institute of Scientific and Technical Information of China (English)

    SONG Ying; LI Meng; LI Ji-cheng; WEI Er-qing

    2006-01-01

    Background: Edaravone had been validated to effectively protect against ischemic injuries. In this study, we investigated the protective effect of edaravone by observing the effects on anti-apoptosis, regulation of Bcl-2/Bax protein expression and recovering from damage to mitochondria after OGD (oxygen-glucose deprivation)-reperfusion. Methods: Viability of PC 12cells which were injured at different time of OGD injury, was quantified by measuring MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining. In addition, PC 12 cells' viability was also quantified after their preincubation in different concentration of edaravone for 30 min followed by (OGD). Furthermore, apoptotic population of PC12 cells that reinsulted from OGD-reperfusion with or without preincubation with edaravone was determined by flow cytometer analysis,electron microscope and Hoechst/PI staining. Finally, change of Bcl-2/Bax protein expression was detected by Westem blot.Results: (1) The viability of PC 12 cells decreased with time (1~12 h) after OGD. We regarded the model of OGD 2 h, then replacing DMEM (Dulbecco's Modified Eagle's Medium) for another 24 h as an OGD-reperfusion in this research. Furthermore,most PC12 cells were in the state of apoptosis after OGD-reperfusion. (2) The viability of PC12 cells preincubated with edaravone at high concentrations (1,0.1, 0.01 μmol/L) increased significantly with edaravone protecting PC 12 cells from apoptosis after OGD-reperfusion injury. (3) Furthermore, edaravone attenuates the damage of OGD-reperfusion on mitochondria and regulated Bcl-2/Bax protein imbalance expression after OGD-reperfusion. Conclusion: Neuroprotective effects of edaravone on ischemic or other brain injuries may be partly mediated through inhibition of Bcl-2/Bax apoptotic pathways by recovering from the damage of mitochondria.

  10. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    Directory of Open Access Journals (Sweden)

    Sho W Suzuki

    Full Text Available Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA. We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  11. Glucocorticoid and estrogen receptors are reduced in mitochondria of lung epithelial cells in asthma.

    Directory of Open Access Journals (Sweden)

    Davina C M Simoes

    Full Text Available Mitochondrial glucocorticoid (mtGR and estrogen (mtER receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS biosynthesis, affecting reactive oxygen species (ROS generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GRα and ERβ in lung tissue. Allergic airway inflammation caused reduction in mtGRα, mtERβ, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GRα and ERβ in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

  12. Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3'UTRs.

    Directory of Open Access Journals (Sweden)

    Marta Martínez-Diez

    Full Text Available Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of beta-F1-ATPase and Hsp60 carried out also at G2/M and, (iii the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (DeltaPsim is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of beta-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3'UTR of the transcript. The 3'UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated beta-F1-ATPase expression in human cancer.

  13. [Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells].

    Science.gov (United States)

    Qiu, Qiang; Wang, Zhen; Jiang, Jian-ming; Li, Dian-dong

    2007-02-01

    Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities. PMID:17518039

  14. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

    Science.gov (United States)

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forte, Trudy M; Ryan, Robert O

    2015-08-21

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid. PMID:26164234

  15. Enhanced cytotoxicity to cancer cells by mitochondria-targeting MWCNTs containing platinum(IV) prodrug of cisplatin.

    Science.gov (United States)

    Yoong, Sia Lee; Wong, Bin Sheng; Zhou, Qi Ling; Chin, Chee Fei; Li, Jian; Venkatesan, Thirumalai; Ho, Han Kiat; Yu, Victor; Ang, Wee Han; Pastorin, Giorgia

    2014-01-01

    Among the arsenal of nano-materials, carbon nanotubes (CNTs) are becoming more prominent due to favorable attributes including their unique shape, which promotes cellular-uptake, and large aspect-ratio that facilitates functionalization of bioactive molecules on their surface. In this study, multi-walled carbon nanotubes (MWCNTs) were functionalized with either mitochondrial-targeting fluorescent rhodamine-110 (MWCNT-Rho) or non-targeting fluorescein (MWCNT-Fluo). Despite structural similarities, MWCNT-Rho associated well with mitochondria (ca. 80% co-localization) in contrast to MWCNT-Fluo, which was poorly localized (ca. 21% co-localization). Additionally, MWCNT-Rho entrapping platinum(IV) pro-drug of cisplatin (PtBz) displayed enhanced potency (IC50 = 0.34 ± 0.07 μM) compared to a construct based on MWCNT-Fluo (IC50 ≥ 2.64 μM). Concurrently, preliminary in vitro toxicity evaluation revealed that empty MWCNT-Rho neither decreased cell viability significantly nor interfered with mitochondrial membrane-potential, while seemingly being partially expelled from cells. Due to its targeting capability and apparent lack of cytotoxicity, MWCNT-Rho complex was used to co-encapsulate PtBz and a chemo-potentiator, 3-bromopyruvate (BP), and the resulting MWCNT-Rho(PtBz+BP) construct demonstrated superior efficacy over PtBz free drug in several cancer cell lines tested. Importantly, a 2-fold decrease in mitochondrial potential was observed, implying that mitochondrial targeting of compounds indeed incurred additional intended damage to mitochondria. PMID:24140044

  16. ING1 induces apoptosis through direct effects at the mitochondria

    DEFF Research Database (Denmark)

    Bose, P; Thakur, S; Thalappilly, S;

    2013-01-01

    translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the...... mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein...

  17. Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells

    Institute of Scientific and Technical Information of China (English)

    Jin-bo WANG; Li-li QI; Shui-di ZHENG; Tian-xing WU

    2009-01-01

    Objective:To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2,Bax,Bad,Bcl-xL,caspase-3,poly ADP-ribose polymerase (PARP),and survivin of HT-29 cells.Methods:HT-29 cells were treated with curcumin (0~80 μmol/L) for 24 h.The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax,Bcl-2,Bci-xL,Bad,caspase-3,PARP,and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR).Results:Curcumin significantly induced the growth inhibition and apoptosis of HT-29 ceils.A decrease in expressions of Bcl-2,Bci-xL and survivin was observed after exposure to 10~80 μmol/L curcumin,while the levels of Bax and Bad increased in the curcumin-treated cells.Curcumin also induced the release of cytochrome c,the activation ofcaspase-3,and the cleavage of PARP in a dose-dependent manner.Conclusion:These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.

  18. Mitochondria and mammalian reproduction

    OpenAIRE

    Ramalho-Santos, J; Amaral, Sandra

    2013-01-01

    Mitochondria are cellular organelles with crucial roles in ATP synthesis, metabolic integration, reactive oxygen species (ROS) synthesis and management, the regulation of apoptosis (namely via the intrinsic pathway), among many others. Additionally, mitochondria in different organs or cell types may have distinct properties that can decisively influence functional analysis. In terms of the importance of mitochondria in mammalian reproduction, and although there are species-specifi...

  19. Cell lines and Salmonella

    NARCIS (Netherlands)

    Jonge R de; Hendriks H; Garssen J; Universteit Utrecht, afdeling; MGB; LPI

    2001-01-01

    In human gastrointestinal disease caused by Salmonella, transepithelial migration of neutrophils follows the attachment of bacteria to epithelial tissue. This migration of neutrophils is stimulated by the release of chemokines, including interleukin-8 (Il -8), from the epithelial cells. We have dev

  20. The mitochondria-independent cytotoxic effect of nelfinavir on leukemia cells can be enhanced by sorafenib-mediated mcl-1 downregulation and mitochondrial membrane destabilization

    Directory of Open Access Journals (Sweden)

    Gingelmaier Andrea

    2010-01-01

    Full Text Available Abstract Background Nelfinavir is an HIV protease inhibitor that has been used for a long period of time to treat HIV-infected individuals. It has recently emerged that nelfinavir could represent a prospective new anti-cancer drug, prompting us to test the effect of nelfinavir on leukemia cells. Methods By combining in vitro and ex vivo studies, the effect of nelfinavir on leukemia cells and non-malignant, bone marrow-derived tissue cells was analyzed. Results At a concentration of 9 μg/ml, nelfinavir induced death of 90% of HL60, IM9, and Jurkat cells. At the same concentration and treatment conditions, less than 10% of aspirated human bone marrow cells showed nelfinavir-induced cell damage. Nelfinavir-induced death of leukemia cells was accompanied by activation of caspases 3, 7, and 8. Despite caspase activation, the upregulation of the anti-apoptotic bcl-2 family member protein mcl-1 that resulted from nelfinavir treatment stabilized the mitochondrial membrane potential, resulting in primarily mitochondria-independent cell death. Pharmacological downregulation of mcl-1 expression by treatment with sorafenib (2 μg/ml significantly enhanced nelfinavir-induced apoptosis even at lower nelfinavir concentrations (5 μg/ml, but did not have additional detrimental effects on non-malignant bone marrow cells. Conclusions The ability of nelfinavir to induce apoptosis in leukemia cells as a single agent in a mitochondria-independent manner might suggest it could be used as a second or third line of treatment for leukemia patients for whom standard mitochondria-directed treatment strategies have failed. Combination treatment with nelfinavir and sorafenib might further enhance the efficacy of nelfinavir even on chemo-resistant leukemia cells.

  1. Do mitochondria play a role in remodelling lace plant leaves during programmed cell death?

    Directory of Open Access Journals (Sweden)

    Lane Stephanie

    2011-06-01

    Full Text Available Abstract Background Programmed cell death (PCD is the regulated death of cells within an organism. The lace plant (Aponogeton madagascariensis produces perforations in its leaves through PCD. The leaves of the plant consist of a latticework of longitudinal and transverse veins enclosing areoles. PCD occurs in the cells at the center of these areoles and progresses outwards, stopping approximately five cells from the vasculature. The role of mitochondria during PCD has been recognized in animals; however, it has been less studied during PCD in plants. Results The following paper elucidates the role of mitochondrial dynamics during developmentally regulated PCD in vivo in A. madagascariensis. A single areole within a window stage leaf (PCD is occurring was divided into three areas based on the progression of PCD; cells that will not undergo PCD (NPCD, cells in early stages of PCD (EPCD, and cells in late stages of PCD (LPCD. Window stage leaves were stained with the mitochondrial dye MitoTracker Red CMXRos and examined. Mitochondrial dynamics were delineated into four categories (M1-M4 based on characteristics including distribution, motility, and membrane potential (ΔΨm. A TUNEL assay showed fragmented nDNA in a gradient over these mitochondrial stages. Chloroplasts and transvacuolar strands were also examined using live cell imaging. The possible importance of mitochondrial permeability transition pore (PTP formation during PCD was indirectly examined via in vivo cyclosporine A (CsA treatment. This treatment resulted in lace plant leaves with a significantly lower number of perforations compared to controls, and that displayed mitochondrial dynamics similar to that of non-PCD cells. Conclusions Results depicted mitochondrial dynamics in vivo as PCD progresses within the lace plant, and highlight the correlation of this organelle with other organelles during developmental PCD. To the best of our knowledge, this is the first report of

  2. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  3. He-Ne laser irradiation causes changes in mitochondria ultrastructure in successive generations of yeast cells

    International Nuclear Information System (INIS)

    Changes in the mitochondria ultrastructural organization in the Torulopsis sphaerica yeast cells, cultivated in 18 hours after irradiation by the He-Ne laser, are studied. Two doses - 460 and 1150 J/m2 were chosen, while irradiation in low doses optimally stimulates the given culture growth (the biomass increases up to 141.2 %). The studies were conducted on the cells of 4-5 generation after irradiation. It is shown, that laser irradiation in the yeast cells effects the ATP-ase synthesis not only through the mechanism of fast respiratory control but also through the synthesis of mitochondrial fermentation complexes (slow respiratory control), which is regulated at the genetic level

  4. Axin is expressed in mitochondria and suppresses mitochondrial ATP synthesis in HeLa cells.

    Science.gov (United States)

    Shin, Jee-Hye; Kim, Hyun-Wook; Rhyu, Im Joo; Kee, Sun-Ho

    2016-01-01

    Many recent studies have revealed that axin is involved in numerous cellular functions beyond the negative regulation of β-catenin-dependent Wnt signaling. Previously, an association of ectopic axin with mitochondria was observed. In an effort to investigate the relationship between axin and mitochondria, we found that axin expression suppressed cellular ATP production, which was more apparent as axin expression levels increased. Also, mitochondrial expression of axin was observed using two axin-expressing HeLa cell models: doxycycline-inducible ectopic axin expression (HeLa-axin) and axin expression enhanced by long-term treatment with XAV939 (HeLa-XAV). In biochemical analysis, axin is associated with oxidative phosphorylation (OXPHOS) complex IV and is involved in defects in the assembly of complex IV-containing supercomplexes. Functionally, axin expression reduced the activity of OXPHOS complex IV and the oxygen consumption rate (OCR), suggesting axin-mediated mitochondrial dysfunction. Subsequent studies using various inhibitors of Wnt signaling showed that the reduction in cellular ATP levels was weaker in cases of ICAT protein expression and treatment with iCRT3 or NSC668036 compared with XAV939 treatment, suggesting that XAV939 treatment affects ATP synthesis in addition to suppressing Wnt signaling activity. Axin-mediated regulation of mitochondrial function may be an additional mechanism to Wnt signaling for regulation of cell growth. PMID:26704260

  5. Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

    KAUST Repository

    Vishal, Chaturvedi

    2011-06-07

    Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/ MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity. the author(s), publisher and licensee Libertas Academica Ltd.

  6. Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chloride-induced hypoxia.

    Science.gov (United States)

    Sun, Lei; Liu, Ning; Liu, Shan-Shan; Xia, Wu-Yan; Liu, Meng-Yao; Li, Lin-Feng; Gao, Jian-Xin

    2015-01-01

    Autophagy has dual functions in cell survival and death. However, the effects of autophagy on cancer cell survival or death remain controversial. In this study, we show that Autophagy can mediate programmed cell death (PCD) of cancer cells in responding to cobalt chloride (CoCl2)-induced hypoxia in a Beclin-1-independent but autophagy protein 5 (ATG5)-dependent manner. Although ATG5 is not directly induced by CoCl2, its constitutive expression is essential for CoCl2-induced PCD. The ATG5-mediated autophagic PCD requires interplays with endoplasmic reticulum (ER) and/or mitochondria. In this process, ATG5 plays a central role in regulating ER stress protein CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and mitochondrial protein second mitochondria derived activator of caspases (Smac). Two pathways for autophagic PCD in cancer cells responding to hypoxia have been identified: ATG5/CHOP/Smac pathway and ATG5/Smac pathway, which are probably dependent on the context of cell lines. The former is more potent than the latter for the induction of PCD at the early stage of hypoxia, although the ultimate efficiency of both pathways is comparable. In addition, both pathways may require ATG5-mediated conversion of LC3-I into LC3-II. Therefore, we have defined two autophagy-mediated pathways for the PCD of cancer cells in hypoxia, which are dependent on ATG5, interplayed with ER and mitochondria and tightly regulated by hypoxic status. The findings provide a new evidence that autophagy may inhibit tumor cell proliferation through trigger of PCD, facilitating the development of novel anti-cancer drugs. PMID:26609472

  7. Automation of cell line development

    OpenAIRE

    Lindgren, Kristina; Salmén, Andréa; Lundgren, Mats; Bylund, Lovisa; Ebler, Åsa; Fäldt, Eric; Sörvik, Lina; Fenge, Christel; Skoging-Nyberg, Ulrica

    2009-01-01

    An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used ...

  8. A newly identified essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast.

    Directory of Open Access Journals (Sweden)

    Laurence Vernis

    Full Text Available A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H(2O(2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

  9. Cissus quadrangularis Linn. Stem Ethanolic Extract Liberates Reactive Oxygen Species and Induces Mitochondria Mediated Apoptosis in KB Cells

    Science.gov (United States)

    Sheikh, Saba; Siddiqui, Sahabjada; Dhasmana, Anupam; Safia; Haque, Ejazul; Kamil, Mohammed; Lohani, Mohtashim; Arshad, Mohammad; Mir, Snober Shabnam

    2015-01-01

    Background: Cissus quadrangularis Linn. (CQ) commonly known as Hadjod (Family: Vitaceae) is usually distributed in India and Sri Lanka and contains several bioactive compounds responsible for various metabolic and physiologic effects. Objective: In this study, the biological effects of CQ ethanolic extract were evaluated by in vitro and supported by in silico analysis on KB oral epidermoid cancer cell line. Materials and Methods: Anti-cancer potential of ethanolic extract of CQ stem against KB oral epidermoid cancer cells was evaluated in terms of morphological analysis, nuclei staining, liberation of reactive oxygen species (ROS), cell cycle arrest, mitochondrial membrane potential (MMP) and p53 and Bcl-2 protein expression which reveal the induction of apoptosis along with supporting in silico analysis. Results: Ethanolic extract of CQ stem contains various bioactive compounds responsible for cancer cell morphological alterations, liberation of ROS, G1 phase cell cycle arrest and decreased MMP along with up-regulation of p53 and down-regulation of Bcl-2. By employing in silico approach, we have also postulated that the CQ extract active constituents sequester Bcl-2 with higher affinity as compared to p53, which may be the reason for induction of growth arrest and apoptosis in KB cells. Conclusion: Our data indicate that the CQ extract has a remarkable apoptotic effect that suggests that it could be a viable treatment option for specific types of cancers. SUMMARY Cissus quadrangularis stem ethanolic extract induces apoptosis and cell cycle arrest at G1 phaseIt liberates (ROS) and mitochondria mediated apoptosisIt upregulates p53 and down-regulates Bcl-2 protein expressionIn silico studies indicates that the active constituents of CQ binds Bcl-2 with higher affinity as compared to p53. PMID:26929569

  10. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells.

    Science.gov (United States)

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including "p38 MAPK Signaling", "p53 Signaling", "14-3-3-mediated Signaling", "p70S6K Signaling" and "Protein Ubiquitination Pathway". This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  11. Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

    Directory of Open Access Journals (Sweden)

    Megumi Kowno

    Full Text Available BACKGROUND: The pluripotent state of embryonic stem (ES cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2, a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1 that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

  12. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme.

    Science.gov (United States)

    Moreadith, R W; Lehninger, A L

    1984-05-25

    Little evidence has been available on the oxidative pathways of glutamine and glutamate, the major respiratory substrates of cancer cells. Glutamate formed from glutamine by phosphate-dependent glutaminase undergoes quantitative transamination by aerobic tumor mitochondria to yield aspartate. However, when malate is also added there is a pronounced decrease in aspartate production and a large formation of citrate and alanine, in both state 3 and 4 conditions. In contrast, addition of malate to normal rat heart, liver, or kidney mitochondria oxidizing glutamate causes a marked increase in aspartate production. Further analysis showed that extramitochondrial malate is oxidized almost quantitatively to pyruvate + CO2 by NAD(P)+-linked malic enzyme, present in the mitochondria of all tumors tested, but absent in heart, liver, and kidney mitochondria. On the other hand intramitochondrial malate generated from glutamate is oxidized quantitatively to oxalacetate by mitochondrial malate dehydrogenase of tumors. Acetyl-CoA derived from extramitochondrial malate via pyruvate and oxalacetate derived from glutamate via intramitochondrial malate are quantitatively converted into citrate, which is extruded. No evidence was found that malic enzyme of tumor mitochondria converts glutamate-derived malate into pyruvate as postulated in other reports. Possible mechanisms for the integration of mitochondrial malic enzyme and malate dehydrogenase activities in tumors are discussed. PMID:6144677

  13. Three dimensional time lapse imaging of live cell mitochondria with photothermal optical lock-in optical coherence microscopy (Conference Presentation)

    Science.gov (United States)

    Sison, Miguel; Chakrabortty, Sabyasachi; Extermann, Jerome; Nahas, Amir; Pache, Christophe; Weil, Tanja; Lasser, Theo

    2016-03-01

    The photothermal optical lock-in optical coherence microscope (poli-OCM) introduced molecular specificity to OCM imaging, which is conventionally, a label-free technique. Here we achieve three-dimensional live cell and mitochondria specific imaging using ~4nm protein-functionalized gold nanoparticles (AuNPs). These nanoparticles do not photobleach and we demonstrate they're suitability for long-term time lapse imaging. We compare the accuracy of labelling with these AuNPs using classical fluorescence confocal imaging with a standard mitochondria specific marker. Furthermore, time lapse poli-OCM imaging every 5 minutes over 1.5 hours period was achieved, revealing the ability for three-dimensional monitoring of mitochondria dynamics.

  14. Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways

    OpenAIRE

    Chia-Jung Li; Shih-Fang Tsang; Chun-Hao Tsai; Hsin-Yi Tsai; Jong-Ho Chyuan; Hsue-Yin Hsu

    2012-01-01

    Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 ...

  15. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

    OpenAIRE

    Jin, Seok Min; Youle, Richard J.

    2013-01-01

    Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPRmt) to increase chaperone production, while the...

  16. Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

    Science.gov (United States)

    Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

    2001-01-01

    Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

  17. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Science.gov (United States)

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments. PMID:25675104

  18. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Directory of Open Access Journals (Sweden)

    Sae Tanaka

    Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  19. Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of genistein on several tumor cell lines were investigated to study the effects of genistein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treatment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were detected in B16 cells, while such effects were not found in K1735M2. Further tests showed that after genistein treatment the protein content and mRNA levels of p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21WAF1/CIP1 increased in both cell lines after treatment.The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoisomerase Ⅱ, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.

  20. [Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria].

    Science.gov (United States)

    Veklich, T O; Kosterin, S O; Shynlova, O P

    2002-01-01

    In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process. PMID:12199098

  1. Interlab Cell Line Collection: Bioresource of Established Human and Animal Cell Lines

    OpenAIRE

    Parodi, Barbara; Aresu, Ottavia; Visconti, Paola; Manniello, Maria Assunta; Strada, Paolo

    2015-01-01

    The Interlab Cell Line Collection (ICLC) was established in 1994 as a core facility of the National Institute of Cancer Research. It supplies: human and animal cell lines; Short Tandem Repeat (STR) profiling of human cell lines; quality control service; mycoplasma detection and eradication service; safe deposit service and patent deposit service of cell lines and hybridomas. The catalogue of services is on-line, and the cell lines are distributed all over the world. 

  2. Protective Effects of Mitochondria-Targeted Peptide MTP-131 on Trabecular Meshwork Cells under Elevated Hydrostatic Pressure

    Directory of Open Access Journals (Sweden)

    Xiaohong Liu

    2013-12-01

    Full Text Available Purpose: To investigate the effects of MTP-131 on trabeculum cells cultured under hydrostatic pressure Methods: Glaucomatous human trabecular meshwork (GTM3 cells were divided into three groups: the MTP-131 group, the hydrostatic group, and the normal group. The MTP-131 group was pretreated with 1 µM MTP-131, and the cells were then cultured under 80 mmHg pressure for 24 hours. The normal group was cultured under normal atmospheric pressure for 24 h, and the hydrostatic group was cultured under 80 mmHg pressure for 24 h. The change in the mitochondrial membrane potential the generation of reactive oxygen species (ROS and the release of cytochrome c from the mitochondria were analyzed with confocal microscopy. The generation of ROS and apoptosis were detected by flow cytometry. The expression of caspase-3 was detected by western blot analysis. Results: In GTM3 cells, culturing under hydrostatic pressure resulted in a decrease in the mitochondrial membrane potential, an elevation of intracellular ROS and a release of cytochrome c from the mitochondria. Pretreatment with MTP-131 prevented the mitochondrial depolarization, decreased the intracellular ROS, prohibited the release of cytochrome c from the mitochondria and inhibited apoptosis. The path of apoptosis is caspase-3 independent. Conclusion: MTP-131 can protect cultured trabecular cells from the damage caused by hydrostatic pressure.

  3. Copper oxide nanoparticles induced mitochondria mediated apoptosis in human hepatocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Maqsood A Siddiqui

    Full Text Available Copper oxide nanoparticles (CuO NPs are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lacking. We explored the possible mechanisms of apoptosis induced by CuO NPs in human hepatocellular carcinoma HepG2 cells. Prepared CuO NPs were spherical in shape with a smooth surface and had an average diameter of 22 nm. CuO NPs (concentration range 2-50 µg/ml were found to induce cytotoxicity in HepG2 cells in dose-dependent manner, which was likely to be mediated through reactive oxygen species generation and oxidative stress. Tumor suppressor gene p53 and apoptotic gene caspase-3 were up-regulated due to CuO NPs exposure. Decrease in mitochondrial membrane potential with a concomitant increase in the gene expression of bax/bcl2 ratio suggested that mitochondria mediated pathway involved in CuO NPs induced apoptosis. This study has provided valuable insights into the possible mechanism of apoptosis caused by CuO NPs at in vitro level. Underlying mechanism(s of apoptosis due to CuO NPs exposure should be further invested at in vivo level.

  4. ZnPcS2P2-based Photodynamic Therapy Induces Mitochondria-dependent Apoptosis in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    Hui-Fang HUANG; Yuan-Zhong CHEN; Yong WU

    2005-01-01

    Mitochondria play a key role in the regulation of apoptosis induced by numerous antitumor chemotherapeutic and other toxic agents. Photodynamic therapy (PDT) exerts significant cellular killing efficacy through either an apoptotic or necrotic cell death pathway. This study investigated the mechanism underlying the killing effects of a novel amphipathic photosensitizer [di-sulfonated di-phthalimidomethyl phthalocyanine zinc (ZnPcS2P2)]-mediated photodynamic therapy (ZnPcS2P2-PDT) on K562 cells. Apoptosis was evident in the post-PDT cells through the TdT-mediated dUTP nick end labeling (TUNEL) method and DNA fragmentation assay. After ZnPcS2P2-PDT, K562 cells underwent mitochondria-dependent apoptosis as evidenced by the release of cytochrome c from mitochondria into cytosol, accompanied by mitochondrial membrane potential (Δ#m) reduction, indicating the opening of the mitochondrial permeability transition pore (PTP). The activities of protease from the caspase family and caspase-3 were also significantly elevated.Furthermore, ZnPcS2P2-PDT down-regulated the expression of chimaeric Bcr-Abl oncoprotein, which is the molecular hallmark of chronic myelogenous leukemia (CML).

  5. Monomethylarsonous acid, but not inorganic arsenic, is a mitochondria-specific toxicant in vascular smooth muscle cells.

    Science.gov (United States)

    Pace, Clare; Banerjee, Tania Das; Welch, Barrett; Khalili, Roxana; Dagda, Ruben K; Angermann, Jeff

    2016-09-01

    Arsenic exposure has been implicated as a risk factor for cardiovascular diseases, metabolic disorders, and cancer, yet the role mitochondrial dysfunction plays in the cellular mechanisms of pathology is largely unknown. To investigate arsenic-induced mitochondrial dysfunction in vascular smooth muscle cells (VSMCs), we exposed rat aortic smooth muscle cells (A7r5) to inorganic arsenic (iAs(III)) and its metabolite monomethylarsonous acid (MMA(III)) and compared their effects on mitochondrial function and oxidative stress. Our results indicate that MMA(III) is significantly more toxic to mitochondria than iAs(III). Exposure of VSMCs to MMA(III), but not iAs(III), significantly decreased basal and maximal oxygen consumption rates and concomitantly increased compensatory extracellular acidification rates, a proxy for glycolysis. Treatment with MMA(III) significantly increased hydrogen peroxide and superoxide levels compared to iAs(III). Exposure to MMA(III) resulted in significant decreases in mitochondrial ATP, aberrant perinuclear clustering of mitochondria, and decreased mitochondrial content. Mechanistically, we observed that mitochondrial superoxide and hydrogen peroxide contribute to mitochondrial toxicity, as treatment of cells with MnTBAP (a mitochondrial superoxide dismutase mimetic) and catalase significantly reduced mitochondrial respiration deficits and cell death induced by both arsenic compounds. Overall, our data demonstrates that MMA(III) is a mitochondria-specific toxicant that elevates mitochondrial and non-mitochondrial sources of ROS. PMID:27327130

  6. Polarographic evaluation of mitochondrial enzymes activity in isolated mitochondria and in permeabilized human muscle cells with inherited mitochondrial defects

    Czech Academy of Sciences Publication Activity Database

    Wenchich, L.; Drahota, Zdeněk; Honzík, T.; Hansíková, H.; Tesařová, M.; Zeman, J.; Houštěk, Josef

    2003-01-01

    Roč. 52, č. 6 (2003), s. 781-788. ISSN 0862-8408 R&D Projects: GA MŠk LN00A079; GA MZd NE6555 Grant ostatní: CZ-FR(CZ) Projekt Barrande 2001-025 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 111100003 Keywords : mitochondria * muscle cells Subject RIV: fb - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 0.939, year: 2003

  7. Effect of chloro- and bromo-derivatives of isocrotonic acid on bioenergetic processes in Ehrlich ascites cells and isolated mitochondria

    International Nuclear Information System (INIS)

    The effect of nine chloro- and bromoderivatives of isocrotonic acid on some bioenergetic processes in both Ehrlich ascites cells and isolated rat liver mitochondria has been investigated. Substances studied in the concentration range 25 to 200 μM significantly inhibited incorporation of both 14C-adenine and 14C-valine into acid-insoluble material of Ehrlich ascites cells. The rate of 14C-precursors incorporation being directly related to the concentration of the inhibitor. γ,γ-bis-4-ethylphenyl-α,β-dichloroisocrotonic acid fully inhibits both aerobic glucose utilization and lactic acid formation at 200 μM concentration. At lower concentrations, however, glycolysis is stimulated. Maximal stimulation of rat liver mitochondrial respiration in state 4 with succinate as substrate was reached at concentrations as low as 10 μM. On the other hand, these substances were able to release the oligomycin-inhibited respiration of rat liver mitochondria. Our data suggest that cytotoxic and cancerostatic action of isocrotonic acid derivatives lies primarily in the exclusion of key processes in the energy metabolism of Ehrlich ascites cells and isolated mitochondria. (author)

  8. Generation of rabbit pluripotent stem cell lines

    OpenAIRE

    Tancos, Z.; Nemes, C.; Polgar, Z.; Gocza, E; Daniel, N; Stout, T. A. E.; P. Maraghechi; Pirity, M.K.; Osteil, P.; Tapponnier, Y.; Markossian, S.; Godet, M.; Afanassieff, M.; Bosze, Z.; Duranthon, V

    2012-01-01

    Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comp...

  9. Telomeres, mitochondria and stem cell aging%端粒、线粒体和干细胞衰老

    Institute of Scientific and Technical Information of China (English)

    黄黎亚; 陈书艳

    2012-01-01

    Stem cells are the special cells which play a key role in the whole life cycle. It is very important to maintain the activity of stem cells, slow down slem cell aging and prolong the life of stem cells. In this paper, the molecular mechanism of stem cell aging is reviewed from the aspects including genome maintenance, telomeres, cell cycle inhibitors and mitochondria. The relationship between telomere-p53 pathway and mitochondria-reactive oxygen species pathway is focused, and the impacts of the two pathways on stem cell aging are introduced.%干细胞是一类在所有生命周期中具有关键性作用的特殊细胞,保持干细胞活力、延缓干细胞衰老及延长干细胞寿命具有重要意义.该文从基因组维护和端粒、细胞周期抑制因子、线粒体等几个方面对干细胞衰老的分子机制作一整体性综述,侧重于端粒-p53通路和线粒体-活性氧自由基途径间的关系及其对干细胞衰老的影响.

  10. Effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells

    Institute of Scientific and Technical Information of China (English)

    Shi-Yong Gao; Qiu-Juan Wang; Yu-Bin Ji

    2006-01-01

    AIM: To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.METHODS: HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy (LCSM). HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca2+]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]i in the cells were observed using LCSM.RESULTS: Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca2+ in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca2+ in the cells at the same time as it lowered the membrane potential of mitochondria.CONCLUSION: Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca2+ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca2+ in the cell, turning on the mechanism for apoptosis.

  11. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  12. A novel synthetic C-1 analogue of 7-deoxypancratistatin induces apoptosis in p53 positive and negative human colorectal cancer cells by targeting the mitochondria: enhancement of activity by tamoxifen.

    Science.gov (United States)

    Ma, Dennis; Tremblay, Phillip; Mahngar, Kevinjeet; Akbari-Asl, Pardis; Collins, Jonathan; Hudlicky, Tomas; McNulty, James; Pandey, Siyaram

    2012-06-01

    The natural compound pancratistatin (PST), isolated from the Hymenocallis littoralis plant, specifically induces apoptosis in many cancer cell lines. Unlike many other chemotherapeutics, PST is not genotoxic and has minimal adverse effects on non-cancerous cells. However, its availability for preclinical and clinical work is limited due to its low availability in its natural source and difficulties in its chemical synthesis. Several synthetic analogues of 7-deoxypancratistatin with different modifications at C-1 were synthesized and screened for apoptosis inducing activity in human colorectal cancer (CRC) cells. We found that a C-1 acetoxymethyl derivative of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), was effective in inducing apoptosis in both p53 positive (HCT 116) and p53 negative (HT-29) human CRC cell lines, demonstrating similar efficacy to that of natural PST. JCTH-4 was able to decrease mitochondrial membrane potential (MMP), increase levels of reactive oxygen species in isolated mitochondria, cause release of the apoptogenic factor cytochrome c (Cyto c) from isolated mitochondria, and induce autophagy in HCT 116 and HT-29 cells. Interestingly, when JCTH-4 was administered with tamoxifen (TAM), there was an enhanced effect in apoptosis induction, reactive oxygen species (ROS) production and Cyto c release by isolated mitochondria, and autophagic induction by CRC cells. Minimal toxicity was exhibited by a normal human fetal fibroblast (NFF) and a normal colon fibroblast (CCD-18Co) cell line. Hence, JCTH-4 is a novel compound capable of selectively inducing apoptosis and autophagy in CRC cells alone and in combination with TAM and may serve as a safer and more effective alternative to current cancer therapies. PMID:21494837

  13. Human Cell Line and Tissue Sample Authentication

    OpenAIRE

    Ewing, Margaret M.; McLaren, Robert S.; Hebble, Kathryn D.; Ready, Kim; Storts, Douglas R.; Hooper, Kyle

    2013-01-01

    Background: Short Tandem Repeat (STR) genotyping analysis is a proven technology for uniquely identifying virtually all human samples. STR genotyping was adopted as the preferred technology for identification of human tissue culture cell lines by the ATCC Standards Development Organization (ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling). We developed new automation-compatible protocols/systems for generating STR profiles from human cell lines or tissue samples...

  14. Smac/DIABLO release from mitochondria and XIAP inhibition are essential to limit clonogenicity of Type I tumor cells after TRAIL receptor stimulation.

    Science.gov (United States)

    Maas, C; Verbrugge, I; de Vries, E; Savich, G; van de Kooij, L W; Tait, S W G; Borst, J

    2010-10-01

    Death receptors, such as Fas/CD95 and TRAIL receptors, engage the extrinsic pathway for caspase activation, but also couple to the intrinsic mitochondrial route. In so-called Type II cells, death receptors require the mitochondrial pathway for apoptotic execution, whereas in Type I cells they reportedly do not. For established tumor cell lines, the Type I/Type II distinction is based on short-term apoptosis assays. We report here that the mitochondrial pathway is essential for apoptotic execution of Type I tumor cells by death receptors, when long-term clonogenicity is taken into account. A blockade of the mitochondrial pathway in Type I tumor cells - by RNA interference for Bid or Bcl-2 overexpression - reduced effector caspase activity and mediated significant clonogenic resistance to TRAIL. Downstream from the mitochondria, Caspase-9 did not contribute to clonogenic death of TRAIL-treated Type I cells. Rather, the release of Smac/DIABLO and the inhibition of XIAP activity proved to be crucial for full effector caspase activity and clonogenic execution. Thus, in Type I cells the intrinsic pathway downstream from death receptors is not redundant, but limits clonogenicity by virtue of Smac/DIABLO release and XIAP inhibition. This finding is relevant for cancer therapy using death receptor agonists. PMID:20395960

  15. A new hypothesis of pathogenesis based on the divorce between mitochondria and their host cells: possible relevance for Alzheimer's disease.

    Science.gov (United States)

    Agnati, L F; Guidolin, D; Baluska, F; Leo, G; Barlow, P W; Carone, C; Genedani, S

    2010-06-01

    On the basis of not only the endosymbiotic theory of eukaryotic cell organization and evolution but also of observations of transcellular communication via Tunneling NanoTubes (TNTs), the hypothesis is put forward that when mitochondria, which were once independently living prokaryote-like organisms, are subjected to detrimental genetic, toxic, or environmental conditions, including age-related endogenous factors, they can regress towards their original independent state. At that point, they can become potentially pathogenic intruders within their eukaryotic host cell. Because of the protoplasmic disequilibrium caused by an altered, or mutated, mitochondral population, certain host cells with a minimal capacity for self-renewal, such as dopaminergic neurons, risk a loss of function and degenerate. It is also proposed that altered mitochondria, as well as their mutated mtDNA, can migrate, via TNTs, into adjacent cells. In this way, neurodegenerative states are propagated between cells (glia and/or neurons) of the Central Nervous System (CNS) and that this leads to conditions such as Alzheimer's and Parkinson's disease. This proposal finds indirect support from observations on rotenone-poisoned glioblastoma cells which have been co-cultured with non-poisoned cells. Immunocytochemical techniques revealed that mitochondria, moving along the TNTs, migrated from the poisoned cells towards the healthy cells. It has also been demonstrated by means of immunocytochemistry that, in glioblastoma cell cultures, Amyloid Precursor Protein (APP) is present in TNTs, hence it may migrate from one cell to neighbouring cells. This datum may be of high relevance for a better understanding of Alzheimer's Disease (AD) since molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the APP are key pathogenic factors in AD, causing mitochondrial dysfunction, free radical generation, oxidative damage, and inflammation

  16. Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death.

    Science.gov (United States)

    Lu, Yapeng; Liu, Siyuan; Wang, Ying; Wang, Dang; Gao, Jing; Zhu, Li

    2016-09-01

    Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells. PMID:27288117

  17. Mitochondria and calcium flux as targets of neuroprotection caused by minocycline in cerebellar granule cells

    OpenAIRE

    Garcia-Martinez, Eva Maria; Sanz-Blasco, Sara; Karachitos, Andonis; Bandez, Miguel J.; Fernandez-Gomez, Francisco J.; Perez-Alvarez, Sergio; Mera, Raquel Maria Melero Fernandez De; Jordan, Maria J.; Aguirre, Norberto; Galindo, Maria F.; Villalobos, Carlos; Navarro, Ana; Kmita, Hanna; Jordán, Joaquín

    2009-01-01

    Abstract Minocycline, an antibiotic of the tetracycline family, has attracted considerable interest for its theoretical therapeutic applications in neurodegenerative diseases. However, the mechanism of action underlying its effect remains elusive. Here we have studied the effect of minocycline under excitotoxic conditions. Fluorescence and bioluminescence imaging studies in rat cerebellar granular neuron cultures using fura-2/AM and mitochondria-targeted aequorin revealed that mino...

  18. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

    Science.gov (United States)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-05-11

    Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 μM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer. PMID:22546556

  19. Licochalcone A-Induced Human Bladder Cancer T24 Cells Apoptosis Triggered by Mitochondria Dysfunction and Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Xuan Yuan

    2013-01-01

    Full Text Available Licochalcone A (LCA, a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigated the mechanisms involved in LCA-induced apoptosis in human bladder cancer T24 cells. LCA significantly inhibited cells proliferation, increased reactive oxygen species (ROS levels, and caused T24 cells apoptosis. Moreover, LCA induced mitochondrial dysfunction, caspase-3 activation, and poly-ADP-ribose polymerase (PARP cleavage, which displayed features of mitochondria-dependent apoptotic signals. Besides, exposure of T24 cells to LCA triggered endoplasmic reticulum (ER stress; as indicated by the enhancement in 78 kDa glucose-regulated protein (GRP 78, growth arrest and DNA damage-inducible gene 153/C/EBP homology protein (GADD153/CHOP expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12. All the findings from our study suggest that LCA initiates mitochondrial ROS generation and induces oxidative stress that consequently causes T24 cell apoptosis via the mitochondria-dependent and the ER stress-triggered signaling pathways.

  20. Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells.

    Science.gov (United States)

    Cuvillier, O; Edsall, L; Spiegel, S

    2000-05-26

    Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells. PMID:10747891

  1. Mitochondria and Cancer.

    Science.gov (United States)

    Zong, Wei-Xing; Rabinowitz, Joshua D; White, Eileen

    2016-03-01

    Decades ago, Otto Warburg observed that cancers ferment glucose in the presence of oxygen, suggesting that defects in mitochondrial respiration may be the underlying cause of cancer. We now know that the genetic events that drive aberrant cancer cell proliferation also alter biochemical metabolism, including promoting aerobic glycolysis, but do not typically impair mitochondrial function. Mitochondria supply energy; provide building blocks for new cells; and control redox homeostasis, oncogenic signaling, innate immunity, and apoptosis. Indeed, mitochondrial biogenesis and quality control are often upregulated in cancers. While some cancers have mutations in nuclear-encoded mitochondrial tricarboxylic acid (TCA) cycle enzymes that produce oncogenic metabolites, there is negative selection for pathogenic mitochondrial genome mutations. Eliminating mtDNA limits tumorigenesis, and rare human tumors with mutant mitochondrial genomes are relatively benign. Thus, mitochondria play a central and multifunctional role in malignant tumor progression, and targeting mitochondria provides therapeutic opportunities. PMID:26942671

  2. Exogenous ether lipids predominantly target mitochondria

    DEFF Research Database (Denmark)

    Kuerschner, Lars; Richter, Doris; Hannibal-Bach, Hans Kristian;

    2012-01-01

    Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high......, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in...

  3. Mitochondria and Endothelial Function

    OpenAIRE

    Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

    2013-01-01

    In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review...

  4. Mitochondria and neuroplasticity

    OpenAIRE

    Cheng, Aiwu; Hou, Yan; Mattson, Mark P.

    2010-01-01

    The production of neurons from neural progenitor cells, the growth of axons and dendrites and the formation and reorganization of synapses are examples of neuroplasticity. These processes are regulated by cell-autonomous and intercellular (paracrine and endocrine) programs that mediate responses of neural cells to environmental input. Mitochondria are highly mobile and move within and between subcellular compartments involved in neuroplasticity (synaptic terminals, dendrites, cell body and th...

  5. Targeting mitochondria in cancer cells using gold nanoparticle-enhanced radiotherapy: A Monte Carlo study

    Energy Technology Data Exchange (ETDEWEB)

    Kirkby, Charles, E-mail: charles.kirkby@albertahealthservices.ca; Ghasroddashti, Esmaeel [Department of Medical Physics, Jack Ady Cancer Centre, Lethbridge, Alberta T1J 1W5 (Canada); Department of Physics and Astronomy, University of Calgary, Calgary, Alberta T2N 1N4 (Canada); Department of Oncology, University of Calgary, Calgary, Alberta T2N 4N2 (Canada)

    2015-02-15

    Purpose: Radiation damage to mitochondria has been shown to alter cellular processes and even lead to apoptosis. Gold nanoparticles (AuNPs) may be used to enhance these effects in scenarios where they collect on the outer membranes of mitochondria. A Monte Carlo (MC) approach is used to estimate mitochondrial dose enhancement under a variety of conditions. Methods: The PENELOPE MC code was used to generate dose distributions resulting from photons striking a 13 nm diameter AuNP with various thicknesses of water-equivalent coatings. Similar dose distributions were generated with the AuNP replaced by water so as to estimate the gain in dose on a microscopic scale due to the presence of AuNPs within an irradiated volume. Models of mitochondria with AuNPs affixed to their outer membrane were then generated—considering variation in mitochondrial size and shape, number of affixed AuNPs, and AuNP coating thickness—and exposed (in a dose calculation sense) to source spectra ranging from 6 MV to 90 kVp. Subsequently dose enhancement ratios (DERs), or the dose with the AuNPs present to that for no AuNPs, for the entire mitochondrion and its components were tallied under these scenarios. Results: For a representative case of a 1000 nm diameter mitochondrion affixed with 565 AuNPs, each with a 13 nm thick coating, the mean DER over the whole organelle ranged from roughly 1.1 to 1.6 for the kilovoltage sources, but was generally less than 1.01 for the megavoltage sources. The outer membrane DERs remained less than 1.01 for the megavoltage sources, but rose to 2.3 for 90 kVp. The voxel maximum DER values were as high as 8.2 for the 90 kVp source and increased further when the particles clustered together. The DER exhibited dependence on the mitochondrion dimensions, number of AuNPs, and the AuNP coating thickness. Conclusions: Substantial dose enhancement directly to the mitochondria can be achieved under the conditions modeled. If the mitochondrion dose can be directly

  6. Targeting mitochondria in cancer cells using gold nanoparticle-enhanced radiotherapy: A Monte Carlo study

    International Nuclear Information System (INIS)

    Purpose: Radiation damage to mitochondria has been shown to alter cellular processes and even lead to apoptosis. Gold nanoparticles (AuNPs) may be used to enhance these effects in scenarios where they collect on the outer membranes of mitochondria. A Monte Carlo (MC) approach is used to estimate mitochondrial dose enhancement under a variety of conditions. Methods: The PENELOPE MC code was used to generate dose distributions resulting from photons striking a 13 nm diameter AuNP with various thicknesses of water-equivalent coatings. Similar dose distributions were generated with the AuNP replaced by water so as to estimate the gain in dose on a microscopic scale due to the presence of AuNPs within an irradiated volume. Models of mitochondria with AuNPs affixed to their outer membrane were then generated—considering variation in mitochondrial size and shape, number of affixed AuNPs, and AuNP coating thickness—and exposed (in a dose calculation sense) to source spectra ranging from 6 MV to 90 kVp. Subsequently dose enhancement ratios (DERs), or the dose with the AuNPs present to that for no AuNPs, for the entire mitochondrion and its components were tallied under these scenarios. Results: For a representative case of a 1000 nm diameter mitochondrion affixed with 565 AuNPs, each with a 13 nm thick coating, the mean DER over the whole organelle ranged from roughly 1.1 to 1.6 for the kilovoltage sources, but was generally less than 1.01 for the megavoltage sources. The outer membrane DERs remained less than 1.01 for the megavoltage sources, but rose to 2.3 for 90 kVp. The voxel maximum DER values were as high as 8.2 for the 90 kVp source and increased further when the particles clustered together. The DER exhibited dependence on the mitochondrion dimensions, number of AuNPs, and the AuNP coating thickness. Conclusions: Substantial dose enhancement directly to the mitochondria can be achieved under the conditions modeled. If the mitochondrion dose can be directly

  7. Effect of Smac in combination with cisplatin on esophageal cancer cell line ECA109

    OpenAIRE

    Hou, Liang; Chen, Kang; Hao, Yingtao; Zhao, Yunpeng; Sun, Qifeng; Zhao, Xiaogang; Peng, Chuanliang

    2015-01-01

    Objective: This study was to investigate inhibiting effect of structurally unique Second mitochondria-derived activator of caspase (Smac) in combination with cisplatin on esophageal cancer cell line ECA109. Methods: PcDNA3.1-Smac (ECA109/Smac group), pcDNA3.1 (ECA109/neo group) and PBS (ECA109 or control group) were transfected into ECA109 cells respectively, and transfected cells which expressed Smac stably were got. Smac protein expression was analyzed by Western blot. The invasive ability ...

  8. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  9. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G2/M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G2/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  10. Chromium(VI)-induced Production of Reactive Oxygen Species, Change of Plasma Membrane Potential and Dissipation of Mitochondria Membrane Potential in Chinese Hamster Lung Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To examine whether Reactive Oxygen Species (ROS) is generated, and whether plasma membrane potential and mitochondrial membrane potential are depolarized in Chinese Hamster Lung (CHL) cell lines exposed to Cr (VI). Methods CHL cells were incubated with Cr(VI) at 10 μmol/L, 2.5 μmol/L, 0.65 μmol/L for 3 and 6 hours, respectively. The production of ROS was performed by using 2,7_dichlorofluorescin diacetate; The changes in plasma membrane potential were estimated using fluorescent cationic dye DiBAC4; And the changes in mitochondria membrane potential were estimated using fluorescent dye Rhodamine 123. Results The ROS levels in CHL cells increased in all treated groups compared with the control group (P<0.01); The plasma membrane potential and mitochondrial membrane potential in CHL cells dissipated after incubated with Cr(VI) at 10 μmol/L for 3 hours and 6 hours (P<0.01), at 2.5 μmol/L for 6 hours (P<0.01 or 0.05). Conclusion Cr(VI) causes the dissipation of plasma membrane potential and mitochondrial membrane potential in CHL cell cultures, and Cr(VI)_induced ROS may play a role in the injuries.

  11. Chromium(VI)—induces Production of Reactive Oxygen Species,Change of Plasma Membrane Potential and Dissipation of Mitochondria Membrane otential in Chinese Hamster Lung Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    XIEYI; ZHUANGZHI-XIONG

    2001-01-01

    Objective:To examine whether Reactive Oxygen Species(ROS) is generated,and whether plasma membrane potential and mitochnodrial membrane potential are depolarized in Chinese Hamster Lung(CHL)cell lines exposed to Cr(VI),Methods:CHL Cells were incubated with Cr(VI) at 10 umol/L,2.5umol/L,0.65umol/L for 3 and 6 hours,respectively.The rpoduction of ROS was performed by using 2,7-dichlorofluorescin discetate;The changes in plasma membrane potential were performed by using 2,7-dichlorofluorescin discetate;The changes in plasma membrane potential were performed by using 2,7-dichlorofluorescin diacetate;The changes in plasma membrane potential were estimated using fluorescent cationic dye DiBAC4;And the changes in mitochondria membrane potential were estimated using fluorescent dye Rhodamine 123,Results:The ROS levels in CHL cells increased in all treated groups compared with the control group(P<0.01);The plasma membrane potential and mitochondrial membrane potential in CHL cells dissipated after incubated with Cr(VI) at 10umol/L for 3 hours and 6 hours(P<0.01),at 2.5umol/L for 6 hours(P<0.01 or 0.05),Conclusion:Cr(VI) causes the dissipation of plasma membrane potential and mitochnodrial membrane otential in CHL cell cultrues,and Cr(VI)-induced ROS may play a role in the injuries.

  12. Multimodal Upconversion Nanoplatform with a Mitochondria-Targeted Property for Improved Photodynamic Therapy of Cancer Cells.

    Science.gov (United States)

    Zhang, Xiaoman; Ai, Fujin; Sun, Tianying; Wang, Feng; Zhu, Guangyu

    2016-04-18

    Upconversion nanoparticles (UCNPs) with the capacity to emit high-energy visible or UV light under low-energy near-infrared excitation have been extensively explored for biomedical applications including imaging and photodynamic therapy (PDT) against cancer. Enhanced cellular uptake and controlled subcellular localization of a UCNP-based PDT system are desired to broaden the biomedical applications of the system and to increase its PDT effect. Herein, we build a multimodal nanoplatform with enhanced therapeutic efficiency based on 808 nm excited NaYbF4:Nd@NaGdF4:Yb/Er@NaGdF4 core-shell-shell nanoparticles that have a minimized overheating effect. The photosensitizer pyropheophorbide a (Ppa) is loaded onto the nanoparticles capped with biocompatible polymers, and the nanoplatform is functionalized with transcriptional activator peptides as targeting moieties. Significantly increased cellular uptake of the nanoparticles and dramatically elevated photocytotoxicity are achieved. Remarkably, colocalization of Ppa with mitochondria, a crucial subcellular organelle as a target of PDT, is proven and quantified. The subsequent damage to mitochondria caused by this colocalization is also confirmed to be significant. Our work provides a comprehensively improved UCNP-based nanoplatform that maintains great biocompatibility but shows higher photocytotoxicity under irradiation and superior imaging capabilities, which increases the biomedical values of UCNPs as both nanoprobes and carriers of photosensitizers toward mitochondria for PDT. PMID:27049165

  13. Mitochondria and Cancer.

    Science.gov (United States)

    Vyas, Sejal; Zaganjor, Elma; Haigis, Marcia C

    2016-07-28

    Mitochondria are bioenergetic, biosynthetic, and signaling organelles that are integral in stress sensing to allow for cellular adaptation to the environment. Therefore, it is not surprising that mitochondria are important mediators of tumorigenesis, as this process requires flexibility to adapt to cellular and environmental alterations in addition to cancer treatments. Multiple aspects of mitochondrial biology beyond bioenergetics support transformation, including mitochondrial biogenesis and turnover, fission and fusion dynamics, cell death susceptibility, oxidative stress regulation, metabolism, and signaling. Thus, understanding mechanisms of mitochondrial function during tumorigenesis will be critical for the next generation of cancer therapeutics. PMID:27471965

  14. Unveiling interactions among mitochondria, caspase-like proteases, and the actin cytoskeleton during plant programmed cell death (PCD.

    Directory of Open Access Journals (Sweden)

    Christina E N Lord

    Full Text Available Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD. PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B, a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1 activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs. The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.

  15. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  16. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy. PMID:23874836

  17. Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria.

    Science.gov (United States)

    Cuvillier, O; Levade, T

    2001-11-01

    Sphingosine 1-phosphate (S-1P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-1P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-alpha (TNF-alpha), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which stimulates sphingosine kinase, the enzyme responsible for S-1P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethylsphingosine (DMS), a specific inhibitor of sphingosine kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-alpha, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-1P cotreatment. Hence, S-1P, likely generated through a protein kinase C- mediated activation of sphingosine kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells. PMID:11675357

  18. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    Science.gov (United States)

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  19. Standards for Cell Line Authentication and Beyond

    Science.gov (United States)

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  20. Establishment of a hamster lymphoma cell line

    Directory of Open Access Journals (Sweden)

    Abe,Shinji

    1974-08-01

    Full Text Available The establishment of a hamster lymphoma cell line was attempted. Simple mincing and trypsinization of lymphoma tissue resulted in a high degree of cell degeneration. The ascitic tumor cells produced by intraperitoneal transplantation of lymphoma tissue gave a better result. These ascitic cells grew and were cultured successively in medium consisting of RPMI 1640 and 20% fetal calf serum. Cells were round and grew in suspension. Accelerated cell growth was observed one month after starting the culture. In the stained preparations, cells were lymphoblastic. Cells were transplantable into new-born hamsters and produced tumors, but not in young adult hamsters.

  1. Near neighbour analysis of variant cell lines derived from the promyeloid cell line HL60.

    OpenAIRE

    Bunce, C. M.; Lord, J M; Wong, A K; Brown, G.

    1988-01-01

    The human promyeloid cell line H60 can be induced to differentiate towards either neutrophils or monocytes. Variant cell lines, derived from HL60, which show reduced capacities for neutrophil and monocyte differentiation can be arranged in a developmental sequence which suggests that the potentials for neutrophil and monocyte differentiation are expressed sequentially by HL60 cells in this order. Analysis of the patterns of total cellular phosphoproteins within HL60 and 5 variant cell lines, ...

  2. Susceptibility of cell lines to avian viruses

    Directory of Open Access Journals (Sweden)

    Simoni Isabela Cristina

    1999-01-01

    Full Text Available The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA. CPE and viral RNA were detected in CER and BHK-21 cells after reovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV.

  3. Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

    International Nuclear Information System (INIS)

    An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

  4. Cytoplasmic hybrid (cybrid cell lines as a practical model for mitochondriopathies

    Directory of Open Access Journals (Sweden)

    Heather M. Wilkins

    2014-01-01

    Full Text Available Cytoplasmic hybrid (cybrid cell lines can incorporate human subject mitochondria and perpetuate its mitochondrial DNA (mtDNA-encoded components. Since the nuclear background of different cybrid lines can be kept constant, this technique allows investigators to study the influence of mtDNA on cell function. Prior use of cybrids has elucidated the contribution of mtDNA to a variety of biochemical parameters, including electron transport chain activities, bioenergetic fluxes, and free radical production. While the interpretation of data generated from cybrid cell lines has technical limitations, cybrids have contributed valuable insight into the relationship between mtDNA and phenotype alterations. This review discusses the creation of the cybrid technique and subsequent data obtained from cybrid applications.

  5. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    International Nuclear Information System (INIS)

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  6. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ying [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Nie, Ling [Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Yin, Yang-Guang [Emergency Department, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Zhou, Shi-Wen, E-mail: Zhoushiwen1956@yahoo.cn [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China)

    2012-03-15

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  7. Time-resolved spectroscopy of mitochondria, cells and tissues under normal and pathological conditions.

    Science.gov (United States)

    Beauvoit, B; Chance, B

    1998-07-01

    In this study, the detailed dependence of light scattering on tissue architecture and intracellular composition has been investigated. Firstly, we simulated the reduced scattering coefficient (mu(s)') of the rat liver using the Mie theory, the Rayleigh-Debye-Gans approximation and electron microscopy data. Then, the reduced scattering coefficient of isolated rat liver mitochondria, isolated hepatocytes and various rat tissues (i.e. perfused liver, brain, muscle, tumors) was measured at 780 nm by using time-resolved spectroscopy and a sample-substitution protocol. The comparison of the isolated mitochondria data with the isolated hepatocyte and whole liver measurements suggests that the mitochondrial compartment is the primary factor for light propagation in hepatic tissue, thus strengthening the relevance of the preliminary theoretical study. Nevertheless, the possibility that other intracellular components, such as peroxisomes and lysosomes, interfere with light propagation in rat liver is discussed. Finally, we demonstrate that light scattering in normal rat tissues and tumors is roughly proportional to the mitochondrial content, according to estimates of the mitochondrial protein content of the tissues. PMID:9746339

  8. Analysis of glutathione and glutathione disulfide in whole cells and mitochondria by postcolumn derivatization high-performance liquid chromatography with ortho-phthalaldehyde.

    Science.gov (United States)

    Lenton, K J; Therriault, H; Wagner, J R

    1999-10-01

    A method is described for the detection of glutathione (GSH) and glutathione disulfide (GSSG) based on a HPLC postcolumn reaction with ortho-phthalaldehyde (OPT) at pH 12 followed by fluorescence detection. Although similar methods have been reported, the high pH of the postcolumn reaction adds considerable selectivity and sensitivity to the measurement of GSH and glutathione disulfide. The limit of detection approaches 100 fmol, which is sufficient to detect whole-cell glutathione disulfide in 10,000 cells or mitochondrial glutathione disulfide in 20 million cells. Using this method, glutathione and glutathione disulfide were measured in human lymphocytes, granulocytes, and cultured Jurkat T cells, as well as in the corresponding samples of mitochondria. The percentage of glutathione disulfide to total glutathione in whole-cell extracts was approximately 1%. In contrast, the percentage was relatively high in mitochondria, with the mitochondria of granulocytes having the highest (25%) followed by those of lymphocytes (15%) and finally by cultured Jurkat T cells (9%). This method extends the analysis of glutathione and glutathione disulfide to mitochondria obtained from a relatively small number of cells. PMID:10527505

  9. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

    KAUST Repository

    Radogna, Flavia

    2015-03-01

    Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

  10. Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

    Science.gov (United States)

    Qian, Lei; Bradford, Andrew M; Cooke, Peter H; Lyons, Barbara A

    2016-07-01

    Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26869103

  11. Ontogenic change in tissue osmolality and developmental sequence of mitochondria-rich cells in Mozambique tilapia developing in freshwater.

    Science.gov (United States)

    Yanagie, Ryohei; Lee, Kyung Mi; Watanabe, Soichi; Kaneko, Toyoji

    2009-10-01

    We investigated a change in tissue fluid osmolality and developmental sequences of mitochondria-rich (MR) cells during embryonic and larval stages of Mozambique tilapia, Oreochromis mossambicus, developing in freshwater. Tissue osmolality, representing body fluid osmolality, ranged from 300 to 370 mOsm/kg during embryonic and larval stages. This suggests that tilapia embryos and larvae are also able to regulate body fluid osmolality to some extent, although the levels are somewhat higher and fluctuate more greatly in embryos and larvae than in adults. Na(+)/K(+)-ATPase-immunoreactive MR cells were first detected in the yolk-sac membrane 3 days before hatching (day -3), followed by their appearance in the body skin on day -2. Subsequently, MR cells in both the yolk-sac membrane and body skin increased in number, and most densely observed on days -1 and 0. Whereas yolk-sac and skin MR cells decreased after hatching, MR cells in turn started developing in the gills after hatching. Thus, the principal site for MR cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gills during larval stages, and tilapia could maintain continuously their ion balance through those MR cells during early life stages. PMID:19589394

  12. Quantitative proteomics reveals oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to rotenone

    OpenAIRE

    Villeneuve, Lance; Tiede, LeAnn M.; Morsey, Brenda; Fox, Howard S

    2013-01-01

    Mitochondria are implicated in a variety of degenerative disorders and aging. Mitochondria are responsive to the oxygen in their environment yet tissue culture is performed at atmospheric (21%) oxygen and not at physiological (1-11%) oxygen levels found in tissues. We employed imaging of mitochondrial probes, mass spectrometry, western blots and ATP assays of the human neuroblastoma cell-line SH-SY5Y; and imaging of mitochondrial probes in human primary neurons in standard non-physiological o...

  13. Capsaicin induces apoptosis in SCC-4 human tongue cancer cells through mitochondria-dependent and -independent pathways.

    Science.gov (United States)

    Ip, Siu-Wan; Lan, Sheng-Hui; Huang, An-Cheng; Yang, Jai-Sing; Chen, Ya-Yin; Huang, Hui-Ying; Lin, Zen-Pin; Hsu, Yuan-Man; Yang, Mei-Due; Chiu, Chang-Fang; Chung, Jing-Gung

    2012-05-01

    Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose-dependent manner in human tongue cancer SCC-4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub-G1 phase), and induced G0/G1 phase arrest in SCC-4 cells. We demonstrated that capsaicin-induced apoptosis is associated with an increase in reactive oxygen species and Ca²⁺ generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ(m)). Treatment with capsaicin induced a dramatic increase in caspase-3 and -9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin-induced apoptosis is involved in the activation of caspase-3 (one of the apoptosis-executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF-4, and GADD153 from mitochondria of SCC-4 cells. PMID:20925121

  14. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    International Nuclear Information System (INIS)

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  15. JWA is required for arsenic trioxide induced apoptosis in HeLa and MCF-7 cells via reactive oxygen species and mitochondria linked signal pathway

    International Nuclear Information System (INIS)

    Arsenic trioxide, emerging as a standard therapy for refractory acute promyelocytic leukemia, induces apoptosis in a variety of malignant cell lines. JWA, a novel retinoic acid-inducible gene, is known to be involved in apoptosis induced by various agents, for example, 12-O-tetradecanoylphorbol 13-acetate, N-4-hydroxy-phenyl-retinamide and arsenic trioxide. However, the molecular mechanisms underlying how JWA gene is functionally involved in apoptosis remain largely unknown. Herein, our studies demonstrated that treatment of arsenic trioxide produced apoptosis in HeLa and MCF-7 cells in a dose-dependent manner and paralleled with increased JWA expression. JWA expression was dependent upon generation of intracellular reactive oxygen species induced by arsenic trioxide. Knockdown of JWA attenuated arsenic trioxide induced apoptosis, and was accompanied by significantly reduced activity of caspase-9, enhanced Bad phosphorylation and inhibited MEK1/2, ERK1/2 and JNK phosphorylations. Arsenic trioxide induced loss of mitochondrial transmembrane potential was JWA-dependent. These findings suggest that JWA may serve as a pro-apoptotic molecule to mediate arsenic trioxide triggered apoptosis via a reactive oxygen species and mitochondria-associated signal pathway

  16. Proteomics of human mitochondria

    DEFF Research Database (Denmark)

    Palmfeldt, Johan; Bross, Peter

    2016-01-01

    Proteomics have passed through a tremendous development in the recent years by the development of ever more sensitive, fast and precise mass spectrometry methods. The dramatically increased research in the biology of mitochondria and their prominent involvement in all kinds of diseases and ageing...... sensitivity of mass spectrometry technology aids in lowering this hurdle and new approaches like generation of induced pluripotent cells from somatic cells allow to produce patient-specific cellular disease models with great potential. We describe which human sample types are accessible, review the status of...... the catalog of human mitochondrial proteins and discuss proteins with dual localization in mitochondria and other cellular compartments. We describe the status and developments of pertinent mass spectrometric strategies, and the use of databases and bioinformatics. Using selected illustrative examples...

  17. Glutathione and mitochondria

    OpenAIRE

    Ribas, Vicent; García-Ruiz, Carmen; Fernández-Checa, José C.

    2014-01-01

    Glutathione (GSH) is the main non-protein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the e...

  18. A TP-FRET-based two-photon fluorescent probe for ratiometric visualization of endogenous sulfur dioxide derivatives in mitochondria of living cells and tissues.

    Science.gov (United States)

    Yang, Xiaoguang; Zhou, Yibo; Zhang, Xiufang; Yang, Sheng; Chen, Yun; Guo, Jingru; Li, Xiaoxuan; Qing, Zhihe; Yang, Ronghua

    2016-08-11

    A ratiometric two-photon fluorescent probe for SO2 derivatives was first proposed based on acedan-merocyanine dyads via a TP-FRET strategy. It was successfully applied to visualization of the fluctuations of enzymatically generated SO2 derivatives in the mitochondria of HepG2 cells and rat liver tissues using two-photon fluorescence microscopy imaging. PMID:27469474

  19. Mitochondria functionality and sperm quality

    OpenAIRE

    Amaral, Alexandra; Lourenço, Bárbara; Marques, Mónica; Ramalho-Santos, J.

    2013-01-01

    Although mitochondria are best known for being the eukaryotic cell powerhouses, these organelles participate in various cellular functions besides ATP production, such as calcium homoeostasis, generation of reactive oxygen species (ROS), the intrinsic apoptotic pathway and steroid hormone biosynthesis. The aim of this review was to discuss the putative roles of mitochondria in mammalian sperm function and how they may relate to sperm quality and fertilisation ability, particularly in...

  20. Mitochondria: Redox Metabolism and Dysfunction

    OpenAIRE

    Jia Kang; Shazib Pervaiz

    2012-01-01

    Mitochondria are the main intracellular location for fuel generation; however, they are not just power plants but involved in a range of other intracellular functions including regulation of redox homeostasis and cell fate. Dysfunction of mitochondria will result in oxidative stress which is one of the underlying causal factors for a variety of diseases including neurodegenerative diseases, diabetes, cardiovascular diseases, and cancer. In this paper, generation of reactive oxygen/nitrogen sp...

  1. Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway.

    Science.gov (United States)

    Wang, Xiaorui; Miao, Junqiu; Yan, Chaoqun; Ge, Rui; Liang, Taigang; Liu, Enli; Li, Qingshan

    2016-10-20

    Dibutyltin (DBT) which was widely used as biocide and plastic stabilizer has been described as a potent neurotoxicant. Chitosan (CS), a natural nontoxic biopolymer, possesses a variety of biological activities including antibacterial, antifungal, free radical scavenging and neuroprotective activities. The present study was undertaken to investigate the protective effects of CS against DBT-induced apoptosis in rat pheochromocytoma (PC12) cells and the underlying mechanisms in vitro. Our results demonstrated that pretreatment with CS significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release induced by DBT in a dose-dependent manner. Meanwhile, DBT-induced cell apoptosis, mitochondrial membrane potential (MMP) disruption, and generation of intracellular reactive oxygen species (ROS) were attenuated by CS. Real-time PCR assay showed that DBT markedly enhanced the mRNA levels of Bax, Bad, cytochrome-c and Apaf-1, reduced the Bcl-2 and Bcl-xL mRNA levels, while these genes expression alteration could be partially reversed by CS treatment. Furthermore, CS also inhibited the DBT-inducted activation of caspase-9, and -3 at mRNA and protein expression levels. Taken together, these results suggested that CS could protect the PC12 cells from apoptosis induced by DBT through inhibition of the mitochondria-dependent pathway. PMID:27474647

  2. Autophagy Protects Against Senescence and Apoptosis via the RAS-Mitochondria in High-Glucose-Induced Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Fei Chen

    2014-04-01

    Full Text Available Backgrounds: Autophagy is an important process in the pathogenesis of diabetes and plays a critical role in maintaining cellular homeostasis. However, the autophagic response and its mechanism in diabetic vascular endothelium remain unclear. Methods and Results: We studied high-glucose-induced renin-angiotensin system (RAS-mitochondrial damage and its effect on endothelial cells. With regard to therapeutics, we investigated the beneficial effect of angiotensin-converting enzyme inhibitors (ACEIs or angiotensin II type 1 receptor blockers (ARBs against high-glucose-induced endothelial responses. High glucose activated RAS, enhanced mitochondrial damage and increased senescence, apoptosis and autophagic-responses in endothelial cells, and these effects were mimicked by using angiotensin II (Ang. The use of an ACEI or ARB, however, inhibited the negative effects of high glucose. Direct mitochondrial injury caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP resulted in similar negative effects of high glucose or Ang and abrogated the protective effects of an ACEI or ARB. Additionally, by impairing autophagy, high-glucose-induced senescence and apoptosis were accelerated and the ACEI- or ARB-mediated beneficial effects were abolished. Furthermore, increases in FragEL™ DNA Fragmentation (TUNEL-positive cells, β-galactosidase activation and the expression of autophagic biomarkers were revealed in diabetic patients and rats, and the treatment with an ACEI or ARB decreased these responses. Conclusions: These data suggest that autophagy protects against senescence and apoptosis via RAS-mitochondria in high-glucose-induced endothelial cells.

  3. Minireview: Selective Degradation of Mitochondria by Mitophagy*

    OpenAIRE

    Kim, Insil; Rodriguez-Enriquez, Sara; Lemasters, John J.

    2007-01-01

    Mitochondria are the essential site of aerobic energy production in eukaryotic cells. Reactive oxygen species (ROS) are an inevitable by-product of mitochondria metabolism and can cause mitochondrial DNA mutations and dysfunction. Mitochondrial damage can also be the consequence of disease processes. Therefore, maintaining a healthy population of mitochondria is essential to the well-being of cells. Autophagic delivery to lysosomes is the major degradative pathway in mitochondrial turnover, a...

  4. Plant Cell Lines in Cell Morphogenesis Research

    Czech Academy of Sciences Publication Activity Database

    Seifertová, Daniela; Klíma, Petr; Pařezová, Markéta; Petrášek, Jan; Zažímalová, Eva; Opatrný, Z.

    Vol. 1080. New York: Humana Press, 2014 - (Žárský, V.; Cvrčková, F.), s. 215-229. (Methods in Molecular Biology). ISBN 978-1-62703-643-6 R&D Projects: GA ČR(CZ) GAP305/11/0797; GA ČR(CZ) GAP305/11/2476 Institutional support: RVO:61389030 Keywords : BY-2 * VBI-0 * Suspension-cultured cells Subject RIV: EB - Genetics ; Molecular Biology

  5. Immunolocation of mitochondria-rich cells in epidermis of the common toad, Bufo bufo L

    DEFF Research Database (Denmark)

    Spies, Ingrid B.M.

    Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation.......Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation....

  6. IN VITRO STUDY ON CYTOTOXIC EFFECTS OF THE ORGANOPHO-SPHOROUS PESTICIDE PROFENOFOS ON THE GILL CELL LINE, FG-9307, OF THE FLOUNDER (PARALICHTHYS OLIVACEUS)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The FG-9307 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy-generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.

  7. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  8. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    International Nuclear Information System (INIS)

    Research highlights: → Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). → Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. → Monoclonal cell lines showed reduced sensitivity for Paclitaxel. → In situ CD133+ cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. → CD133+ and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133+ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  9. The Research Progress of Mitochondria and Cell Regulation%线粒体与细胞调控的研究进展

    Institute of Scientific and Technical Information of China (English)

    汤浩(综述); 林春龙(审校)

    2015-01-01

    线粒体是一种细胞器,广泛存在于除红细胞以外的大多数细胞体内,同时也是细胞进行氧化磷酸化、三羧酸循环及氧化呼吸链的主要场所,产生 ATP,提供细胞活动所需能量,同时线粒体也参与细胞代谢的多个环节,如细胞增殖、凋亡等。近年来,线粒体与细胞调控对疾病的发生、发展成为潜在的研究热点。因此,明确线粒体在细胞调控中的作用机制,以便更好地认识及指导对相关性疾病的治疗具有重要意义。%Mitochondria is a cell organelle,which widely exists in the majority of the cells except red blood cells,at the same time,it is an important place for cell to carry out oxidative phosphorylation ,three tri-carboxylic acid cycle and oxidative respiratory chain , producing ATP for the requirement of cell activity , moreover,mitochondria also participates in multiple processes of cell metabolism ,such as cell proliferation, apoptosis,etc.In recent years,the mitochondria and cell regulation on the occurrence of diseases has become a potential research hotspot.Therefore clarifying the mechanism of cell regulation in mitochondria has a great significance for understanding and guiding the treatment of associated diseases.

  10. Isoflurane preconditioning increases B-cell lymphoma-2 expression and reduces cytochrome c release from the mitochondria in the ischemic penumbra of rat brain

    OpenAIRE

    Li, Liaoliao; Peng, Longyun; Zuo, Zhiyi

    2008-01-01

    We and others have shown that prior exposure to the volatile anesthetic isoflurane induces ischemic tolerance in the brain. Our results also suggest that isoflurane preconditioning reduces cell apoptosis in the penumbral region of rat brain. We designed this study to determine whether isoflurane preconditioning decreased mitochondria-dependent cell apoptosis. Adult male Sprague-Dawley rats were exposed to or not exposed to 2% isoflurane for 30 min at 24 h before the permanent middle cerebral ...

  11. Nitro-Arachidonic Acid Prevents Angiotensin II-Induced Mitochondrial Dysfunction in a Cell Line of Kidney Proximal Tubular Cells.

    Directory of Open Access Journals (Sweden)

    Beatriz Sánchez-Calvo

    Full Text Available Nitro-arachidonic acid (NO2-AA is a cell signaling nitroalkene that exerts anti-inflammatory activities during macrophage activation. While angiotensin II (ANG II produces an increase in reactive oxygen species (ROS production and mitochondrial dysfunction in renal tubular cells, little is known regarding the potential protective effects of NO2-AA in ANG II-mediated kidney injury. As such, this study examines the impact of NO2-AA on ANG II-induced mitochondrial dysfunction in an immortalized renal proximal tubule cell line (HK-2 cells. Treatment of HK-2 cells with ANG II increases the production of superoxide (O2●-, nitric oxide (●NO, inducible nitric oxide synthase (NOS2 expression, peroxynitrite (ONOO- and mitochondrial dysfunction. Using high-resolution respirometry, it was observed that the presence of NO2-AA prevented ANG II-mediated mitochondrial dysfunction. Attempting to address mechanism, we treated isolated rat kidney mitochondria with ONOO-, a key mediator of ANG II-induced mitochondrial damage, in the presence or absence of NO2-AA. Whereas the activity of succinate dehydrogenase (SDH and ATP synthase (ATPase were diminished upon exposure to ONOO-, they were restored by pre-incubating the mitochondria with NO2-AA. Moreover, NO2-AA prevents oxidation and nitration of mitochondrial proteins. Combined, these data demonstrate that ANG II-mediated oxidative damage and mitochondrial dysfunction is abrogated by NO2-AA, identifying this compound as a promising pharmacological tool to prevent ANG II-induced renal disease.

  12. Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344.

    Science.gov (United States)

    Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D; Townsend, Danyelle M; Tew, Kenneth D

    2016-08-01

    ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. PMID:27255112

  13. Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

    DEFF Research Database (Denmark)

    Matic, S.; Geisler, D.A.; Møller, I.M.;

    2005-01-01

    The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage...... cofactor concentrations. Possible uses and limitations of this method for plant cell research are discussed....... of NAD(P)(+). After the addition of cofactors and substrates, activities of cytosolic as well as mitochondrial respiratory enzymes could be directly determined inside the permeabilized cells. However, at an AlaM concentration at which the cytoplasmic enzymes were maximally accessible, the vacuole...

  14. Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis.

    OpenAIRE

    Shitara, H; Kaneda, H.; Sato, A; Inoue, K.; Ogura, A.; Yonekawa, H.; Hayashi, J I

    2000-01-01

    Exclusion of paternal mitochondria in fertilized mammalian eggs is very stringent and ensures strictly maternal mtDNA inheritance. In this study, to examine whether elimination was specific to sperm mitochondria, we microinjected spermatid or liver mitochondria into mouse embryos. Congenic B6-mt(spr) strain mice, which are different from C57BL/6J (B6) strain mice (Mus musculus domesticus) only in possessing M. spretus mtDNA, were used as mitochondrial donors. B6-mt(spr) mice and a quantitativ...

  15. Mitochondria-Rich Cells as Experimental Model in Studies of Epithelial Chloride Channels

    DEFF Research Database (Denmark)

    Willumsen, Niels J.; Amstrup, Jan; Møbjerg, Nadja;

    2002-01-01

    -actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl- pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via ß-adrenergic receptors, ion selectivity, genistein stimulation and...... inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor...

  16. Mitochondria-dependent apoptosis induced by a novel amphipathic photochemotherapeutic agent ZnPcS2P2 in HL60 cells

    Institute of Scientific and Technical Information of China (English)

    Hui-fang HUANG; Yuan-zhong CHEN; Yong WU

    2005-01-01

    Aim: To investigate the mechanism underlying the killing effects of a novel amphipathic photosensitizer, disulfonated diphthalimidomethyl phthalocyanine zinc (ZnPcS2P2), mediated photodynamic therapy (ZnPc-PDT) in human myelogenous leukemia HL60 cells. Methods: After incubation for 5 h with 0.5 μmol/L ZnPcS2P2, the HL60 cells were exposed to a light source of 670 nm wavelength.Thereafter, the cells were detected at different time intervals after PDT. The characteristics of apoptosis were detected by observation of ultrastructure assay,DNA fragmentation assay and terminal deoxynucleotidyl transferase deoxyuridine nick-end labeling method (TUNEL). Mitochondria-dependent apoptosis was determined by the detection of mitochondrial membrane potential (Δψm), activities of caspase family protease and of caspase-3, cytosol cytochrome c. Proteins Bcl-2and Bax were detected by immunoblot analysis. Results: Evident characteristics of apoptosis were observed post-ZnPc-PDT with ultrastructure assay, DNA fragmentation assay and TUNEL staining. TUNEL assay showed that apoptotic rates in the cells collected from 6 h, 12 h and 24 h after PDT were 9.6%, 24.4%, and 33.0%,respectively. HL60 cells underwent mitochondria-dependent apoptosis as a result of cytochrome c release from mitochondria into cytosol accompanied by a reduction of Δψm. The activities of caspase family protease and of caspase-3were elevated. Furthermore, ZnPc-PDT could remarkably down-regulate the Bcl-2pro-apoptotic protein and up-regulate the anti-apoptotic Bax protein. Conclusion:ZnPc-PDT could induce mitochondria-dependent apoptosis in HL60 cells.

  17. Mitochondria functionality and sperm quality.

    Science.gov (United States)

    Amaral, Alexandra; Lourenço, Bárbara; Marques, Mónica; Ramalho-Santos, João

    2013-01-01

    Although mitochondria are best known for being the eukaryotic cell powerhouses, these organelles participate in various cellular functions besides ATP production, such as calcium homoeostasis, generation of reactive oxygen species (ROS), the intrinsic apoptotic pathway and steroid hormone biosynthesis. The aim of this review was to discuss the putative roles of mitochondria in mammalian sperm function and how they may relate to sperm quality and fertilisation ability, particularly in humans. Although paternal mitochondria are degraded inside the zygote, sperm mitochondrial functionality seems to be critical for fertilisation. Indeed, changes in mitochondrial integrity/functionality, namely defects in mitochondrial ultrastructure or in the mitochondrial genome, transcriptome or proteome, as well as low mitochondrial membrane potential or altered oxygen consumption, have been correlated with loss of sperm function (particularly with decreased motility). Results from genetically engineered mouse models also confirmed this trend. On the other hand, increasing evidence suggests that mitochondria derived ATP is not crucial for sperm motility and that glycolysis may be the main ATP supplier for this particular aspect of sperm function. However, there are contradictory data in the literature regarding sperm bioenergetics. The relevance of sperm mitochondria may thus be associated with their role in other physiological features, particularly with the production of ROS, which in controlled levels are needed for proper sperm function. Sperm mitochondria may also serve as intracellular Ca²⁺ stores, although their role in signalling is still unclear. PMID:23901129

  18. On Message Ribonucleic Acids Targeting to Mitochondria

    OpenAIRE

    Dave, Kunjan R.; Bhattacharya, Sanjoy K.; Di Ding

    2009-01-01

    Mitochondria are subcellular organelles that provide energy for a variety of basic cellular processes in eukaryotic cells. Mitochondria maintain their own genomes and many of their endosymbiont genes are encoded by nuclear genomes. The crosstalk between the mitochondrial and nuclear genomes ensures mitochondrial biogenesis, dynamics and maintenance. Mitochondrial proteins are partly encoded by nucleus and synthesized in the cytosol and partly in the mitochondria coded by mitochondrial genome....

  19. Proton pump activity of mitochondria-rich cells: The interpretation of external proton-concentration gradients

    DEFF Research Database (Denmark)

    Jensen, Lars Jørn; Sørensen, Jens N.; Larsen, Erik Hviid;

    1997-01-01

    Active Cl*O- transport, reheogenic H*O+ pump, unstirred layer, mathematics of diffusion, proton concentration-profiles outside epithelial cells......Active Cl*O- transport, reheogenic H*O+ pump, unstirred layer, mathematics of diffusion, proton concentration-profiles outside epithelial cells...

  20. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the...

  1. Gambogic acid induces mitochondria-dependent apoptosis by modulation of Bcl-2 and Bax in mantle cell lymphoma JeKo-1 cells

    Institute of Scientific and Technical Information of China (English)

    Jingyan Xu; Min Zhou; Jian Ouyang; Jing Wang; Qiguo Zhang; Yong Xu; Yueyi Xu

    2013-01-01

    Objective:To study the mechanisms in gambogic acid (GA)-induced JeKo-1 human Mantle Cell Lymphoma cell apoptosis in vitro.Methods:The proliferation of GA-treated JeKo-1 cells was measured by CCK-8 assay and Ki-67 immunocytochemical detection.Apoptosis,cell cycle and mitochondrial membrane potential were measured by flow cytometric analysis.Caspase-3,-8 and-9 were detected by colorimetric assay.Bcl-2 and Bax were analyzed by Western blotting.Results:GA inhibited cell growth in a time-and dose-dependent manner.GA induces apoptosis in JeKo-1 cells but not in normal bone marrow cells,which was involved in reducing the membrane potential of mitochondria,activating caspases-3,-8 and-9 and decreasing the ratio of Bcl-2 and Bax without cell cycle arresting.Conclusions:GA induced apoptosis in human MCL JeKo-1 cells by regulating Bcl-2/Bax and activating caspase-3,-8 and-9 via mitochondrial pathway without affecting cell cycle.

  2. Plumbagin induces the apoptosis of human tongue carcinoma cells through the mitochondria-mediated pathway

    OpenAIRE

    Qiu, Jia-Xuan; He, Yuan-qiao; Wang, Yong; Xu, Ru-liang; Qin, You; Shen, Xiang; Zhou, Shu-Feng; Mao, Zong-Fu

    2013-01-01

    Background Plumbagin, a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been proven to possess anti-tumor activity both in vitro and in vivo. However, its anti-tumor properties for human tongue carcinoma have not been reported. This study aimed to investigate the inhibitory effect and the underlying mechanism of plumbagin on the growth of human tongue carcinoma cells. Material/Methods Cell proliferation ability was detected by EdU incorporation assay and colony form...

  3. Empowering self-renewal and differentiation: the role of mitochondria in stem cells

    OpenAIRE

    Rehman, Jalees

    2010-01-01

    Stem cells are characterized by their multi-lineage differentiation potential (pluripotency) and their ability for self-renewal, which permits them to proliferate while avoiding lineage commitment and senescence. Recent studies demonstrate that undifferentiated, pluripotent stem cells display lower levels of mitochondrial mass and oxidative phosphorylation, and instead preferentially use non-oxidative glycolysis as a major source of energy. Hypoxia is a potent suppressor of mitochondrial oxid...

  4. Morphological and quantitative changes in mitochondria, plastids, and peroxisomes during the log-to-stationary transition of the growth phase in cultured tobacco BY-2 cells.

    Science.gov (United States)

    Toyooka, Kiminori; Sato, Mayuko; Wakazaki, Mayumi; Matsuoka, Ken

    2016-03-01

    We developed a wide-range and high-resolution transmission electron microscope acquisition system and obtained giga-pixel images of tobacco BY-2 cells during the log and stationary phases of cell growth. We demonstrated that the distribution and ultrastructure of compartments involved in membrane traffic (i.e., Golgi apparatus, multivesicular body, and vesicle cluster) change during the log-to-stationary transition. Mitochondria, peroxisomes, and plastids were also enumerated. Electron densities of mitochondria and peroxisomes were altered during the growth-phase shift, while their numbers were reduced by nearly half. Plastid structure dramatically changed from atypical to spherical with starch granules. Nearly the same number of plastids was observed in both log and stationary phases. These results indicate that mechanisms regulating organelle populations differ from organelle to organelle. PMID:26855065

  5. A reversible and highly selective fluorescence "on-off-on" probe for detecting nickel ion in the mitochondria of living cells.

    Science.gov (United States)

    Yu, Tingting; Tian, Xiaohe; Li, Hong; Li, Wei; Zhu, Weiju; Zhou, Hongping; Tian, Yupeng; Wu, Jieying

    2016-08-15

    A simple molecular fluorescent probe L has been designed, synthesized and characterized. The probe upon interaction with different metals ions show high selectivity and sensitivity for Ni(2+) through fluorescence "turn-off" response in acetonitrile, and the fluorescence could be recovered when chelating agent TPEN is added in this system, viz an "on-off-on" process. Its recognition mechanism has been established by fluorescence spectra, (1)H NMR study. Moreover, to further explore its utility in biological system, L is selected as a probe for Ni(2+) indicator in live cells and shows sensitivity on Ni(2+) intracellular distribution. Fluorescence co-localization studies demonstrate that L is a specific mitochondria-targeted fluorescent probe with excellent cell membrane permeability. Furthermore, a rapidly reversible fluorescence imaging of probe L detecting Ni(2+) in the mitochondria is successfully achieved. These facts would make this probe a great potential for detecting Ni(2+) in biology. PMID:27043479

  6. Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control1

    OpenAIRE

    Li, Ching-Hao; Cheng, Yu-Wen; Liao, Po-Ling; Kang, Jaw-Jou

    2010-01-01

    p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC)-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Di...

  7. Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

    OpenAIRE

    Ching-Hao Li; Yu-Wen Cheng; Po-Ling Liao; Jaw-Jou Kang

    2010-01-01

    p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC)-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Di...

  8. Copper Oxide Nanoparticles Induced Mitochondria Mediated Apoptosis in Human Hepatocarcinoma Cells

    OpenAIRE

    Maqsood A Siddiqui; Alhadlaq, Hisham A; Ahmad, Javed; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed; Ahamed, Maqusood

    2013-01-01

    Copper oxide nanoparticles (CuO NPs) are heavily utilized in semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics and heat transfer fluids. It has been reported that liver is one of the target organs for nanoparticles after they gain entry into the body through any of the possible routes. Recent studies have shown cytotoxic response of CuO NPs in liver cells. However, the underlying mechanism of apoptosis in liver cells due to CuO NPs exposure is largely lack...

  9. Regulatory networks define phenotypic classes of human stem cell lines

    OpenAIRE

    Müller, Franz-Josef; Louise C. Laurent; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Philip H. Schwartz; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The...

  10. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds...

  11. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    International Nuclear Information System (INIS)

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  12. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jason Z. [Virginia Tech CRC, Blacksburg, VA (United States); Ke, Yuebin [Shenzhen Center for Disease Control and Prevention, Shenzhen 518055 (China); Misra, Hara P. [Virginia Tech CRC, Blacksburg, VA (United States); Trush, Michael A. [Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD (United States); Li, Y. Robert [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Virginia Tech-Wake Forest University SBES, Blacksburg, VA (United States); Department of Biology, University of North Carolina at Greensboro, NC (United States); Zhu, Hong, E-mail: zhu@campbell.edu [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Jia, Zhenquan, E-mail: z_jia@uncg.edu [Department of Biology, University of North Carolina at Greensboro, NC (United States)

    2014-12-15

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  13. High prevalence of side population in human cancer cell lines

    OpenAIRE

    Boesch, Maximilian; Zeimet, Alain G; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther; Sopper, Sieghart; Wolf, Dominik

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems.

  14. Role of mitochondria in drug-induced cholestatic injury.

    Science.gov (United States)

    Kass, George E N; Price, Shirley C

    2008-02-01

    Mitochondria have multiple functions in eukaryotic cells and are organized into dynamic tubular networks that continuously undergo changes through coordinated fusion and fission and migration through the cytosol. Mitochondria integrate cell-signaling networks, especially those involving the intracellular messenger Ca(2+), into the regulation of metabolic pathways. Recently, it has become clear that mitochondria are central to the three main cell death pathways, namely necrosis, apoptosis, and autophagic cell death. This article discusses the role of mitochondria in drug-induced cholestatic injury to the liver. The role of mitochondria in the cellular adaptation against the toxic effects of bile acids is discussed also. PMID:18242496

  15. Mitochondria-acting hexokinase II peptides carried by short-length carbon nanotubes with increased cellular uptake, endosomal evasion, and enhanced bioactivity against cancer cells

    Science.gov (United States)

    Yoong, Sia Lee; Lau, Wei Liang; Liu, Ang Yu; Prendergast, D'arcy; Ho, Han Kiat; Yu, Victor Chun Kong; Lee, Chengkuo; Ang, Wee Han; Pastorin, Giorgia

    2015-08-01

    Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study, multi-walled carbon nanotubes (MWCNTs) were used to effectively deliver HKII(pep) across cellular barriers without compromising their bioactivity. The peptide was conjugated on either oxidized MWCNTs or 2,2'-(ethylenedioxy)bis(ethylamine)-functionalized MWCNTs, yielding MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep), respectively. Both conjugates were shown to be internalized by breast cancer MCF-7 cells using confocal microscopy. Moreover, these nanoconjugates seemed to have escaped from endosomes and be in the vicinity of mitochondria. The WST-1 cytotoxicity assay conducted on MCF-7 and colon carcinoma HCT116 cells revealed that MWCNT-peptide conjugates were significantly more effective in curbing cancer cell growth compared to a commercially available cell permeable HKII fusion peptide. In addition, both nanoconjugates displayed an enhanced ability in eliciting apoptosis and depleting the ATP level in HCT116 cells compared to the mere HKII peptide. Importantly, hexokinase II release from mitochondria was demonstrated in MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep) treated cells, highlighting that the structure and bioactivity of HKII(pep) were not compromised after covalent conjugation to MWCNTs.Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study

  16. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    International Nuclear Information System (INIS)

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  17. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Park, Jae Hyeon [Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2012-09-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  18. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  19. Cu(II)-disulfide complexes with superoxide dismutase- and catalase-like activities protect mitochondria and whole cells against oxidative stress.

    Science.gov (United States)

    Aliaga, Margarita E; Sandoval-Acuña, Cristián; López-Alarcón, Camilo; Fuentes, Jocelyn; Speisky, Hernan

    2014-10-01

    Mitochondria are a major subcellular site of superoxide (O2(-)) formation. Conditions leading to an uncontrolled production, accumulation and/or conversion of O2(-) into hydrogen peroxide result in an increment in the intramitochondrial oxidative tone which, ultimately leads to the loss of cell viability. Recently, we reported on the ability of a series of Cu(II)-disulfide complexes to act simultaneously as SOD- and catalase-like molecules. In the present study, we addressed the potential of such compounds to protect mitochondria and cells against the oxidative stress and the cytolytic damage induced by diclofenac. Exposure of Caco-2 cells to diclofenac (250µM, 20min) led to a near 80% inhibition of mitochondrial complex I activity and almost doubled the rate of mitochondrial O2(-) production (assessed by Mitosox). A comparable increment was seen in whole cells when the oxidative tone was assessed through the largely hydrogen peroxide-dependent dichlorofluorescein (DCFH) oxidation. The increment in mitochondrial O2(-) production was totally and concentration-dependently prevented by the addition of the complexes formed between Cu(II) and the disulfides of glutathione, homocysteine, or a-dehydro-lipoic acid (20µM each); comparatively, the Cu(II)-cystine complex exerted a weaker protection. A comparable protection pattern was seen at the whole cell level, as these complexes were also effective in preventing the increment in DCFH oxidation. The mitochondrial and whole cell antioxidant protection also translated into a full protection against the cytolytic effects of diclofenac (45min). Results from the present study indicate that the here-tested Cu(II)-disulfides complexes are able to effectively protect cells against the oxidative and the lytic effects of O2(-)-overproducing mitochondria, suggesting a potential for these type of compounds to act as SOD- and catalase-like molecules under oxidative-stress conditions. Supported by FONDECYT #1110018. PMID:26461399

  20. The studies of DNA metabolic dynamics in embryonic cell line and non-embryonic cell line of Onobrychis viciaefolia scop

    International Nuclear Information System (INIS)

    The hypocotyls of aseptic seedlings of Onobrychis viciaefolia Scop. were used as explants for inducing callus. The embryonic cell line (E-line) and non-embryonic cell line (NE line) were established. The DNA synthesis dynamics in both cell lines were studied by autoradiography. The results showed that: DNA synthesis in E-line was active and confined to embryonic cell or cell masses and then the cells divided quickly and formed somatic embryos at different stages. The changes of DNA metabolism took place in a certain pattern and the maximum rate of DNA synthesis occured during the formation of globular embryo. There was a clear relationship between the difference of DNA synthesis rate and the polarity of the embryo. In NE-line, the beginning of cell division and the forming of callus were also based on DNA replication but were much deoxythymidine. There was a significant difference in DNA synthesis dynamics between the two cell lines

  1. Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

    International Nuclear Information System (INIS)

    Highlights: • Identification and quantification of Cu,Zn-superoxide dismutase in mice hepatic cells. • IDA-ICP-MSis applied to obtain a high degree of accuracy, precision and sensibility. • This methodology reduces the time of analysis and avoids clean-up procedures. • The application of this method to Hg-exposed mice reveals perturbations in Cu,Zn-SOD. - Abstract: In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min

  2. Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

    Energy Technology Data Exchange (ETDEWEB)

    García-Sevillano, M.A.; García-Barrera, T. [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Navarro, F. [International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, Huelva 21007 (Spain); Gómez-Ariza, J.L., E-mail: ariza@uhu.es [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain)

    2014-09-09

    Highlights: • Identification and quantification of Cu,Zn-superoxide dismutase in mice hepatic cells. • IDA-ICP-MSis applied to obtain a high degree of accuracy, precision and sensibility. • This methodology reduces the time of analysis and avoids clean-up procedures. • The application of this method to Hg-exposed mice reveals perturbations in Cu,Zn-SOD. - Abstract: In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min

  3. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    Passage of cell cultures may adversely influence cell susceptibility to virus infection through selection of cell clones that thrive in vitro but may not necessarily display high sensitivity to virus infection. Susceptibility to a given virus can therefore vary not only between cell lines and......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  4. Detection algorithm for the validation of human cell lines.

    Science.gov (United States)

    Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

    2012-09-15

    Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

  5. Transport of Calcium Ions into Mitochondria.

    Science.gov (United States)

    Xu, Zhaolong; Zhang, Dayong; He, Xiaolan; Huang, Yihong; Shao, Hongbo

    2016-06-01

    To uptake calcium ions of mitochondria is of significant functional connotation for cells, because calcium ions in mitochondria are involved in energy production, regulatory signals transfer, and mitochondrial permeability transition pore opening and even programmed cell death of apoptosis, further playing more roles in plant productivity and quality. Cytoplasmic calcium ions access into outer mitochondrial membrane (OMM) from voltage dependent anion-selective channel (VDAC) and were absorbed into inner mitochondrial membrane (IMM) by mitochondrial calcium uniporter (MCU), rapid mitochondrial calcium uptake (RaM) or mitochondrial ryanodine receptor (mRyR). Although both mitochondria and the mechanisms of calcium transport have been extensively studied, but there are still long-standing or even new challenges. Here we review the history and recent discoveries of the mitochondria calcium ions channel complex involved calcium assimilation, and discuss the role of calcium ions into mitochondria. PMID:27252588

  6. Chloride transport in a glioma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Wolpaw, E.W.

    1984-01-01

    Maintenance of the extracellular environment is a major function of central nervous system astroglia. The transport of Cl/sup -/ across the cell membrane may be an integral part of this function, since Cl/sup -/ transport has been implicated in homeostasis of cell volume, pH, and extracellular K/sup +/ concentration. The work presented here investigated Cl/sup -/ transport in the glioma cell line LRM55. Results indicate that LRM55 cells are a good model for astroglia and that these cells contain three Cl/sup -/ transporters; a Cl/sup -//HCO/sub 3//sup -/ exchanger, a K/sup +//Cl/sup -/ cotransporter, and a Cl/sup -//SO/sub 4//sup 2 -/ exchanger. Ion transport studies measured the fluxes of Cl/sup -/ (as /sup 36/Cl/sup -/), K/sup +/ (as /sup 86/Rb/sup +/), and SO/sub 4//sup 2 -/ (as /sup 35/SO/sub 4//sup 2 -/). Cl/sup -/ flux was trans-simulated by Cl/sup -/ or HCO/sub 3//sup -/ and was inhibited by SITS or furosemide. External K/sup +/ stimulated Cl/sup -/ influx and external Cl/sup -/ stimulated Rb/sup +/ influx. Furosemide, but not SITS, inhibited the K/sup +//Cl/sup -/ cotransporter. High K/sup +/ medium increased cell volume and Cl/sup -/ content. Steady-state Cl/sup -/ concentration was at least twice that predicted from passive equilibration according to the Nernst equation. SO/sub 4//sup 2 -/ flux was trans-stimulated by SO/sub 4//sup 2 -/ or by Cl/sup -/. Cl/sup -/ was a competitive inhibitor of SO/sub 4//sup 2 -/ influx, but SO/sub 4//sup 2 -/ had no detectable effect on Cl/sup -/ influx or efflux. SO/sub 4//sup 2 -/ flux was inhibited by SITS or furosemide.

  7. Uptake of 99mTc-tetrofosmin by breast cancer, sarcoma and melanoma cell lines

    International Nuclear Information System (INIS)

    99mTc-tetrofosmin was developed for myocardial perfusion imaging. However, it has been suggested that this cationic tracer may be of value as a tumor-imaging agent. Very little is however known about the mechanism of uptake of 99mTc-tetrofosmin in heart tissue of tumor cells. We studied in-vitro the uptake of this radiopharmaceutical in breast cancer-, sarcoma- and melanoma cell lines. The influence of density of tumor cells, temperature- and time of incubation on the radioactivity uptake were analysed. In these tumor cell lines the uptake of 99mTc-tetrofosmin was highest at a cellular density of 1.106 cell/ml and at a temperature of incubation of 37 deg. C. This indicates that the uptake of 99mTc-tetrofosmin by these tumor cells occurs by a metabolism-dependent process, probably related to mitochondria, besides cation channel transport. 99mTc-tetrofosmin scintigraphy might thus be useful for detecting and imaging at a very early clinical stage these malignant tumors. (author)

  8. ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

    International Nuclear Information System (INIS)

    The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases

  9. ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, D. [School of Life Sciences, Jilin University, Changchun (China); The State Engineering Laboratory of AIDS Vaccine, Jilin University, Changchun (China); Guo, T.Q. [School of Life Sciences, Jilin University, Changchun (China); Wang, Z.Y. [State Key Laboratory of Theoretical and Computational Chemistry, Jilin University, Changchun (China); Lu, J.H.; Liu, D.P.; Meng, Q.F.; Xie, J. [School of Life Sciences, Jilin University, Changchun (China); Zhang, X.L. [Faculty of ScienceNational University of Singapore (Singapore); Liu, Y. [School of Life Sciences, Jilin University, Changchun (China); Teng, L.S. [School of Life Sciences, Jilin University, Changchun (China); The State Engineering Laboratory of AIDS Vaccine, Jilin University, Changchun (China)

    2014-07-25

    The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.

  10. Electroacupuncture inhibits apoptosis in annulus fibrosis cells through suppression of the mitochondria-dependent pathway in a rat model of cervical intervertebral disc degradation

    Directory of Open Access Journals (Sweden)

    Jun Liao

    2012-01-01

    Full Text Available The purpose of this study was to investigate whether treatment with electroacupuncture (EA inhibited mitochondria-dependent apoptosis in annulus fibrosis (AF cells in a rat model of cervical intervertebral disc degradation induced by unbalanced dynamic and static forces. Forty Sprague-Dawley rats were used in this study, of which 30 underwent surgery to induce cervical intervertebral disc degradation, 10 rats received EA at acupoints Dazhui (DU 14 and Shousanli (LI 10. TUNEL staining was measured to assess apoptosis in AF cells, immunohistochemistry was used to examine Bcl-2 and Bax expression, colorimetric assays were used to determine caspase 9 and caspase 3 activities and RT-PCR and western blotting were used to assess the mRNA and protein expression of Crk and ERK2. Treatment with EA reduced the number of AF-positive cells in TUNEL staining, increased Bcl-2-positive cells and decreased Bax-positive cells in immunohistochemical staining, significantly inhibited the activation of caspases-9 and -3, and enhanced the mRNA and protein expression of Crk and ERK2. Our data show that EA inhibits AF cell apoptosis via the mitochondria-dependent pathway and up-regulates Crk and ERK2 expression. These results suggest that treatment with may be a good alternative therapy for preventing cervical spondylosis.

  11. Mitochondria, Energy and Cancer: The Relationship with Ascorbic Acid

    Science.gov (United States)

    González, Michael J.; Rosario-Pérez, Glorivee; Guzmán, Angélica M.; Miranda-Massari, Jorge R.; Duconge, Jorge; Lavergne, Julio; Fernandez, Nadia; Ortiz, Norma; Quintero, Ana; Mikirova, Nina; Riordan, Neil H.; Ricart, Carlos M.

    2012-01-01

    Ascorbic Acid (AA) has been used in the prevention and treatment of cancer with reported effectiveness. Mitochondria may be one of the principal targets of ascorbate's cellular activity and it may play an important role in the development and progression of cancer. Mitochondria, besides generating adenosine triphosphate (ATP), has a role in apoptosis regulation and in the production of regulatory oxidative species that may be relevant in gene expression. At higher concentrations AA may increase ATP production by increasing mitochondrial electron flux, also may induce apoptotic cell death in tumor cell lines, probably via its pro-oxidant action In contrast, at lower concentrations AA displays antioxidant properties that may prevent the activation of oxidant-induced apoptosis. These concentration dependent activities of ascorbate may explain in part the seemingly contradictory results that have been reported previously. PMID:23565030

  12. Mitochondria-involved apoptosis induced by MPPa mediated photodynamic therapy

    International Nuclear Information System (INIS)

    Numerous new photosensitizers are now in various stages of trials demonstrating the broad applicability of Photodynamic therapy (PDT). However, only a handful of photosensitizers have received regulatory approval. Lack of effective photosensitizers has become a major limit for extensive application of PDT. Our previous study showed MPPa to be a good photosensitizer candidature, MPPa-PDT can lead PC-3M cell line to death mainly via apoptotic way both in vitro and in vivo, and part of the mechanism was investigated. Mitochondria may play a key role in the process, in order to further elucidate the mechanism, we investigated the level of ROS, GSH, NO, Ca2+, mitochondrial membrane potential, as well as cytochrome C. All in all, ROS production, depletion of GSH, and the activation of ROS downstream, such as mitochondria depolarization, cytochrome C release, were detected in our study. The results provide a mechanism by which oxidative stress provokes apoptosis of PC-3M cells

  13. Distinctions and similarities of cell bioenergetics and the role of mitochondria in hypoxia, cancer, and embryonic development

    Czech Academy of Sciences Publication Activity Database

    Ježek, Petr; Plecitá-Hlavatá, Lydie; Smolková, Katarína; Rossignol, R.

    2010-01-01

    Roč. 42, č. 5 (2010), s. 604-622. ISSN 1357-2725 R&D Projects: GA AV ČR(CZ) IAA500110701; GA MZd(CZ) NR9183; GA MŠk(CZ) ME09029 Institutional research plan: CEZ:AV0Z50110509 Keywords : mitochondria * bioenergetics * hypoxia Subject RIV: ED - Physiology Impact factor: 4.956, year: 2010

  14. DNA profiling and characterization of animal cell lines.

    Science.gov (United States)

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

  15. Do mitochondria recombine in humans?

    OpenAIRE

    Eyre-Walker, A

    2000-01-01

    Until very recently, mitochondria were thought to be clonally inherited through the maternal line in most higher animals. However, three papers published in 2000 claimed population-genetic evidence of recombination in human mitochondrial DNA. Here I review the current state of the debate. I review the evidence for the two main pathways by which recombination might occur: through paternal leakage and via a mitochondrial DNA sequence in the nuclear genome. There is no strong evidence for either...

  16. The establishment of mitochondria: Paracoccus and Rhodopseudomonas.

    Science.gov (United States)

    Whatley, F R

    1981-01-01

    Many aerobic bacteria (both facultative and obligate) possess a number of those biochemical features of mitochondria which are concerned with energy metabolism. However, only restricted number, notably Paracoccus denitrificans and Rhodopseudomonas spheroides, have the majority of these features. The theory of endosymbiosis proposes that a primitive eukaryote took up bacteria to yield mitochondria. The present-day Paracoccus then resembles the ancestral bacterium in many respects the primitive amoeba, Pelomyxa palustris, which lacks mitochondria but contains a permanent population of unique symbiotic bacteria, has many of the characteristics of a present-day transitional form. The evolution of mitochondria from endosymbiotic bacteria would involve their integration with the host cell both biochemically and structurally: a number of the intermediate steps are discussed. Attention is drawn to the existence in some ciliates of hydrogenosomes, which function as anaerobic mitochondria. PMID:6264827

  17. The new research on mitochondria

    Institute of Scientific and Technical Information of China (English)

    JI Yu-bin; ZHANG He; JI Chen-feng; LIU Hong-juan

    2008-01-01

    Apoptosis, also known as programmed cell death, is the removal of damaged body organizations,aging or redundant cells in a suicide, has to maintain the health of the body, the normal development of the nervous system, the immune system to maintain the normal function of such areas is of great significance. The morphological characteristics of apoptosis are the cytoplasm concentrated, condensed nuclear chromatin, DNA fragments of a large-scale, the cell membrane invagination and foam formation of apoptotic bodies. There are two classic apoptosis ways which are generally accepted by majority of the scholars currently: Mitochondrial pathway and Death receptor pathway. Mitochondrial membrane is a two-tier structure surrounded the cystic, between the external cavity and internal cavity which is called the Room, surrounded by the internal cavity known as the mitochondria internal room or mitochondrial matrix. Mitochondria with the functions of control cell survival and death: mitochondria play an important role in physiological such as oxidative phosphorylation, electronic transfer, storage Ca2+, energy metabolism, anti-oxidation activity and so on, to provide the basic energy to the various activities of cell life. Study found that mitochondria contain some of the material is closely related to apoptosis, such as Cyt-C, Smac/Diablo, AIF, Ca2+, ROS and so on. In the signal to stimulate apoptosis, mitochondrial membrane permeability, resulting in a series of key changes, including Cyt-C, Smac/Diablo release, decline of the mitochondrial membrane potential (Δψm), the state of the redox within cells, the intervention of Bcl gene family and so on. Different signal transduction ultimately focuses on the mitochondria to activate or inhibit these incidents, then the corresponding signal transduction to control apoptosis. Therefore, the mitochondria in the incidence of apoptosis play an important role. In recent years, the study confirmed that apoptosis imbalance can cause a

  18. Biochemistry of Mitochondria

    Directory of Open Access Journals (Sweden)

    Filiz Koc

    2003-02-01

    Full Text Available Mitochondria are energy source of cells. They have external and internal membranes, cristas and matrix. External membranes consist of specialized transport proteins. They have monoamine oxidase and citokrome-c reductase which both play role in KREBS cycle as catalyst and many enzymes which are necessary for phospholipid and phosphoric acid synthesis. Enzymes of electron transport chain and oxidative phosphorylation are located in the internal membranes. Also, here, there are transport systems for specific substances, such as ATP, ADP, P1, pyruvate, succinate, malate, citrate, and -ketoglutarate . Matrix; having gel-like consistency, contains a large number of enzymes. [Archives Medical Review Journal 2003; 12(0.100: 1-13

  19. Development and characterization of a new human hepatic cell line

    Science.gov (United States)

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  20. The Failure of Mitochondria Leads to Neurodegeneration: Do Mitochondria Need A Jump Start?

    OpenAIRE

    Lee, Junghee; Boo, Jung Hyun; Ryu, Hoon

    2009-01-01

    Mitochondria are the power engine generating biochemical energy in the cell. Mitochondrial dysfunction and bioenergy deficiency is closely linked to the pathogenesis of neurodegenerative disorders. Mitochondria play a variety of roles by integrating extracellular signals and executing important intracellular events in neuronal survival and death. In this context, the regulation of mitochondrial function via therapeutic approaches may exert some salutary and neuroprotective mechanisms. Underst...

  1. Distinct differentiation characteristics of individual human embryonic stem cell lines

    Directory of Open Access Journals (Sweden)

    Knuutila Sakari

    2006-08-01

    Full Text Available Abstract Background Individual differences between human embryonic stem cell (hESC lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61 from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

  2. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    KAUST Repository

    Hou, Xu

    2015-03-12

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)\\'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  3. Ursodeoxycholic acid suppresses mitochondria-dependent programmed cell death induced by sodium nitroprusside in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Although ursodeoxycholic acid (UDCA) and its highly water-soluble formula (Yoo's solution; YS) have been shown to prevent neuronal damage, the effects of UDCA or YS against Parkinson's disease (PD)-related dopaminergic cell death has not been studied. This study investigated the protective effects of UDCA and YS on sodium nitroprusside (SNP)-induced cytotoxicity in human dopaminergic SH-SY5Y cells. Both UDCA (50–200 μM) and YS (100–200 μM) dose-dependently prevented SNP (1 mM)-induced cell death. Results showed that both UDCA and YS effectively attenuated the production of total reactive oxygen species (ROS), peroxynitrite (ONOO−) and nitric oxide (NO), and markedly inhibited the mitochondrial membrane potential (MMP) loss and intracellular reduced glutathione (GSH) depletion. SNP-induced programmed cell death events, such as nuclear fragmentation, caspase-3/7 and -9 activation, Bcl-2/Bax ratio decrease, and cytochrome c release, were significantly attenuated by both UDCA and YS. Furthermore, selective inhibitor of phosphatidylinositiol-3-kinase (PI3K), LY294002, and Akt/PKB inhibitor, triciribine, reversed the preventive effects of UDCA on the SNP-induced cytotoxicity and Bax translocation. These results suggest that UDCA can protect SH-SY5Y cells under programmed cell death process by regulating PI3K-Akt/PKB pathways.

  4. Analysis of three marine fish cell lines by rapd assay.

    Science.gov (United States)

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  5. Reactive biomolecular divergence in genetically altered yeast cells and isolated mitochondria as measured by biocavity laser spectroscopy : a rapid diagnostic method for studying cellular responses to stress and disease.

    Energy Technology Data Exchange (ETDEWEB)

    Yaffe, Michael P. (University of California, San Diego, CA); Gourley, Paul Lee; Copeland, Robert Guild; McDonald, Anthony Eugene; Hendricks, Judy K.; Naviaux, Robert K. (Univesity of California, San Diego, CA)

    2006-12-01

    We report an analysis of four strains of baker's yeast (Saccharomyces cerevisiae) using biocavity laser spectroscopy. The four strains are grouped in two pairs (wild type and altered), in which one strain differs genetically at a single locus, affecting mitochondrial function. In one pair, the wild-type rho+ and a rho0 strain differ by complete removal of mitochondrial DNA (mtDNA). In the second pair, the wild-type rho+ and a rho- strain differ by knock-out of the nuclear gene encoding Cox4, an essential subunit of cytochrome c oxidase. The biocavity laser is used to measure the biophysical optic parameter Deltalambda, a laser wavelength shift relating to the optical density of cell or mitochondria that uniquely reflects its size and biomolecular composition. As such, Deltalambda is a powerful parameter that rapidly interrogates the biomolecular state of single cells and mitochondria. Wild-type cells and mitochondria produce Gaussian-like distributions with a single peak. In contrast, mutant cells and mitochondria produce leptokurtotic distributions that are asymmetric and highly skewed to the right. These distribution changes could be self-consistently modeled with a single, log-normal distribution undergoing a thousand-fold increase in variance of biomolecular composition. These features reflect a new state of stressed or diseased cells that we call a reactive biomolecular divergence (RBD) that reflects the vital interdependence of mitochondria and the nucleus.

  6. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  7. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan;

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

  8. Targeting mitochondria with small molecules: the preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide

    OpenAIRE

    Cairns, Andrew G; McQuaker, Stephen J.; Murphy, Michael P; Hartley, Richard C.

    2015-01-01

    Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP-conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow ...

  9. tRNA Biology in Mitochondria

    Directory of Open Access Journals (Sweden)

    Thalia Salinas-Giegé

    2015-02-01

    Full Text Available Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

  10. Modulation of ganglioside expression in human melanoma cell lines

    International Nuclear Information System (INIS)

    Cell surface gangliosides in human melanoma cell lines were modulated by pretreatment and adaptation to 6-thioguanine and 5-bromo-deoxyuridine. Chemo- and radiation sensitivities were compared in original cell lines and modulated cells by the human tumor colony-forming assay. Modulated cells showed decreased expression of cell surface GM2 and GD2 gangliosides. This reduction was correlated with increased resistance to bleomycin, vincristine, cisplatin and radiation treatment. These results suggest that cell surface GM2 and GD2 ganglioside expression in human melanoma cells is intimately associated with several cellular biological properties, such as drug or radiation sensitivity and cellular differentiation. (author)

  11. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

    OpenAIRE

    Hollander, John M.; Thapa, Dharendra; Shepherd, Danielle L.

    2014-01-01

    Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial su...

  12. Lipid droplets interact with mitochondria using SNAP23

    DEFF Research Database (Denmark)

    Jägerström, Sara; Polesie, Sam; Wickström, Ylva;

    2009-01-01

    peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic...... factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell....

  13. DNA content analysis of insect cell lines by flow cytometry

    OpenAIRE

    Léry, Xavier; Charpentier, Guy; Belloncik, Serge

    1999-01-01

    The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cel...

  14. Routine enterovirus diagnosis in a human rhabdomyosarcoma cell line

    OpenAIRE

    Bell, Eleanor J.; Cosgrove, Bonnie P.

    1980-01-01

    For many years a substitute cell line has been sought to replace monkey kidney cell cultures for the diagnosis of enterovirus infections. Reports by various workers have shown that the RD cell line, derived from a human rhabdomyosarcoma, will support the replication of most of the prototype strains of enterovirus. The present study shows that, with the exception of the group B coxsackieviruses, RD cells are more sensitive than cynomolgus monkey kidney cultures for the isolation of a wide vari...

  15. Getting mitochondria to center stage

    Energy Technology Data Exchange (ETDEWEB)

    Schatz, Gottfried, E-mail: gottfried.schatz@unibas.ch

    2013-05-10

    The question of how eukaryotic cells assemble their mitochondria was long considered to be inaccessible to biochemical investigation. This attitude changed about fifty years ago when the powerful tools of yeast genetics, electron microscopy and molecular biology were brought to bear on this problem. The rising interest in mitochondrial biogenesis thus paralleled and assisted in the birth of modern biology. This brief recollection recounts the days when research on mitochondrial biogenesis was an exotic effort limited to a small group of outsiders.

  16. Getting mitochondria to center stage

    International Nuclear Information System (INIS)

    The question of how eukaryotic cells assemble their mitochondria was long considered to be inaccessible to biochemical investigation. This attitude changed about fifty years ago when the powerful tools of yeast genetics, electron microscopy and molecular biology were brought to bear on this problem. The rising interest in mitochondrial biogenesis thus paralleled and assisted in the birth of modern biology. This brief recollection recounts the days when research on mitochondrial biogenesis was an exotic effort limited to a small group of outsiders

  17. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    Directory of Open Access Journals (Sweden)

    Akagi,Tadaatsu

    1982-10-01

    Full Text Available Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL line (BALL-1. The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1 and T-ALL (TALL-1 cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or normal B cell line (KO-HL-3, it reacted only with BALL-1 cells and did not react with other leukemia/lymphoma and normal B cell lines. The serum absorbed with tonsillar cells reacted only with BALL-1 and some B cell lines. Thus we were able to obtain antisera with specificity to B cell antigen, B-ALL antigen, and B cell line antigen.

  18. Permissiveness of human hepatoma cell lines for HCV infection

    Directory of Open Access Journals (Sweden)

    Sainz Bruno

    2012-01-01

    Full Text Available Abstract Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc, albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread. Results We found that while the early events in HCV infection (i.e. entry plus replication initiation are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56 expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection.

  19. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Human Subjects. Paperwork Reduction Act: This notice contains collection of information requirements... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of...

  20. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  1. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  2. The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria.

    Science.gov (United States)

    Ruan, Haihua; Zhang, Zhen; Tian, Li; Wang, Suying; Hu, Shuangyan; Qiao, Jian-Jun

    2016-09-16

    Microbial pathogens enter host cells by injecting effector proteins of the Type III secretion system (T3SS), which facilitate pathogen translocation across the host cell membrane. These effector proteins exert their effects by modulating a variety of host innate immune responses, thereby facilitating bacterial replication and systemic infection. Salmonella enterica serovar typhimurium (S.typhimurium) is a clinically important pathogen that causes food poisoning and gastroenteritis. The SopB effector protein of S. typhimurium, encoded by Salmonella pathogenicity islands (SPI)-1 T3SS, protects host epithelial cells from infection-induced apoptosis. However, how SopB influences apoptosis induction remains unclear. Here, we investigated the mechanism of SopB action in host cells. We found that SopB inhibits infection-induced apoptosis by attenuating the production of reactive oxygen species (ROS) in mitochondria, the crucial organelles for apoptosis initiation. Further investigation revealed that SopB binds to cytosolic tumor necrosis factor receptor associated factor 6 (TRAF6) and forms a trap preventing the mitochondrial recruitment of TRAF6, an essential event for ROS generation within mitochondria. By studying the response of Traf6(+/+) and Traf6(-/-)mouse embryonic fibroblasts to S. typhimurium infection, we found that TRAF6 promoted apoptosis by increasing ROS accumulation, which led to increased Bax/Bcl-2 ratio, Bax recruitment to mitochondrial membrane, and release of Cyt c into the cytoplasm. These findings show that SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial ROS generation. PMID:27473656

  3. GREG cells, a dysferlin-deficient myogenic mouse cell line

    International Nuclear Information System (INIS)

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (∼ 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1–43 dye. Under the wounding conditions used, the majority (∼ 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  4. GREG cells, a dysferlin-deficient myogenic mouse cell line

    Energy Technology Data Exchange (ETDEWEB)

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children' s National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: zimmerbj@mail.nih.gov [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  5. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  6. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Cherbas, L.; Willingham, A.; Zhang, D.; Yang, L.; Zou, Y.; Eads, B. D.; Carlson, J. W.; Landolin, J. M.; Kapranov, P.; Dumais, J.; Samsonova, A.; Choi, J. -H.; Roberts, J.; Davis, C. A.; Tang, H.; van Baren, M. J.; Ghosh, S.; Dobin, A.; Bell, K.; Lin, W.; Langton, L.; Duff, M. O.; Tenney, A. E.; Zaleski, C.; Brent, M. R.; Hoskins, R. A.; Kaufman, T. C.; Andrews, J.; Graveley, B. R.; Perrimon, N.; Celniker, S. E.; Gingeras, T. R.; Cherbas, P.

    2010-12-22

    Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what

  7. Fruit Extract from Pyropolyporus fomentarius (L. ex Fr.) Teng Induces Mitochondria-Dependent Apoptosis in Leukemia Cells but Enhances Immunomodulatory Activities of Splenic Lymphocytes.

    Science.gov (United States)

    Li, Yixiang; Zhou, Yuetao; Wang, Xiaobing; Wang, Pan; Xiao, Yaping; Cheng, Xiaoxia; Zhang, Yanhua; Liu, Yilin; Liu, Quanhong

    2016-01-01

    Pyropolyporus fomentarius (L. ex Fr.) Teng is a unique woody mushroom due to its medicinal value with numerous pharmacological activities. This study presented the potential antitumor and immunomodulatory properties of ethanol extract of P. fomentarius. The results showed that P. fomentarius extract inhibited the viability of murine leukemia L1210 cells in a dose-dependent manner with IC50 value of 69.35 μg/ml. Flow cytometry analysis demonstrated that the extract induced apoptosis in L1210 cells. Additionally, the decline of mitochondrial membrane potential was observed as well as the changes of caspase-3, caspase-9, Bcl-2, and Bax, suggesting that proapoptosis effect of the extract involved mitochondria-related pathway. Simultaneously, the P. fomentarius extract significantly enhanced the proliferation and activation of splenic lymphocytes in a dose-dependent manner. This P. fomentarius extract has potential applications as a natural antitumor agent with immunomodulatory activity. PMID:27115601

  8. Creatine Prevents the Structural and Functional Damage to Mitochondria in Myogenic, Oxidatively Stressed C2C12 Cells and Restores Their Differentiation Capacity.

    Science.gov (United States)

    Barbieri, Elena; Guescini, Michele; Calcabrini, Cinzia; Vallorani, Luciana; Diaz, Anna Rita; Fimognari, Carmela; Canonico, Barbara; Luchetti, Francesca; Papa, Stefano; Battistelli, Michela; Falcieri, Elisabetta; Romanello, Vanina; Sandri, Marco; Stocchi, Vilberto; Ciacci, Caterina; Sestili, Piero

    2016-01-01

    Creatine (Cr) is a nutritional supplement promoting a number of health benefits. Indeed Cr has been shown to be beneficial in disease-induced muscle atrophy, improve rehabilitation, and afford mild antioxidant activity. The beneficial effects are likely to derive from pleiotropic interactions. In accord with this notion, we previously demonstrated that multiple pleiotropic effects, including preservation of mitochondrial damage, account for the capacity of Cr to prevent the differentiation arrest caused by oxidative stress in C2C12 myoblasts. Given the importance of mitochondria in supporting the myogenic process, here we further explored the protective effects of Cr on the structure, function, and networking of these organelles in C2C12 cells differentiating under oxidative stressing conditions; the effects on the energy sensor AMPK, on PGC-1α, which is involved in mitochondrial biogenesis and its downstream effector Tfam were also investigated. Our results indicate that damage to mitochondria is crucial in the differentiation imbalance caused by oxidative stress and that the Cr-prevention of these injuries is invariably associated with the recovery of the normal myogenic capacity. We also found that Cr activates AMPK and induces an upregulation of PGC-1α expression, two events which are likely to contribute to the protection of mitochondrial quality and function. PMID:27610211

  9. Derivation of Genea052 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  10. Derivation of Genea047 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination.

  11. Derivation of Genea015 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  12. Derivation of human embryonic stem cell line Genea023

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  13. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  14. Derivation of Genea042 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  15. DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase.

    Science.gov (United States)

    Wintersberger, U; Blutsch, H

    1976-09-01

    The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected. PMID:786635

  16. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  17. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  18. Non-cytotoxic copper overload boosts mitochondrial energy metabolism to modulate cell proliferation and differentiation in the human erythroleukemic cell line K562.

    Science.gov (United States)

    Ruiz, Lina M; Jensen, Erik L; Rossel, Yancing; Puas, German I; Gonzalez-Ibanez, Alvaro M; Bustos, Rodrigo I; Ferrick, David A; Elorza, Alvaro A

    2016-07-01

    Copper is integral to the mitochondrial respiratory complex IV and contributes to proliferation and differentiation, metabolic reprogramming and mitochondrial function. The K562 cell line was exposed to a non-cytotoxic copper overload to evaluate mitochondrial dynamics, function and cell fate. This induced higher rates of mitochondrial turnover given by an increase in mitochondrial fusion and fission events and in the autophagic flux. The appearance of smaller and condensed mitochondria was also observed. Bioenergetics activity included more respiratory complexes, higher oxygen consumption rate, superoxide production and ATP synthesis, with no decrease in membrane potential. Increased cell proliferation and inhibited differentiation also occurred. Non-cytotoxic copper levels can modify mitochondrial metabolism and cell fate, which could be used in cancer biology and regenerative medicine. PMID:27094959

  19. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  20. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  1. Generation and characterization of human insulin-releasing cell lines

    OpenAIRE

    Joffé Elisa; Machado Marcel CC; Buchanan Cecilia; Terra Letícia F; Stigliano Iván; Krogh Karin; Peters Maria G; Labriola Leticia; Puricelli Lydia; Sogayar Mari C

    2009-01-01

    Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which w...

  2. Generation and characterization of human insulin-releasing cell lines

    Directory of Open Access Journals (Sweden)

    Joffé Elisa

    2009-06-01

    Full Text Available Abstract Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

  3. Generation and characterization of human insulin-releasing cell lines

    Science.gov (United States)

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  4. Radiobiological characteristics of cancer stem cells from esophageal cancer cell lines

    OpenAIRE

    Wang, Jian-Lin; Yu, Jing-Ping; Zhi-qiang SUN; Sun, Su-Ping

    2014-01-01

    AIM: To study the cancer stem cell population in esophageal cancer cell lines KYSE-150 and TE-1 and identify whether the resulting stem-like spheroid cells display cancer stem cells and radiation resistance characteristics.

  5. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a...

  6. Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: Effects on DNA, mitochondria, AhR binding and spindle organization

    International Nuclear Information System (INIS)

    Highlights: → PM2.5 induces mitotic arrest in BEAS-2B cells. → PM2.5 induces DNA damage and activates DNA damage response. → AhR regulated genes (Cyp1A1, Cyp1B1 and AhRR) are upregulated after PM exposure. → Mitotic spindle assembly is perturbed in PM exposed cells. - Abstract: Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in 'classical' apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.

  7. Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria

    Science.gov (United States)

    Kong, Byung-Whi; Lassiter, Kentu; Piekarski-Welsher, Alissa; Dridi, Sami; Reverter-Gomez, Antonio; Hudson, Nicholas James; Bottje, Walter Gay

    2016-01-01

    As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE. Funding provided by USDA-NIFA (#2013–01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville). PMID:27244447

  8. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  9. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  10. ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

    Directory of Open Access Journals (Sweden)

    Lilia Espada

    Full Text Available In this study we demonstrate that accumulation of reactive oxygen species (ROS is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell

  11. HIV-1 Vpr triggers mitochondrial destruction by impairing Mfn2-mediated ER-mitochondria interaction.

    Directory of Open Access Journals (Sweden)

    Chih-Yang Huang

    Full Text Available Human immunodeficiency virus 1 (HIV-1 viral protein R (Vpr has been shown to induce host cell death by increasing the permeability of mitochondrial outer membrane (MOM. The mechanism underlying the damage to the mitochondria by Vpr, however, is not clearly illustrated. In this study, Vpr that is introduced, via transient transfection or lentivirus infection, into the human embryonic kidney cell line HEK293, human CD4(+ T lymphoblast cell line SupT1, or human primary CD4(+ T cells serves as the model system to study the molecular mechanism of Vpr-mediated HIV-1 pathogenesis. The results show that Vpr injures MOM and causes a loss in membrane potential (MMP by posttranscriptionally reducing the expression of mitofusin 2 (Mfn2 via VprBP-DDB1-CUL4A ubiquitin ligase complex, gradually weakening MOM, and increasing mitochondrial deformation. Vpr also markedly decreases cytoplasmic levels of dynamin-related protein 1 (DRP1 and increases bulging in mitochondria-associated membranes (MAM, the specific regions of endoplasmic reticulum (ER which form physical contacts with the mitochondria. Overexpression of Mfn2 and DRP1 significantly decreased the loss of MMP and apoptotic cell death caused by Vpr. Furthermore, by employing time-lapse confocal fluorescence microscopy, we identify the transport of Vpr protein from the ER, via MAM to the mitochondria. Taken together, our results suggest that Vpr-mediated cellular damage may occur on an alternative protein transport pathway from the ER, via MAM to the mitochondria, which are modulated by Mfn2 and DRP1.

  12. Mitochondria in Neurodegeneration

    OpenAIRE

    Lezi, E; Swerdlow, Russell H.

    2012-01-01

    Many neurodegenerative diseases demonstrate abnormal mitochondrial morphology and biochemical dysfunction. Alterations are often systemic rather than brain-limited. Mitochondrial dysfunction may arise as a consequence of abnormal mitochondrial DNA, mutated nuclear proteins that interact directly or indirectly with mitochondria, or through unknown causes. In most cases it is unclear where mitochondria sit in relation to the overall disease cascades that ultimately causes neuronal dysfunction a...

  13. Characterization of an epithelial cell line from bovine mammary gland.

    Science.gov (United States)

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  14. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    Science.gov (United States)

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  15. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  16. Genetic mapping of paternal sorting of mitochondria in cucumber

    Science.gov (United States)

    Mitochondria are organelles that have their own DNA; serve as the powerhouses of eukaryotic cells; play important roles in stress responses, programmed cell death, and ageing; and in the vast majority of eukaryotes, are maternally transmitted. Strict maternal transmission of mitochondria makes it di...

  17. In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

    Science.gov (United States)

    Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R; Shen, Han-Ming; Lin, Qingsong

    2016-01-01

    To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death. PMID:26915414

  18. Ethanol induced mitochondria injury and permeability transition pore opening: Role of mitochondria in alcoholic liver disease

    Institute of Scientific and Technical Information of China (English)

    Ming Yan; Ping Zhu; Hui-Min Liu; Hai-Tao Zhang; Li Liu

    2007-01-01

    AIM: To observe changes of mitochondria and investigate the effect of ethanol on mitochondrial permeability transition pore (PTP), mitochondrial membrane potential (MMP, Δψm) and intracellular calcium concentration in hepatocytes by establishing an animal model of alcoholic liver disease (ALD).METHODS: Fourty adult male Wistar rats were randomly divided into two groups, the model group (20) was administered alcohol intragastrically plus an Oliver oil diet to establish an ALD model, and the control group (20) was given an equal amount of normal saline. The ultramicrostructural changes of mitochondria were observed under electron microscopy. Mitochondria of liver was extracted, and patency of PTP, mitochondrial membrane potential (Δψm), mitochondrial mass and intracellular calcium concentration of isolated hepacytes were detected by flow cytometry using rhodamine123 (Rh123), Nonyl-Acridine Orange and calcium fluorescent probe Fluo-3/AM, respectively.RESULTS: Membrane and cristae were broken or disappeared in mitochondria in different shapes under electron microscopy. Some mitochondria showed U shape or megamitochondrion. In the model group, liver mitochondria PTP was broken, and mitochondria swelled, the absorbance at 450 nm, A540 decreased (0.0136 ± 0.0025 vs 0.0321 ± O.0013,model vs control,P<O.01);mitochondria transmembrane potential (239.4638 ± 12.7263 vs 377.5850 ± 16.8119,P<0.01) was lowered;mitochondrial mass (17.4350 ± 1.9880 vs 31.6738 ± 3.4930,P<0.01);and [Ca2+]i was increased in liver cells (7.0020 ± 0.5008 vs 10.2050 ± 0.4701,P<0.01).CONCLUSION:Chronic alcohol intake might lead to broken mitochondria PTP,decreased mitochondria membrane potential and injury,and elevated intracellular Ca2+ production.Ethanol-induced chondriosome injury may be an important mechanism of alcoholic diseases.

  19. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    Science.gov (United States)

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. PMID:20623584

  20. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  1. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    Science.gov (United States)

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells. PMID:310907

  2. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  3. Alcohol Withdrawal and Cerebellar Mitochondria.

    Science.gov (United States)

    Jung, Marianna E

    2015-08-01

    Cerebellar disorders trigger the symptoms of movement problems, imbalance, incoordination, and frequent fall. Cerebellar disorders are shown in various CNS illnesses including a drinking disorder called alcoholism. Alcoholism is manifested as an inability to control drinking in spite of adverse consequences. Human and animal studies have shown that cerebellar symptoms persist even after complete abstinence from drinking. In particular, the abrupt termination (ethanol withdrawal) of long-term excessive ethanol consumption has shown to provoke a variety of neuronal and mitochondrial damage to the cerebellum. Upon ethanol withdrawal, excitatory neurotransmitter molecules such as glutamate are overly released in brain areas including cerebellum. This is particularly relevant to the cerebellar neuronal network as glutamate signals are projected to Purkinje neurons through granular cells that are the most populated neuronal type in CNS. This excitatory neuronal signal may be elevated by ethanol withdrawal stress, which promotes an increase in intracellular Ca(2+) level and a decrease in a Ca(2+)-binding protein, both of which result in the excessive entry of Ca(2+) to the mitochondria. Subsequently, mitochondria undergo a prolonged opening of mitochondrial permeability transition pore and the overproduction of harmful free radicals, impeding adenosine triphosphate (ATP)-generating function. This in turn provokes the leakage of mitochondrial molecule cytochrome c to the cytosol, which triggers a cascade of adverse cytosol reactions. Upstream to this pathway, cerebellum under the condition of ethanol withdrawal has shown aberrant gene modifications through altered DNA methylation, histone acetylation, or microRNA expression. Interplay between these events and molecules may result in functional damage to cerebellar mitochondria and consequent neuronal degeneration, thereby contributing to motoric deficit. Mitochondria-targeting research may help develop a powerful new

  4. Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria.

    Science.gov (United States)

    Kong, Byung-Whi; Lassiter, Kentu; Piekarski-Welsher, Alissa; Dridi, Sami; Reverter-Gomez, Antonio; Hudson, Nicholas James; Bottje, Walter Gay

    2016-01-01

    As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P Analysis (IPA) program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P analysis also identified mitochondrial dysfunction and oxidative phosphorylation as the number 1 and 5 differentially expressed canonical pathways (up-regulated in high FE) in the proteomic dataset. Upstream analysis (based on DE of downstream molecules) predicted that insulin receptor, insulin like growth receptor 1, nuclear factor, erythroid 2-like 2, AMP activated protein kinase (α subunit), progesterone and triiodothyronine would be activated in the high FE phenotype whereas rapamycin independent companion of target of rapamycin, mitogen activated protein kinase 4, and serum response factor would be inhibited in the high FE phenotype. The results provide additional insight into the fundamental molecular landscape of feed efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE. Funding provided by USDA-NIFA (#2013-01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville). PMID:27244447

  5. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  6. The membrane targeted apoptosis modulators erucylphosphocholine and erucylphosphohomocholine increase the radiation response of human glioblastoma cell lines in vitro

    International Nuclear Information System (INIS)

    Alkylphosphocholines constitute a novel class of antineoplastic synthetic phospholipid derivatives that induce apoptosis of human tumor cell lines by targeting cellular membranes. We could recently show that the first intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC) is a potent inducer of apoptosis in highly resistant human astrocytoma/glioblastoma cell lines in vitro. ErPC was shown to cross the blood brain barrier upon repeated intravenous injections in rats and thus constitutes a promising candidate for glioblastoma therapy. Aim of the present study was to analyze putative beneficial effects of ErPC and its clinically more advanced derivative erucylphosphohomocholine (erucyl-N, N, N-trimethylpropanolaminphosphate, ErPC3, Erufosine™ on radiation-induced apoptosis and eradication of clonogenic tumor cells in human astrocytoma/glioblastoma cell lines in vitro. While all cell lines showed high intrinsic resistance against radiation-induced apoptosis as determined by fluorescence microscopy, treatment with ErPC and ErPC3 strongly increased sensitivity of the cells to radiation-induced cell death (apoptosis and necrosis). T98G cells were most responsive to the combined treatment revealing highly synergistic effects while A172 showed mostly additive to synergistic effects, and U87MG cells sub-additive, additive or synergistic effects, depending on the respective radiation-dose, drug-concentration and treatment time. Combined treatment enhanced therapy-induced damage of the mitochondria and caspase-activation. Importantly, combined treatment also increased radiation-induced eradication of clonogenic T98G cells as determined by standard colony formation assays. Our observations make the combined treatment with ionizing radiation and the membrane targeted apoptosis modulators ErPC and ErPC3 a promising approach for the treatment of patients suffering from malignant glioma. The use of this innovative treatment concept in an in vivo xenograft

  7. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

    Full Text Available Abstract Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus decoloratus, Rhipicephalus (Boophilus microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG as cryoprotectant was compared with dimethylsulfoxide (DMSO supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B. microplus, R. (B. decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B. decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B. microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B. decoloratus, R. (B. microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

  8. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway

    International Nuclear Information System (INIS)

    Cerebral endothelial cells (CECs) are crucial components of the blood-brain barrier. Oxidized low-density lipoprotein (oxLDL) can induce cell injuries. In this study, we attempted to evaluate the effects of oxLDL on mouse CECs and its possible mechanisms. Mouse CECs were isolated from brain tissues and identified by immunocytochemical staining of vimentin and Factor VIII. oxLDL was prepared from LDL oxidation by copper sulfate. Exposure of mouse CECs to oxLDL decreased cell viability in concentration- and time-dependent manners. oxLDL time-dependently caused shrinkage of cell morphologies. Administration of oxLDL to CECs induced DNA fragmentation in concentration- and time-dependent manners. Analysis of the cell cycle revealed that oxLDL concentration- and time-dependently increased the proportion of CECs which underwent apoptosis. Analysis of confocal microscopy and immunoblot revealed that oxLDL significantly increased cellular and mitochondrial Bax levels as well as the translocation of this proapoptotic protein from the cytoplasm to mitochondria. In parallel with the increase in the levels and translocation of Bax, oxLDL time-dependently decreased the mitochondrial membrane potential. Exposure of mouse CECs to oxLDL decreased the amounts of mitochondrial cytochrome c, but enhanced cytosolic cytochrome c levels. The amounts of intracellular reactive oxygen species were significantly augmented after oxLDL administration. Sequentially, oxLDL increased activities of caspase-9, -3, and -6 in time-dependent manners. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, significantly decreased caspase-6 activity and the oxLDL-induced DNA fragmentation and cell apoptosis. This study showed that oxLDL induces apoptotic insults to CECs via signal-transducing events, including enhancing Bax translocation, mitochondrial dysfunction, cytochrome c release, increases in intracellular reactive oxygen species, and cascade activation of caspase-9, -3, and -6. Therefore, ox

  9. Magnetic resonance spectroscopy in tumor cell lines research

    International Nuclear Information System (INIS)

    MRS can be used non-invasively to study the several trace metabolites and energy metabolism in vivo. By quantitatively analyzing the compounds changes we could detect abnormal metabolism in tumor and its surrounding tissue, and estimate tumor infiltration in vivo and vitro. In recent years, MRS has been applied in cell line research and is becoming a promising method. In this article we summarized the applications of MRS in cell lines in studying diagnosis, treatment, and tumor mechanisms. (authors)

  10. Characterization of xenoantiserum produced against B cell acute lymphoblastic leukemia cell line

    OpenAIRE

    Akagi,Tadaatsu; Sonobe, Hiroshi; Miyoshi, Isao; Yoshimoto,Shizuo

    1982-01-01

    Antiserum was produced in white rabbit by intravenously injecting living cells of a B cell acute lymphoblastic leukemia (ALL) line (BALL-1). The reactivity of the antiserum against various lymphoid cell lines was examined by membrane immunofluorescence after appropriate absorption. Serum absorbed with non-T, non-B (NALL-1) and T-ALL (TALL-1) cells recognized B cell antigens distinct from Ia-like antigens on both normal and neoplastic B cells. After further absorption with tonsillar cells or n...

  11. On Message Ribonucleic Acids Targeting to Mitochondria

    Directory of Open Access Journals (Sweden)

    Kunjan R. Dave

    2009-12-01

    Full Text Available Mitochondria are subcellular organelles that provide energy for a variety of basic cellular processes in eukaryotic cells. Mitochondria maintain their own genomes and many of their endosymbiont genes are encoded by nuclear genomes. The crosstalk between the mitochondrial and nuclear genomes ensures mitochondrial biogenesis, dynamics and maintenance. Mitochondrial proteins are partly encoded by nucleus and synthesized in the cytosol and partly in the mitochondria coded by mitochondrial genome. The efficiency of transport systems that transport nuclear encoded gene products such as proteins and mRNAs to the mitochondrial vicinity to allow for their translation and/or import are recently receiving wide attention. There is currently no concrete evidence that nuclear encoded mRNA is transported into the mitochondria, however, they can be transported onto the mitochondrial surface and translated at the surface of mitochondria utilizing cytosolic machinery. In this review we present an overview of the recent advances in the mRNA transport, with emphasis on the transport of nuclear-encoded mitochondrial protein mRNA into the mitochondria.

  12. Mitochondria-targeted peptide antioxidants: Novel neuroprotective agents

    OpenAIRE

    Szeto, Hazel H.

    2006-01-01

    Increasing evidence suggests that mitochondrial dysfunction and oxidative stress play a crucial role in the majority of neurodegenerative diseases. Mitochondria are a major source of intracellular reactive oxygen species (ROS) and are particularly vulnerable to oxidative stress. Oxidative damage to mitochondria has been shown to impair mitochondrial function and lead to cell death via apoptosis and necrosis. Because dysfunctional mitochondria will produce more ROS, a feed-forward loop is set ...

  13. Reliable in vitro studies require appropriate ovarian cancer cell lines.

    Science.gov (United States)

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  14. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  15. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    International Nuclear Information System (INIS)

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT

  16. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xian-Ying; Chen, Wei [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Fan, Jun-Ting [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Song, Ran; Wang, Lu [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Gu, Yan-Hong [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zeng, Guang-Zhi [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Shen, Yan; Wu, Xue-Feng [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Tan, Ning-Hua, E-mail: nhtan@mail.kib.ac.cn [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Sun, Yang, E-mail: yangsun@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China)

    2013-02-15

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT.

  17. Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

    Directory of Open Access Journals (Sweden)

    Ching-Hao Li

    2010-02-01

    Full Text Available p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Disruption of cardiolipin biosynthesis by phosphatidylglycero-phosphate synthase (PGS or CDP-diacylglycerol synthase 2 (CDS-2 short hairpin RNA (shRNA transfection eliminated the MMC-induced translocation of mitochondrial p53. The elimination of mitochondrial p53 translocation also reduced Bcl-xL and Bcl-2 mitochondrial distribution. In HEK 293T models with saturated p53 expression, the mitochondrial partition of p53, Bcl-xL, and Bcl-2 obviously decreased in their PGS shRNA- or CDS-2 shRNA-expressing stable clones. In p53-null H1299 models, both the mitochondrial partitions of Bcl-xL and Bcl-2 were strongly reduced in relation to the HEK 293T models. The Bcl-xL mitochondrial partition was elevated in H1299 models expressing pCEP4-p53wt suggesting the direct carrier role of p53 in transporting Bcl-xL to the mitochondria. We also found that the cytosolic pool of Bcl-xL and Bcl-2 remained unaffected in the low-dose MMC treatment but decreased in the high-dose MMC treatment. The cytosolic pool of Bcl-2 and Bcl-xL directly regulated their amounts in p53-dependent mitochondrial distribution. In the low-dose MMC treatment, the increased mitochondrial p53, Bcl-xL, and Bcl-2 could attenuate apoptosis. However, in the high-dose MMC treatment, only the p53 translocated to the mitochondria and resulted in apoptosis progression. On the basis of this study, we thought mitochondrial p53 might regulate apoptosis in a biphasic manner.

  18. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  19. K-Ras and mitochondria: Dangerous liaisons

    Institute of Scientific and Technical Information of China (English)

    Jiri Neuzil; Jakub Rohlena; Lan-Feng Dong

    2012-01-01

    It is well documented that the KRAS oncogene efficiently transforms non-malignant cells,and there is some evidence for the role of mitochondria in this process.Now Peng Huang and colleagues show that K-Ras induction results early on in mitochondria assuming the phenotype consistent with the so-called Warburg effect,i.e.,increased glycolysis and attenuated oxidative phosphorylation.Thus the K-Ras protein capable of swift induction of phenotypic changes typical of cancer cells,yet these changes are reversible,and for cells to irreversibly reach their full malignant potential a much longer K-Ras expression is required,implicating mitochondria in the longer-term effects of the oncogene.

  20. Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

    LENUS (Irish Health Repository)

    Anoopkumar-Dukie, S

    2009-10-01

    Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax\\/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

  1. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines.

    Science.gov (United States)

    Kirkegaard, Jeannette S; Ravassard, Philippe; Ingvarsen, Signe; Diedisheim, Marc; Bricout-Neveu, Emilie; Grønborg, Mads; Frogne, Thomas; Scharfmann, Raphael; Madsen, Ole D; Rescan, Claude; Albagli, Olivier

    2016-03-01

    It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices. PMID:26901817

  2. The toxicity of rifampicin polylactic acid nanoparticles against Mycobacterium bovis BCG and human macrophage THP-1 cell line

    Science.gov (United States)

    Erokhina, M.; Rybalkina, E.; Barsegyan, G.; Onishchenko, G.; Lepekha, L.

    2015-11-01

    Tuberculosis is rapidly becoming a major health problem. The rise in tuberculosis incidence stimulates efforts to develop more effective delivery systems for the existing antituberculous drugs while decreasing the side effects. The nanotechnology may provide novel drug delivery tools allowing controlled drug release. Rifampicin is one of the main antituberculous drugs, characterized by high toxicity, and Poly (L-lactic acid) (PLLA) is a biodegradable polymer used for the preparation of encapsulated drugs. The aim of our work was to evaluate the toxicity of rifampicin-PLLA nanoparticles against Mycobacterium bovis BCG using human macrophage THP-1 cell line. Our data demonstrate that rifampicin-PLLA is effective against M. bovis BCG in the infected macrophages. The drug is inducing the dysfunction of mitochondria and apoptosis in the macrophages and is acting as a potential substrate of Pgp thereby modulating cell chemosensitivity. The severity of the toxic effects of the rifampicin-PLLA nanoparticles is increasing in a dose-dependent manner. We suggest that free rifampicin induces death of M. bovis BCG after PLLA degradation and diffusion from phago-lysosomes to cytoplasm causing mitochondria dysfunction and affecting the Pgp activity.

  3. In vitro radiosensitivity of human leukemia cell lines

    International Nuclear Information System (INIS)

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad[1.17 Gy]), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad[1.65 Gy]), erythroleukemia; 45 (n . 1.1, D0 . 147 rad[1.47 Gy]), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad[0.76 Gy]), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established

  4. Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

    Directory of Open Access Journals (Sweden)

    Belka Claus

    2009-10-01

    Full Text Available Abstract Background Programmed cell death (PCD is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD. Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER. Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA, the ER (Bcl-2 cb5, both (Bcl-2 WT or the cytosol/nucleus (Bcl-2 ΔTM and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.

  5. Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

    International Nuclear Information System (INIS)

    Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide

  6. Effect of dehydrodidemnin B on human colon carcinoma cell lines.

    Science.gov (United States)

    Lobo, C; García-Pozo, S G; Núñez de Castro, I; Alonso, F J

    1997-01-01

    Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. In the present study, a new member, dehydrodidemnin B (DDB), isolated from the mediterranean tunicate Aplidium albicans, was used. The effect of the drug on human colon cultured cell lines was tested using multiple approaches: proliferation studies, long term survival after three hours of exposure to DDB by means of a clonogenic assay and the decrease of the protooncogen, ornithine decarboxylase, activity. A dehydrodidemnin B concentration of 10(-8) M completely inhibited cell growth. The IC50 obtained using the MTT proliferation test, indicated that the most proliferative cell line (CT-2) was the most sensitive to the drug. Using a clonogenic assay a clear dose-response was obtained for the three cell lines used; HT-29 cell line showed the minimum survival after 3 hours of dehydrodidemnin B treatment. A dose-dependent decrease in ornithine decarboxylase activity was also observed in three cell lines assayed. The data presented indicate that the dehydrodidemnin B is a potent cytotoxic agent on rapidly dividing human colon cancer cells. PMID:9066673

  7. Transcription profiles of non-immortalized breast cancer cell lines

    International Nuclear Information System (INIS)

    Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research

  8. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian;

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology to an...... epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more...

  9. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    Science.gov (United States)

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  10. Solid Oxide Fuel Cell Systems PVL Line

    Energy Technology Data Exchange (ETDEWEB)

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability

  11. Drosophila Follicle Stem Cells are regulated by proliferation and niche adhesion as well as mitochondria and ROS

    OpenAIRE

    Wang, Zhu A.; Huang, Jianhua; Kalderon, Daniel

    2012-01-01

    The mechanisms underlying adult stem cell behavior are likely to be diverse and have not yet been investigated systematically. Here we conducted an unbiased genetic screen using Drosophila ovarian follicle stem cells (FSCs) to probe essential functions regulating self-renewal of epithelial stem cells. Surprisingly, we find that niche adhesion emerge as the most commonly affected essential stem cell property, and that proliferation is critical for stem cell maintenance. We also find that PI3K ...

  12. Steroid hormone secretion in inflammatory breast cancer cell lines.

    Science.gov (United States)

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were 90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  13. Mitochondria-rich cell activity in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels.

    Science.gov (United States)

    Lin, Li-Yih; Hwang, Pung-Pung

    2004-03-01

    Mitochondria-rich cells (MRCs) in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae were examined by Na+/K(+)-ATPase immunocytochemistry and vital staining for glycoproteins following acclimation to high (7.5-7.9 mmol l(-1)), normal (0.48-0.52 mmol l(-1)) or low (0.002-0.007 mmol l(-1)) ambient Cl- levels. With a combination of concanavalin-A (Con-A)-Texas-Red conjugate staining (larvae exposed to the dye in vivo in the water) and a monoclonal antibody raised against Na+/K(+)-ATPase, MRCs were easily recognized and presumed to be active when Con-A-positive (i.e. with their apical membrane in contact with the water) or inactive when Con-A-negative. The proportion of active cells gradually increased during a 48-h acclimation to low-Cl- medium but decreased during acclimation to high-Cl- medium. Total densities of MRCs did not change when ambient chloride levels were altered. Furthermore, in live larvae exposed to changes in ambient Cl-, yolk-sac MRCs, vitally stained with DASPEI and subsequently traced in time, did not significantly alter turnover. The polymorphism of the apical membrane compartment of the MRCs represents structural modification of the active MRCs. Yolk-sac pavement cells labeled with the membrane marker FM1-43 (fluorescent lipophilic tracer) were shown to cover active MRCs in larvae transferred from normal to high ambient Cl- levels, thereby inactivating the MRCs. PMID:15010484

  14. Corticotropin-releasing hormone and extracellular mitochondria augment IgE-stimulated human mast-cell vascular endothelial growth factor release, which is inhibited by luteolin

    Directory of Open Access Journals (Sweden)

    Asadi Shahrzad

    2012-05-01

    Full Text Available Abstract Background Autism spectrum disorders (ASDs are neurodevelopmental disorders characterized by varying degrees of dysfunctional social abilities, learning deficits, and stereotypic behaviors. Many patients with ASDs have ‘allergy-like’ symptoms and respond disproportionally to stress. We have previously shown that the peptide neurotensin (NT is increased in the serum of young children with autism and that can stimulate extracellular secretion of mitochondrial (mtDNA which was also increased in the serum of these children. Methods Human mast cells were stimulated by corticotropin-releasing hormone (CRH, mitochondrial DNA, IgE/anti-IgE, either for 24 hours to measure vascular endothelial growth factor (VEGF release by ELISA or for 6 hours or quantitative PCR. Results CRH augmented IgE/anti-IgE-induced human mast-cell release of VEGF and it also induced the expression of IgE receptor (FcεRI on mast cells. Moreover, sonicated mitochondria also augmented VEGF release, and this effect was blocked by the natural flavone luteolin. Conclusion These results indicate that stress and infection-mimicking extracellular mitochondrial components augment allergic inflammation that may be involved in the early pathogenesis of ASDs. Moreover, luteolin inhibits these processes and may be helpful in the treatment of ASDs.

  15. Baicalein Triggers Mitochondria-Mediated Apoptosis and Enhances the Antileukemic Effect of Vincristine in Childhood Acute Lymphoblastic Leukemia CCRF-CEM Cells

    Directory of Open Access Journals (Sweden)

    Yun-Ju Chen

    2013-01-01

    Full Text Available Acute lymphoblastic leukemia (ALL accounts for approximately 75% of childhood leukemia, and chemotherapy remains the mainstay therapy. Baicalein is an active flavonoid used in traditional Chinese medicine and has recently been found to have anticancer, anti-inflammatory, and antiallergic properties. This study aims to investigate the molecular apoptotic mechanisms of baicalein in CCRF-CEM leukemic cells and to evaluate the combined therapeutic efficacy of baicalein with several commonly used chemotherapeutic drugs in CCRF-CEM cells. Our results demonstrate that baicalein induces mitochondria-dependent cleavage of caspases-9 and -3 and PARP with concomitant decreases in IAP family proteins, survivin, and XIAP. Furthermore, our results present for the first time that baicalein triggers a convergence of the intrinsic and extrinsic apoptotic pathways via the death receptor-caspase 8-tBid signaling cascade in CCRF-CEM cells. In addition, we also present for the first time that the combination of baicalein and vincristine results in a synergistic therapeutic efficacy. Overall, this combination strategy is recommended for future clinical trials in the treatment of pediatric leukemia owing to baicalein’s beneficial effects in alleviating the vomiting, nausea, and skin rashes caused by chemotherapy.

  16. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    Science.gov (United States)

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  17. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326. ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  18. Frequency and distribution of Notch mutations in tumor cell lines

    International Nuclear Information System (INIS)

    Deregulated Notch signaling is linked to a variety of tumors and it is therefore important to learn more about the frequency and distribution of Notch mutations in a tumor context. In this report, we use data from the recently developed Cancer Cell Line Encyclopedia to assess the frequency and distribution of Notch mutations in a large panel of cancer cell lines in silico. Our results show that the mutation frequency of Notch receptor and ligand genes is at par with that for established oncogenes and higher than for a set of house-keeping genes. Mutations were found across all four Notch receptor genes, but with notable differences between protein domains, mutations were for example more prevalent in the regions encoding the LNR and PEST domains in the Notch intracellular domain. Furthermore, an in silico estimation of functional impact showed that deleterious mutations cluster to the ligand-binding and the intracellular domains of NOTCH1. For most cell line groups, the mutation frequency of Notch genes is higher than in associated primary tumors. Our results shed new light on the spectrum of Notch mutations after in vitro culturing of tumor cells. The higher mutation frequency in tumor cell lines indicates that Notch mutations are associated with a growth advantage in vitro, and thus may be considered to be driver mutations in a tumor cell line context. The online version of this article (doi:10.1186/s12885-015-1278-x) contains supplementary material, which is available to authorized users

  19. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    International Nuclear Information System (INIS)

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion

  20. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  1. Female sex bias in human embryonic stem cell lines.

    Science.gov (United States)

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat; Eiges, Rachel

    2012-02-10

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  2. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  3. Citrinin induces apoptosis via a mitochondria-dependent pathway and inhibition of survival signals in embryonic stem cells, and causes developmental injury in blastocysts.

    Science.gov (United States)

    Chan, Wen-Hsiung

    2007-06-01

    The mycotoxin CTN (citrinin), a natural contaminant in foodstuffs and animal feeds, has cytotoxic and genotoxic effects on various mammalian cells. CTN is known to cause cell injury, including apoptosis, but the precise regulatory mechanisms of CTN action, particularly in stem cells and embryos, are currently unclear. In the present paper, I report that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Experiments in embryonic stem cells (ESC-B5) showed that CTN induces apoptosis via ROS (reactive oxygen species) generation, increased Bax/Bcl-2 ratio, loss of MMP (mitochondrial membrane potential), induction of cytochrome c release, and activation of caspase 3. In this model, CTN triggers cell death via inactivation of the HSP90 [a 90 kDa isoform of the HSP (heat-shock protein) family proteins]/multichaperone complex and subsequent degradation of Ras and Raf-1, further inhibiting anti-apoptotic processes, such as the Ras-->ERK (extracellular-signal-regulated kinase) signal transduction pathway. In addition, CTN causes early developmental injury in mouse ESCs and blastocysts in vitro. Lastly, using an in vivo mouse model, I show that consumption of drinking water containing 10 muM CTN results in blastocyst apoptosis and early embryonic developmental injury. Collectively, these findings show for the first time that CTN induces ROS and mitochondria-dependent apoptotic processes, inhibits Ras-->ERK survival signalling via inactivation of the HSP90/multichaperone complex, and causes developmental injury in vivo. PMID:17331071

  4. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  5. Grape seed proanthocyanidin extract induced mitochondria-associated apoptosis in human acute myeloid leukaemia 14.3D10 cells

    Institute of Scientific and Technical Information of China (English)

    HU Hong; QIN Yi-min

    2006-01-01

    @@ The important aim in cancer treatment is the selective killing of cancer cells without damaging healthy cells and the most commonly used chemotherapy for eliminating cancer is achieved by activating the mitochondrial apoptotic pathway.1 Therefore, identification of new agents that can selectively kill cancer cells becomes crucial in cancer clinical treatment.

  6. Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts.

    Science.gov (United States)

    Roham, Pratiksha H; Kharat, Kiran R; Mungde, Priyanka; Jadhav, Mahadev A; Makhija, Surinder J

    2016-01-01

    Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D). PMID:26908199

  7. A reaction to mitochondria in action

    Institute of Scientific and Technical Information of China (English)

    Mark P Mattson

    2011-01-01

    In most cells and under most conditions,mitochondria produce the majority of the readily available energy (in the form of ATP) to sustain a myriad of biochemical processes necessary for the survival and proper function of the cell.Excitable cells,such as neurons and muscle cells,require very high amounts of ATP to maintain and restore the ion gradients across membranes as they experience frequent bouts of depolarization-induced Na+ and Ca2* influx [ 1 ].

  8. Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix.

    Science.gov (United States)

    Luecke, Nina; Templin, Christian; Muetzelburg, Marika Victoria; Neumann, Detlef; Just, Ingo; Pich, Andreas

    2010-03-15

    Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration. PMID:20127908

  9. Protective effect of whey protein hydrolysates on H₂O₂-induced PC12 cells oxidative stress via a mitochondria-mediated pathway.

    Science.gov (United States)

    Jin, Man-Man; Zhang, Li; Yu, Hui-Xin; Meng, Jun; Sun, Zhen; Lu, Rong-Rong

    2013-11-15

    Whey protein hydrolysates (WPHs) were prepared with pepsin and trypsin. A PC12 cell model was built to observe the protective effect of WPHs against H2O2-induced oxidative stress. The results indicated that WPHs reduced apoptosis by 14% and increased antioxidant enzyme activities. Flow cytometry was used to assess the accumulation of reactive oxygen species (ROS), Ca(2+) levels and the mitochondrial membrane potential (MMP). The results showed that WPHs suppressed ROS elevation and Ca(2+) levels and stabilised MMP by 16%. The anti-apoptosis/pro-apoptosis proteins Bcl-2/Bax and poly (ADP-ribose) polymerase (PARP) were investigated by Western-blot analysis, which indicated that WPHs increased the expression of Bcl-2 while inhibiting the expression of Bax and the degradation of PARP. WPHs also blocked Caspase-3 activation by 62%. The results demonstrate that WPHs can significantly protect PC12 cells against oxidative stress via a mitochondria-mediated pathway. These findings indicate the potential benefits of WPHs as valuable food antioxidative additives. PMID:23790857

  10. Human cell lines: A promising alternative for recombinant FIX production.

    Science.gov (United States)

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins. PMID:26802680

  11. APR-246 (PRIMA-1(MET)) strongly synergizes with AZD2281 (olaparib) induced PARP inhibition to induce apoptosis in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Deben, Christophe; Lardon, Filip; Wouters, An; Op de Beeck, Ken; Van den Bossche, Jolien; Jacobs, Julie; Van Der Steen, Nele; Peeters, Marc; Rolfo, Christian; Deschoolmeester, Vanessa; Pauwels, Patrick

    2016-06-01

    APR-246 (PRIMA-1(Met)) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis. We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background. PMID:26975633

  12. Nilotinib reduced the viability of human ovarian cancer cells via mitochondria-dependent apoptosis, independent of JNK activation.

    Science.gov (United States)

    Chen, Tze-Chien; Yu, Ming-Chih; Chien, Chih-Chiang; Wu, Ming-Shun; Lee, Yu-Chieh; Chen, Yen-Chou

    2016-03-01

    Nilotinib (AMN) induces apoptosis in various cancer cells; however the effect of AMN on human ovarian cancer cells is still unclear. A reduction in cell viability associated with the occurrence of apoptotic characteristics was observed in human SKOV-3 ovarian cancer cells under AMN but not sorafenib (SORA) or imatinib (STI) stimulation. Activation of apoptotic pathway including increased caspase (Casp)-3 and poly(ADP-ribose) polymerase 1 (PARP1) protein cleavage by AMN was detected with disrupted mitochondrial membrane potential (MMP) accompanied by decreased Bcl-2 protein and increased cytosolic cytochrome (Cyt) c/cleaved Casp-9 protein expressions was found, and AMN-induced cell death was inhibited by peptidyl Casp inhibitors, VAD, DEVD and LEHD. Increased phosphorylated c-Jun N-terminal kinase (JNK) protein expression was detected in AMN- but not SORA- or STI-treated SKOV-3 cells, and the JNK inhibitors, SP600125 and JNKI, showed slight but significant enhancement of AMN-induced cell death in SKOV-3 cells. The intracellular peroxide level was elevated by AMN and H2O2, and N-acetylcysteine (NAC) prevented H2O2- but not AMN-induced peroxide production and apoptosis in SKOV-3 cells. AMN induction of apoptosis with increased intracellular peroxide production and JNK protein phosphorylation was also identified in human A2780 ovarian cancer cells, cisplatin-resistant A2780CP cells, and clear ES-2 cells. The evidence supporting AMN effectively reducing the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis is provided. PMID:26549707

  13. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  14. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  15. Comparison of repair capability of four human tumor cell lines

    International Nuclear Information System (INIS)

    A fast and reliable method for assessment of repair capacity of cells based on the restoration of transcription of the gene for the green fluorescent protein carried by a plasmid vector is presented. Repair capacity of the cells counted under fluorescent microscope 24 hours following transcription. This approach has been applied to compare the rates of repair of different types of DNA lesions in four human tumor cell lines. The analysis of the obtained results show that there are considerable differences in the repair capacity of the different cell lines towards the different types of lesions. It should be noted that the difference in repair capacity of the host cells are better evident when plasmids with lower rather that higher number of lesions per unit length of plasmid DNA are used. This is probably because the egfp genes with fewer lesions can be fully repaired while egfp genes with more lesions remain inactive even after some of the lesions have been repaired

  16. Smac/DIABLO release from mitochondria and XIAP inhibition are essential to limit clonogenicity of Type I tumor cells after TRAIL receptor stimulation

    OpenAIRE

    Borst, Jannie; Maas, Chiel; Verbrugge, Inge; de Vries, Evert; Savich, Gleb; Van De Kooij, Lambertus W; Tait, Stephen W.

    2010-01-01

    Abstract Death receptors such as Fas/CD95 and TRAIL receptors engage the extrinsic pathway for caspase activation, but also couple to the intrinsic mitochondrial route. In so-called Type II cells, death receptors require the mitochondrial pathway for apoptotic execution, whereas in Type I cells they reportedly do not. For established tumor cell lines, the Type I/Type II distinction is based on short-term apoptosis assays. We report here that the mitochondrial pathway is essential f...

  17. Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

    Science.gov (United States)

    Blomberg, K

    1994-02-10

    A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium. PMID:8308301

  18. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  19. Alloreactive cloned T cell lines. I. Interactions between cloned amplifier and cytolytic T cell lines

    OpenAIRE

    1980-01-01

    Several T cell clones have been derived by limiting dilution of secondary mixed leukocyte culture cells stimulated by H-2- and M locus (Mls)-disparate spleen cells. When examined for the expression of cytolytic activity and the ability to proliferate, these cell clones can be classified into two major categories. One type of cell is noncytolytic; when cultured with irradiated spleen cells, such clones proliferate in response to Mls determinants. Some, but not all, of these clones express Lyt-...

  20. Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection

    Science.gov (United States)

    Cowan, Douglas B.; Yao, Rouan; Akurathi, Vamsidhar; Snay, Erin R.; Thedsanamoorthy, Jerusha K.; Zurakowski, David; Ericsson, Maria; Friehs, Ingeborg; Wu, Yaotang; Levitsky, Sidney; del Nido, Pedro J.; Packard, Alan B.

    2016-01-01

    We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid

  1. Human mesenchymal stem cells reprogram adult cardiomyocytes toward a progenitor-like state through partial cell fusion and mitochondria transfer : Cell fusion-mediated cardiomyocyte reprogramming.

    OpenAIRE

    Acquistapace, Adrien; Bru, Thierry; Lesault, Pierre-François; Figeac, Florence; Coudert, Amélie,; Le Coz, Olivier; Christov, Christo; Baudin, Xavier; Auber, Fréderic; Yiou, René; Dubois-Randé, Jean-Luc; Rodriguez, Anne-Marie

    2011-01-01

    International audience Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes. However, the respective physiological impact of these two processes remains unknown in part because of the lack of knowledge of the resulting hybrid cells. To ...

  2. The polyphenols resveratrol and epigallocatechin-3-gallate restore the severe impairment of mitochondria in hippocampal progenitor cells from a Down syndrome mouse model.

    Science.gov (United States)

    Valenti, Daniela; de Bari, Lidia; de Rasmo, Domenico; Signorile, Anna; Henrion-Caude, Alexandra; Contestabile, Andrea; Vacca, Rosa Anna

    2016-06-01

    Mitochondrial dysfunctions critically impair nervous system development and are potentially involved in the pathogenesis of various neurodevelopmental disorders, including Down syndrome (DS), the most common genetic cause of intellectual disability. Previous studies from our group demonstrated impaired mitochondrial activity in peripheral cells from DS subjects and the efficacy of epigallocatechin-3-gallate (EGCG) - a natural polyphenol major component of green tea - to counteract the mitochondrial energy deficit. In this study, to gain insight into the possible role of mitochondria in DS intellectual disability, mitochondrial functions were analyzed in neural progenitor cells (NPCs) isolated from the hippocampus of Ts65Dn mice, a widely used model of DS which recapitulates many major brain structural and functional phenotypes of the syndrome, including impaired hippocampal neurogenesis. We found that, during NPC proliferation, mitochondrial bioenergetics and mitochondrial biogenic program were strongly compromised in Ts65Dn cells, but not associated with free radical accumulation. These data point to a central role of mitochondrial dysfunction as an inherent feature of DS and not as a consequence of cell oxidative stress. Further, we disclose that, besides EGCG, also the natural polyphenol resveratrol, which displays a neuroprotective action in various human diseases but never tested in DS, restores oxidative phosphorylation efficiency and mitochondrial biogenesis, and improves proliferation of NPCs. These effects were associated with the activation of PGC-1α/Sirt1/AMPK axis by both polyphenols. This research paves the way for using nutraceuticals as a potential therapeutic tool in preventing or managing some energy deficit-associated DS clinical manifestations. PMID:26964795

  3. ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, Hui-Chen, E-mail: d93548008@ntu.edu.tw; Chen, Chung-Ming, E-mail: chung@ntu.edu.tw [National Taiwan University, Institute of Biomedical Engineering (China); Hsieh, Wen-Yuan, E-mail: hsiehw@itri.org.tw [Industrial Technology Research Institute, Biomedical Technology and Device Research Labs (China); Chen, Ching-Yun, E-mail: chingyun523@gmail.com; Liu, Chia-Ching, E-mail: d95548005@ntu.edu.tw; Lin, Feng-Huei, E-mail: double@ntu.edu.tw [National Taiwan University, Institute of Biomedical Engineering (China)

    2015-02-15

    Superparamagnetic nanoparticles (Fe{sub 3}O{sub 4}, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future.

  4. ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

    International Nuclear Information System (INIS)

    Superparamagnetic nanoparticles (Fe3O4, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future

  5. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  6. GPC3 reduces cell proliferation in renal carcinoma cell lines

    OpenAIRE

    Valsechi, Marina Curado; Oliveira, Ana Beatriz Bortolozo; Conceição, André Luis Giacometti; Stuqui, Bruna; Candido, Natalia Maria; Provazzi, Paola Jocelan Scarin; de Araújo, Luiza Ferreira; Silva, Wilson Araújo; Calmon, Marilia Freitas; Rahal, Paula

    2014-01-01

    Background Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is downregulated in several cancers, which can result in uncontrolled cell growth and can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma. ...

  7. Mouse DRG Cell Line with Properties of Nociceptors.

    Directory of Open Access Journals (Sweden)

    Ciara Doran

    Full Text Available In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  8. The effect of gallic acid on Jurkat cell line

    Directory of Open Access Journals (Sweden)

    Sourani Zahra

    2015-10-01

    Full Text Available Introduction: Acute lymphoblastic leukemia (ALL is the most prevalent leukemia in children. Fruits and plants have a wide range of biological functions including anti-proliferative effect. Gallic acid (GA, is a polyhydroxyphenolic compound that is widely distributed in the natural plants. The aim of the present study was the evaluation of the effect of GA on proliferation inhibition of Jurkat cells, the lymphoblastic leukemia cell line. Methods: Jurkat cell line was cultured in blood cells culture media in a standard conditions with different concentrations of GA (0-100 μm for 24, 48 and 72 hours. The effect of GA on cell viability was measured using MTS assay. Results: Decline of cell viability to less than 50% was observed at 60, 50 and 30 μm concentrations after 24, 48 and 72 hours incubation time, respectively. Conclusion: The results demonstrated that the polyphenolic compound, GA with antioxidant capability is effective in proliferation inhibition in Jurkat lymphoblastic leukemia cell line with a time and dose dependent manner. Therefore, GA may be a potential compound for cancer prevention and treatment.

  9. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  10. Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.

    OpenAIRE

    Le Doux, J M; Morgan, J.R.; Snow, R G; Yarmush, M. L.

    1996-01-01

    Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited ampho...

  11. Isolation of Mitochondria from Potato Tubers

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Salvato, Fernanda; Chen, Mingjie;

    2014-01-01

    vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where the...

  12. BCL-2 activation by ionizing radiation in a glioblastoma cell line

    International Nuclear Information System (INIS)

    was transcriptionally active, as evidenced by induction of p21/WAF1. However, neither Bcl-2 nor Mcl-1 were induced under these conditions, demonstrating that, at least in this cell line, the presence of functional p53 was necessary for the repression of Bcl-2 and Bcl-xL. In our search for genes induced by ionizing radiation which might mediate Bcl-2 expression we have identified the mitochondrially-encoded cytochrome c oxidase subunits I and II, and the NADH dehydrogenase subunit 4, as having increased mRNA expression following IR. Their differential expression was confirmed by Northern analyses which demonstrated also a time and dose dependent expression. Known oxidants gave a similar activation with radiation while antioxidants prevented this increase, consistent with a role of oxidative radicals in mitochondrial gene expression. Conclusion: These results indicate that ionizing radiation can activate cell survival factors in glioblastoma cells and that this activation is prevented when functional p53 is present. In our search for differentially-induced genes which might mediate activation of Bcl-2 we have cloned and characterized 3 mitocondrially-encoded gene products. This study provides the first indication that irradiation might activate cell survival signals, which might have a role in determining radiation sensitivity. It also identifies mitochondria as a potential target or intermediate in radiation signaling

  13. The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Highlights: ► Zebularine inhibited cell growth of gastric cancer in a time- and dose-dependent manner. ► Chromatin condensation and nuclear fragmentation were induced. ► Zebularine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by zebularine. -- Abstract: DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

  14. Mitochondria in vascular health and disease.

    Science.gov (United States)

    Dromparis, Peter; Michelakis, Evangelos D

    2013-01-01

    The eukaryote's mitochondrial network is perhaps the cell's most sophisticated and dynamic responsive sensing system. Integrating metabolic, oxygen, or danger signals with inputs from other organelles, as well as local and systemic signals, mitochondria have a profound impact on vascular function in both health and disease. This review highlights recently discovered aspects of mitochondrial function (oxygen sensing, inflammation, autophagy, and apoptosis) and discusses their role in diseases of both systemic and pulmonary vessels. We also emphasize the role of mitochondria as therapeutic targets for vascular disease. We highlight the intriguing similarities of mitochondria-driven molecular mechanisms in terms of both pathogenesis and therapies in very diverse diseases, such as atherosclerosis, pulmonary hypertension, and cancer, to support the foundation of a new field in medicine: mitochondrial medicine. PMID:23157555

  15. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    Science.gov (United States)

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  16. Protective Effects of Mitochondria-Targeted Peptide MTP-131 on Trabecular Meshwork Cells under Elevated Hydrostatic Pressure

    OpenAIRE

    Xiaohong Liu; Bingqian Liu; Huiling Hu; Ying Liu; Fei Deng; Jian Ge

    2013-01-01

    Purpose: To investigate the effects of MTP-131 on trabeculum cells cultured under hydrostatic pressure Methods: Glaucomatous human trabecular meshwork (GTM3) cells were divided into three groups: the MTP-131 group, the hydrostatic group, and the normal group. The MTP-131 group was pretreated with 1 µM MTP-131, and the cells were then cultured under 80 mmHg pressure for 24 hours. The normal group was cultured under normal atmospheric pressure for 24 h, and the hydrostatic group was cultured...

  17. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  18. Interaction of Insulin-like Growth Factor-binding Protein-3 and BAX in Mitochondria Promotes Male Germ Cell Apoptosis

    OpenAIRE

    Jia, Yue; Lee, Kuk-Wha; Swerdloff, Ronald; Hwang, David; Cobb, Laura J.; Sinha Hikim, Amiya; Lue, Yan He; Cohen, Pinchas; Wang, Christina

    2009-01-01

    Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonis...

  19. Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

    OpenAIRE

    Oliveira, Cláudia S. F.; Pereira, Helena Paula Fernandes; Alves, Sara Cristina Sequeira; Castro, Lisandra Marisa Flores; Baltazar, Fátima; Chaves, Susana; Preto, Ana; Côrte-Real, Manuela

    2015-01-01

    Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as p...

  20. Regulation of Mitochondria-dependent Apoptosis in Non-Small Cell Lung Cancer: Implications for Cancer Therapy

    OpenAIRE

    Checinska, A.

    2007-01-01

    Summary The work described in this thesis focuses on the characterization of the mechanism(s) underlying the deregulated activation of the intrinsic or mitochondrial apoptotic pathway in non-small cell lung cancer (NSCLC) cells. Apoptosis suppression might contribute to the chemoresistance often observed in NSCLC patients, and elucidation of the molecular causes of this resistance may provide clues for the development of targeted apoptosis-based therapies. In this introduction, lung cancer an...

  1. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    Science.gov (United States)

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  2. Detection of immunotoxicity using T-cell based cytokine reporter cell lines ('Cell Chip')

    International Nuclear Information System (INIS)

    Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype 'Cell Chip' to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included

  3. Redox interplay between mitochondria and peroxisomes

    Directory of Open Access Journals (Sweden)

    Celien eLismont

    2015-05-01

    Full Text Available Reduction-oxidation or ‘redox’ reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from ‘omics’ technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of

  4. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    International Nuclear Information System (INIS)

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines

  5. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    OpenAIRE

    Mintz, K. P.; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found...

  6. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  7. Establishment and applications of male germ cell and Sertoli cell lines.

    Science.gov (United States)

    Wang, Hong; Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; He, Zuping

    2016-08-01

    Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine. PMID:27069011

  8. Structural differentiation of apical openings in active mitochondria-rich cells during early life stages of Nile tilapia (Oreochromis niloticus L.) as a response to osmotic challenge.

    Science.gov (United States)

    Fridman, S; Rana, K J; Bron, J E

    2013-10-01

    This study examines the structural differentiation of the apical crypts of mitochondria-rich cells (MRCs) in Nile tilapia as a response to osmotic challenge. Larvae were transferred from freshwater at 3 days post-hatch to 12.5 and 20 ppt and were sampled at 24- and 48-h post-transfer. Scanning electron microscopy allowed quantification of MRCs, based on apical crypt appearance and surface area, resulting in a morphological classification of 'sub-types', that is, Type I or absorptive (surface area range 5.2-19.6 μm(2)), Type II or active absorptive form (surface area range 1.1-15.7 μm(2)), Type III or weakly functioning form (surface area range 0.08-4.6 μm(2)) and Type IV or active secreting form (surface area range 4.1-11.7 μm(2)). Mucus cell crypts were discriminated from those of MRCs based on the presence of globular extensions and quantified. Density and frequency of MRCs and mucus cells varied significantly according to the experimental salinity and time post-transfer; in freshwater-adapted larvae, all types were present except Type IV but, following transfer to elevated salinities, Type I and Type II disappeared and appeared to be replaced by Type IV crypts. Type III crypt density remained constant following transfer. Transmission electron microscopy with immunogold labelling, using a novel pre-fixation technique with anti-Na(+)/K(+)-ATPase, allowed complementary ultrastructural visualisation of specific localisation of the antibodies on active MRCs, permitting a review of MRC apical morphology and related Na(+)/K(+)-ATPase binding sites. PMID:23307174

  9. Biological characteristics of side population cells in a self-established human ovarian cancer cell line

    Science.gov (United States)

    WEI, ZHENTONG; LV, SHUANG; WANG, YISHU; SUN, MEIYU; CHI, GUANGFAN; GUO, JUN; SONG, PEIYE; FU, XIAOYU; ZHANG, SONGLING; LI, YULIN

    2016-01-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

  10. Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro

    International Nuclear Information System (INIS)

    Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged 'live' during particle exposure or after exposure within fixed cells. Comparisons between the uptake of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.

  11. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  12. Part II. Mitochondrial mutational status of high nitric oxide adapted cell line BT-20 (BT-20-HNO) as it relates to human primary breast tumors.

    Science.gov (United States)

    De Vitto, H; Mendonça, B S; Elseth, K M; Vesper, B J; Portari, E A; Gallo, C V M; Paradise, W A; Rumjanek, F D; Radosevich, J A

    2013-02-01

    Mitochondria combine hydrogen and oxygen to produce heat and adenosine triphosphate (ATP). As a toxic by-product of oxidative phosphorylation (OXPHOS), mitochondria generate reactive oxygen species (ROS). These free radicals may cause damage to mitochondrial DNA (mtDNA) and other molecules in the cell. Nitric oxide (NO) plays an important role in the biology of human cancers, including breast cancer; however, it is still unclear how NO might affect the mitochondrial genome. The aim of the current study is to determine the role of mtDNA in the breast oncogenic process. Using DNA sequencing, we studied one breast cancer cell line as a model system to investigate the effects of oxidative stress. The BT-20 cell line was fully adapted to increasing concentrations of the NO donor DETA-NONOate and is referred to as BT-20-HNO, a high NO (HNO) cell line. The HNO cell line is biologically different from the "parent" cell line from which it originated. Moreover, we investigated 71 breast cancer biopsies and the corresponding noncancerous breast tissues. The free radical NO was able to generate somatic mtDNA mutations in the BT-20-HNO cell line that were missing in the BT-20 parent cell line. We identified two somatic mutations, A4767G and G13481A, which changed the amino acid residues. Another two point mutations were identified in the mtDNA initiation replication site at nucleotide 57 and at the 'hot spot' cytidine-rich D300-310 segment. Furthermore, the NO regulated the mtDNA copy number and selected different mtDNA populations by clonal expansion. Interestingly, we identified eight somatic mutations in the coding regions of mtDNAs of eight breast cancer patients (8/71, 11.2 %). All of these somatic mutations changed amino acid residues in the highly conserved regions of mtDNA which potentially leads to mitochondrial dysfunctions. The other two somatic mtDNA mutations in the displacement loop (D-loop) region [303:315 C(7-8)TC(6) and nucleotide 57] were distributed among 14

  13. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    Science.gov (United States)

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  14. Involvement of mitochondria in apoptosis of cancer cells induced by photodynamic therapy Envolvimento da mitocôndria em apoptose de células cancerígenas induzida por terapia fotodinâmica

    OpenAIRE

    Joselito Nardy Ribeiro; André Romero da Silva; Renato Atilio Jorge

    2004-01-01

    Photodynamic therapy (PDT), a promising cancer treatment that employs a combination of a photosensitizing chemical, oxygen, and visible light, induces apoptosis in tumor cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. Many of the photosensitizers currently in clinical or pre-clinical studies of PDT are localized in or have a major influence on mitochondria. The treatment with PDT causes mitochondrial damage and induces apoptosis through the release of...

  15. Why Do Older Women Have Poor Implantation Rates? A Possible Role of the Mitochondria

    OpenAIRE

    Bartmann, Ana Karina; Romão, Gustavo Salata; da Silveira Ramos, Ester; Ferriani, Rui Alberto

    2004-01-01

    Mitochondria are organelles responsible for oxidative phosphorylation, the main energy source for all eukaryotic cells. In oocytes and embryos, it seems that mitochondria provide sufficient energy for fecundation by supporting spindle formation during meiosis II, and for implantation. Since mitochondria are inherited from mother to child, it is important that oocyte mitochondria should be intact. Older women seem to have more mitochondrial DNA mutations, which can be responsible for poor impl...

  16. 9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    International Nuclear Information System (INIS)

    The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

  17. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  18. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    Science.gov (United States)

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. PMID:26016689

  19. Induced pluripotent stem cell lines derived from equine fibroblasts.

    Science.gov (United States)

    Nagy, Kristina; Sung, Hoon-Ki; Zhang, Puzheng; Laflamme, Simon; Vincent, Patrick; Agha-Mohammadi, Siamak; Woltjen, Knut; Monetti, Claudio; Michael, Iacovos Prodromos; Smith, Lawrence Charles; Nagy, Andras

    2011-09-01

    The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species. PMID:21347602

  20. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    Science.gov (United States)

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  1. Every Single Cell Clones from Cancer Cell Lines Growing Tumors In Vivo May Not Invalidate the Cancer Stem Cell Concept

    OpenAIRE

    Li, Fengzhi

    2009-01-01

    We present the result of our research on the tumorigenic ability of single cell clones isolated from an aggressive murine breast cancer cell line in a matched allografting mouse model. Tumor formation is basically dependent on the cell numbers injected per location. We argue that in vivo tumor formation from single cell clones, isolated in vitro from cancer cell lines, may not provide conclusive evidence to disprove the cancer stem cell (CSC) theory without additional data.

  2. Dynamic effects of autophagy on arsenic trioxide-induced death of human leukemia cell line HL60 cells

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Zhong-qin LIANG; Bo GAO; Yan-li JIA; Zheng-hong QIN

    2008-01-01

    Aim: To evaluate the contribution of an autophagic mechanism to the As2O3-induced death of human acute myeloid leukaemia cell line HL60 cells. Methods: The growth inhibition of HL60 cells induced by As2O3 was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazohum bromide colorimetric assay. The ac-tivation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As2O3-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Results: After treatment with As2O3, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutraliz-ing agent NH4C11 h before As2O3 potentiated the As2O3-induced death of HL60 cells. In contrast, 3-MA attenuated As2O3-induced death when administered 30 min after As2O3. 3-MA and NH4Cl also inhibited As2O3-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As2O3, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As2O3 was suggested by the collapse of mitochondrial membrane potential, re-lease of cytochrome c from mitochondria, and the activation of caspase-3. Pre-treatment with 3-MA prior to As2O3 amplified these apoptotic signals, while post-treatment with 3-MA 30 min after As2O3 attenuated the apoptotic pathways. Conclusion: Autophagy plays complex roles in the As2O3-induced death of HL60 cells; it inhibits As2O3-induced apoptosis in the initiation stage, but amplifies the AS2O3-mediated apoptotic program if it is persistently activated.

  3. Mitochondria and the evolutionary roots of cancer

    International Nuclear Information System (INIS)

    Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell. (paper)

  4. Mitochondria and the evolutionary roots of cancer

    Science.gov (United States)

    Davila, Alfonso F.; Zamorano, Pedro

    2013-04-01

    Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell.

  5. ROS-dependent mitochondria molecular mechanisms underlying antitumor activity of Pleurotus abalonus acidic polysaccharides in human breast cancer MCF-7 cells.

    Directory of Open Access Journals (Sweden)

    Xiaolong Shi

    Full Text Available BACKGROUND: A greater reduction in cancer risk associated with mushroom diet rich in fungus polysaccharides is generally accepted. Meanwhile, edible Pleurotus abalonus as a member of Abalone mushroom family is a popular nutritional supplement that purportedly prevents cancer occurrence. However, these anecdotal claims are supported by limited studies describing tumor-inhibitory responses to the promising polysaccharides, and the molecular mechanisms underlying these properties have not yet been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We here fractionated the crude polysaccharide preparation from the fruiting bodies of P. abalonus into three fractions, namely PAP-1, PAP-2 and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7 cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of 3.68×10(5 Da, was the most active in inhibiting MCF-7 cancer cells with an IC50 of 193 µg/mL. The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm, the increase of Bax/Bcl-2 ratio, caspase-9/3 activation, and poly(ADP-ribose polymerase (PARP degradation, as well as intracellular ROS production. PAP-3 also induced up-regulation of p53, and cell cycle arrest at the S phase. The incubation of MCF-7 cells with antioxidant superoxide dismutase (SOD and N-acetylcysteine (NAC significantly attenuated the ROS generation and apoptosis caused by PAP-3, indicating that intracellular ROS plays a pivotal role in cell death. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the polysaccharides, especially acidic PAP-3, are very important nutritional ingredients responsible for, at least in part, the anticancer health benefits of P. abalonus via ROS-mediated mitochondrial apoptotic

  6. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  7. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line.

    OpenAIRE

    Sherwood, J B; Shouval, D

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for greater than 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates b...

  8. Evaluation Of Lenalidomide Activity On Glioblastoma Cell Lines In Vitro

    OpenAIRE

    Mut, Melike; Gregory POLAR; Carpenter, Joan E.; Gerard REDPATH; Larner, James; Schiff, David; Shaffrey, Mark E.

    2007-01-01

    Purpose: Thalidomide analog, Lenalidomide (Revlimid®) is a chemotherapeutic agent. In this study, lenalidomide was used in human glioblastoma multiforme (GBM) cell lines to determine its pro-apoptotic, anti-proliferative and radiosensitizing properties. Methods: The GBM cells were treated with lenalidomide [0, 1, 5, 30, 60 µM] before ultravioletB (UVB) [0, 50, 100 mj] or γ -irradiation [0, 5, 20 Gy], and kept in drug for 5 days. Viable cell numbers were determined by trypan blue exclusion. Th...

  9. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines.

    OpenAIRE

    Liebmann, J. E.; Cook, J. A.; Lipschultz, C.; Teague, D.; Fisher, J; Mitchell, J B

    1993-01-01

    The cytotoxicity of paclitaxel against eight human tumour cell lines has been studied with in vitro clonogenic assays. The fraction of surviving cells fell sharply after exposure for 24 h to paclitaxel concentrations ranging from 2 to 20 nM; the paclitaxel IC50 was found to range between 2.5 and 7.5 nM. Increasing the paclitaxel concentration above 50 nM, however, resulted in no additional cytotoxicity after a 24 h drug exposure. Cells incubated in very high concentrations of paclitaxel (10,0...

  10. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    Science.gov (United States)

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  11. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  12. Organochlorines and metals induce changes in the mitochondria-rich cells of fish gills: an integrative field study involving chemical, biochemical and morphological analyses.

    Science.gov (United States)

    Fernandes, M N; Paulino, M G; Sakuragui, M M; Ramos, C A; Pereira, C D S; Sadauskas-Henrique, H

    2013-01-15

    Through integrating chemical, biochemical and morphological analyses, this study investigated the effects of multiple pollutants on the gill mitochondria-rich cells (MRCs) in two fish species, Astyanax fasciatus and Pimelodus maculatus, collected from five sites (FU10, FU20, FU30, FU40 and FU50) in the Furnas Hydroelectric Power Station reservoir. Water analyses revealed aluminum, iron and zinc as well as organochlorine (aldrin/dieldrin, endosulfan, heptachlor/heptachlor epoxide and metolachlor) contamination at all of the sites, with the exception of FU10. Copper, chrome, iron and zinc were detected in the gills of both species, and aldrin/dieldrin, endosulfan and heptachlor/heptachlor epoxide were detected in the gills of fish from all of the sites, with the exception of FU10. Fish collected at FU20, FU30 and FU50 exhibited numerous alterations in the surface architecture of their pavement cells and MRCs. The surface MRC density and MRC fractional area were lower in fish from FU20, FU30, FU40 and FU50 than in those from the reference site (FU10) in the winter, and some variability between the sites was observed in the summer. The organochlorine contamination at FU20 and FU50 was associated with variable changes in the MRCs and inhibition of Na(+)/K(+)-ATPase (NKA) activity, especially in P. maculatus. At FU30, the alterations in the MRCs were associated with the contaminants present, especially metals. A multivariate analysis demonstrated a positive association between the biological responses of both species and environmental contamination, indicating that under realistic conditions, a mixture of organochlorines and metals affected the MRCs by inhibiting NKA activity and inducing morphological changes, which may cause an ionic imbalance. PMID:23220410

  13. Effects of irradiation on cytokine production in glioma cell lines

    International Nuclear Information System (INIS)

    The effects of irradiation on cytokine production in glioma cell lines, NP1, NP2 and NP3, were studied. Culture supernatants were collected after 6, 24, 48 or 72 hours and the concentrations of interleukin (IL)-6 and IL-8 measured by enzyme-linked immunosorbent assay. Spontaneous and IL-1β-stimulated productions were analyzed. Some cells were given a single dose of Lineac irradiation (10 or 20 Gy). Production of IL-6 (with or without IL-1β stimulation) increased gradually to a maximum after 72 hours, more in the 20 Gy-irradiated cells than 10 Gy cells (p<0.01). Production of IL-8 increased gradually to a maximum after 48 or 72 hours. Spontaneous production of IL-8 increased more in 20 Gy-irradiated cells than 10 Gy cells after 6 and 24 hours (p<0.01), but increased more in 10 Gy cells than 20 Gy cells after 48 and 72 hours (p<0.01). The production of IL-8 stimulated by IL-1β increased more in 10 Gy cells than 20 Gy cells 24 hours later (p<0.01). IL-6 and IL-8 production differed in the response to irradiation. Our data suggest that bidirectional communication between the immune system and glioma cells changes after radiotherapy. (author)

  14. Silencing of Pokemon Enhances Caspase-Dependent Apoptosis via Fas- and Mitochondria-Mediated Pathways in Hepatocellular Carcinoma Cells

    OpenAIRE

    Yu-Qin Zhang; Chuan-Xing Xiao; Bi-Yun Lin; Ying Shi; Yun-Peng Liu; Jing-Jing Liu; Bayasi Guleng; Jian-Lin Ren

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induce...

  15. PGC-1 family coactivators and cell fate: roles in cancer, neurodegeneration, cardiovascular disease and retrograde mitochondria-nucleus signalling.

    OpenAIRE

    Jones, A W; Z. Yao; Vicencio, J. M.; Karkucinska-Wieckowska, A.; Szabadkai, G.

    2012-01-01

    Over the past two decades, a complex nuclear transcriptional machinery controlling mitochondrial biogenesis and function has been described. Central to this network are the PGC-1 family coactivators, characterised as master regulators of mitochondrial biogenesis. Recent literature has identified a broader role for PGC-1 coactivators in both cell death and cellular adaptation under conditions of stress, here reviewed in the context of the pathology associated with cancer, neurodegeneration and...

  16. Heterogeneity of a human T-lymphoblastoid cell line

    Energy Technology Data Exchange (ETDEWEB)

    Snow, K.; Judd, W.

    1987-08-01

    A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

  17. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  18. The genomic landscape of epithelioid sarcoma cell lines and tumours.

    Science.gov (United States)

    Jamshidi, Farzad; Bashashati, Ali; Shumansky, Karey; Dickson, Brendan; Gokgoz, Nalan; Wunder, Jay S; Andrulis, Irene L; Lazar, Alexander J; Shah, Sohrab P; Huntsman, David G; Nielsen, Torsten O

    2016-01-01

    We carried out whole genome and transcriptome sequencing on four tumour/normal pairs of epithelioid sarcoma. These index cases were supplemented with whole transcriptome sequencing of three additional tumours and three cell lines. Unlike rhabdoid tumour (the other major group of SMARCB1-negative cancers), epithelioid sarcoma shows a complex genome with a higher mutational rate, comparable to that of ovarian carcinoma. Despite this mutational burden, SMARCB1 mutations remain the most frequently recurring event and are probably critical drivers of tumour formation. Several cases show heterozygous SMARCB1 mutations without inactivation of the second allele, and we explore this further in vitro. Finding CDKN2A deletions in our discovery cohort, we evaluated CDKN2A protein expression in a tissue microarray. Six out of 16 cases had lost CDKN2A in greater than or equal to 90% of cells, while the remaining cases had retained the protein. Expression analysis of epithelioid sarcoma cell lines by transcriptome sequencing shows a unique profile that does not cluster with any particular tissue type or with other SWI/SNF-aberrant lines. Evaluation of the levels of members of the SWI/SNF complex other than SMARCB1 revealed that these proteins are expressed as part of a residual complex, similarly to previously studied rhabdoid tumour lines. This residual SWI/SNF is susceptible to synthetic lethality and may therefore indicate a therapeutic opportunity. PMID:26365879

  19. Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...

  20. BCL-2 family proteins as regulators of mitochondria metabolism.

    Science.gov (United States)

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26827940

  1. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Science.gov (United States)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  2. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

  3. Imatinib mesylate induces mitochondria-dependent apoptosis and inhibits invasion of human pigmented villonodular synovitis fibroblast-like synovial cells.

    Science.gov (United States)

    Chen, Kang; Ren, Qiao; Han, Xiao-Rui; Zhang, Xiao-Nan; Wei, Bo; Bai, Xi-Zhuang

    2016-01-01

    Pigmented villonodular synovitis (PVNS) is a rare sarcoma-like disorder characterized by synovial lesions proliferation and invasion to articular cartilage for which no effective treatments are available. Imatinib mesylate (IM) is known to exert antitumor activity in some tumors, but its effects on PVNS fibroblast-like synoviocytes (PVNS-FLS) and the specific mechanism involved remain to be established. In the present study, the in vitro effects of IM on cell proliferation and survival rates were investigated in PVNS-FLS. Apoptosis induction was assessed via acridine orange/ethidium bromide (AO)/(EB) and Annexin V/PI staining as well as western blotting. The invasion ability of PVNS-FLS was evaluated by Transwell invasion chambers. IM significantly inhibited survival and invasion ability of PVNS-FLS in a dose- and time-dependent manner. The drug-treated cell groups exhibited markedly higher apoptosis, which was blocked upon pretreatment with the specific caspase-9 inhibitor Z-LEHD-FMK. Expression of cleaved caspase-9 was significantly increased and the Bcl-2 family and caspase-3 were activated following treatment with IM. Our results collectively demonstrated that IM has a strong antiproliferative effect on PVNS-FLS in vitro, attributable to induction of mitochondrial-dependent apoptosis in association with activation of caspase-9/-3 and the Bcl-2/Bax family, and exhibits significant inhibition on the invasion ability of PVNS-FLS, suggesting that IM may be useful as a novel treatment of this disease. PMID:26499059

  4. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  5. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria

    2011-10-01

    Full Text Available Abstract Background Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. Methods Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84 were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. Results Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids, where it was found that echovirus 12, 17 and 26 easily infected the spheroids. Conclusions We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors.

  6. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  7. Apoptosis in Raji cell line induced by influenza A virus

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  8. The role of mitochondria in neurodegenerative diseases: mitochondria as a therapeutic target in Alzheimer’s disease

    OpenAIRE

    Reddy, P. Hemachandra

    2009-01-01

    Mitochondria are cytoplasmic organelles responsible for life and death. Extensive evidence from animal and clinical studies suggests that mitochondria play a critical role in aging, cancer, diabetes and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Several lines of research suggest that mitochondrial oxidative damage is an important cellular change in most late-onset neurodegenerative diseases. Further, emerging evidence suggests that ...

  9. Metabolic integration during the evolutionary origin of mitochondria

    Institute of Scientific and Technical Information of China (English)

    DENNIS G SEARCY

    2003-01-01

    Although mitochondria provide eukaryotic cells with certain metabolic advantages, in other ways they may be disadvantageous. For example, mitochondria produce reactive oxygen species that damage both nucleocytoplasm and mitochondria, resulting in mutations, diseases, and aging. The relationship of mitochondria to the cytoplasm is best understood in the context of evolutionary history. Although it is clear that mitochondria evolved from symbiotic bacteria, the exact nature of the initial symbiosis is a matter of continuing debate. The exchange of nutrients between host and symbiont may have differed from that between the cytoplasm and mitochondria in modern cells. Speculations about the initial relationships include the following. (1) The pre-mitochondrion may have been an invasive, parasitic bacterium. The host did not benefit. (2) The relationship was a nutritional syntrophy based upon transfer of organic acids from host to symbiont. (3) The relationship was a syntrophy based upon H2 transfer from symbiont to host, where the host was a methanogen. (4) There was a syntrophy based upon reciprocal exchange of sulfur compounds.The last conjecture receives support from our detection in eukaryotic cells of substantial H2S-oxidizing activity in mitochondria, and sulfur-reducing activity in the cytoplasm.

  10. Isolation of rat adrenocortical mitochondria

    International Nuclear Information System (INIS)

    Highlights: ► A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. ► The purified isolated mitochondria show excellent morphological integrity. ► The properties of oxidative phosphorylation are excellent. ► The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

  11. Isolation of rat adrenocortical mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Solinas, Paola [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Fujioka, Hisashi [Electron Microscopy Facility, Department of Pharmacology, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Tandler, Bernard [Department of Biological Sciences, School of Dental Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Hoppel, Charles L., E-mail: charles.hoppel@case.edu [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

  12. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Institute of Scientific and Technical Information of China (English)

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI

    2011-01-01

    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  13. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    Science.gov (United States)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  14. RBE of neutrons for induction of cell reproductive death and chromosome aberrations in three cell lines

    International Nuclear Information System (INIS)

    The authors have compared the RBE values for induction of dicentrics and centric rings with those for cell inactivation and with the mean or effective quality factors (Q) recommended for radiation protection. The induction of cell reproductive death and chromosome aberrations has been investigated in plateau phase cultures of established lines of a rat rhabdomyosarcoma, a rat ureter carcinoma and Chinese hamster cells for single doses of 300 kV X-rays and 0.5, 4.2 and 15 MeV neutrons. The different cell lines show considerable variations in sensitivity and the RBE values obtained are presented in tabular form. The mean RBE values for the rat rhabdomyosarcoma cells are lower than those for the other two relatively resistant cell lines. Those for the Chinese hamster cells extrapolated to levels according to low doses of X-rays are in good agreement with the quoted Q values. (Auth./C.F.)

  15. Cell line profiling to improve monoclonal antibody production.

    Science.gov (United States)

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest

  16. Mitochondria and Cancer: Past, Present, and Future

    Directory of Open Access Journals (Sweden)

    M. L. Verschoor

    2013-01-01

    Full Text Available The area of mitochondrial genomics has undergone unprecedented growth over the past several years. With the advent of the age of omics, investigations have reached beyond the nucleus to encompass the close biological communication and finely coordinated interactions between mitochondria and their nuclear cell mate. Application of this holistic approach, to all metabolic interactions within the cell, is providing a more complete understanding of the molecular transformation of the cell from normal to malignant behavior, before histopathological indications are evident. In this review the surging momentum in mitochondrial science, as it relates to cancer, is described in three progressive perspectives: (1 Past: the historical contributions to current directions of research; (2 Present: Contemporary findings, results and approaches to mitochondria and cancer, including the role of next generation sequencing and proteomics; (3 Future: Based on the present body of knowledge, the potential assets and benefits of mitochondrial research are projected into the near future.

  17. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S;

    2007-01-01

    antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin......UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  18. In vitro chemosensitivity of head and neck cancer cell lines

    Directory of Open Access Journals (Sweden)

    Schuler PJ

    2010-08-01

    Full Text Available Abstract Background Systemic treatment of head and neck squamous cell carcinoma (HNSCC includes a variety of antineoplastic drugs. However, drug-resistance interferes with the effectiveness of chemotherapy. Preclinical testing models are needed in order to develop approaches to overcome chemoresistance. Methods Ten human cell lines were obtained from HNSCC, including one with experimentally-induced cisplatin resistance. Inhibition of cell growth by seven chemotherapeutic agents (cisplatin, carboplatin, 5- fluorouracil, methotrexate, bleomycin, vincristin, and paclitaxel was measured using metabolic MTT-uptake assay and correlated to clinically-achievable plasma concentrations. Results All drugs inhibited cell growth in a concentration-dependent manner with an IC50 comparable to that achievable in vivo. However, response curves for methotrexate were unsatisfactory and for paclitaxel, the solubilizer cremophor EL was toxic. Cross-resistance was observed between cisplatin and carboplatin. Conclusion Chemosensitivity of HNSCC cell lines can be determined using the MTT-uptake assay. For DNA-interfering cytostatics and vinca alkaloids this is a simple and reproducible procedure. Determined in vitro chemosensitivity serves as a baseline for further experimental approaches aiming to modulate chemoresistance in HNSCC with potential clinical significance.

  19. The Expression of a Novel Mitochondrially-Encoded Gene in Gonadic Precursors May Drive Paternal Inheritance of Mitochondria.

    Science.gov (United States)

    Milani, Liliana; Ghiselli, Fabrizio; Pecci, Andrea; Maurizii, Maria Gabriella; Passamonti, Marco

    2015-01-01

    Mitochondria have an active role in germ line development, and their inheritance dynamics are relevant to this process. Recently, a novel protein (RPHM21) was shown to be encoded in sperm by the male-transmitted mtDNA of Ruditapes philippinarum, a species with Doubly Uniparental Inheritance (DUI) of mitochondria. In silico analyses suggested a viral origin of RPHM21, and we hypothesized that the endogenization of a viral element provided sperm mitochondria of R. philippinarum with the ability to invade male germ line, thus being transmitted to the progeny. In this work we investigated the dynamics of germ line development in relation to mitochondrial transcription and expression patterns using qPCR and specific antibodies targeting the germ line marker VASPH (R. philippinarum VASA homolog), and RPHM21. Based on the experimental results we conclude that both targets are localized in the primordial germ cells (PGCs) of males, but while VASPH is detected in all PGCs, RPHM21 appears to be expressed only in a subpopulation of them. Since it has been predicted that RPHM21 might have a role in cell proliferation and migration, we here suggest that PGCs expressing it might gain advantage over others and undertake spermatogenesis, accounting for RPHM21 presence in all spermatozoa. Understanding how foreign sequence endogenization and co-option can modify the biology of an organism is of particular importance to assess the impact of such events on evolution. PMID:26339998

  20. Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines.

    Science.gov (United States)

    Musk, S R; Hatton, D H; Bouffler, S D; Margison, G P; Johnson, R T

    1989-07-01

    The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated. An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation. The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations. In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity. The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS. PMID:2544312

  1. A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells.

    Science.gov (United States)

    Greenwood-Goodwin, Midori; Yang, Jiwei; Hassanipour, Mohammad; Larocca, David

    2016-01-01

    Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy. PMID:27109637

  2. HIV-1 latency in actively dividing human T cell lines

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2008-04-01

    Full Text Available Abstract Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.

  3. Responding to hypoxia: lessons from a model cell line.

    Science.gov (United States)

    Seta, K A; Spicer, Z; Yuan, Y; Lu, G; Millhorn, D E

    2002-08-20

    Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells. PMID:12189251

  4. Boldine: a potential new antiproliferative drug against glioma cell lines.

    Science.gov (United States)

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent. PMID:19050827

  5. Role of mitochondria in parvovirus pathology.

    Directory of Open Access Journals (Sweden)

    Jonna Nykky

    Full Text Available Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association.

  6. Fluorouracil Selectively Enriches Stem-like Leukemic Cells in a Leukemic Cell Line

    Directory of Open Access Journals (Sweden)

    Ling Zhang, Song Yang, Yu-Juan He, Hui-Yuan Shao, Li Wang, Hui Chen, Yu-Jie Gao, Feng-Xian Qing, Xian-Chun Chen, Liu-Yang Zhao, Shi Tan

    2010-01-01

    Full Text Available Recent studies have reported that cancer stem cells (CSCs could be isolated from solid cancer cell lines, in which the purity of CSCs was higher than that from tumor tissues. Separation of CSCs from leukemic cell lines was rarely reported. In this study, CD34+CD38- stem-like cell subsets in human KG-1a leukemic cell line were enriched by cytotoxic agent 5-fluorouracil (5-FU. After 4 days incubation of KG-1a cell line with 5-FU (50 μg/ml, the CD34+CD38- subpopulation of cell lines was enriched more than 10 times. The enriched cells had proliferate potential in vitro, low level of RNA transcription and Hoechst 33342 dye efflux ability, accompanied by high expression of ATP-binding cassette transporter protein ABCG2. Our findings suggest that treatment with 5-FU offers an easy method to isolate leukemic stem-like subpopulation. It can facilitate studies of leukemic stem cell biology and the development of new therapeutic strategies.

  7. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    Science.gov (United States)

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment. PMID:26358937

  8. Alzheimer, mitochondria and gender.

    Science.gov (United States)

    Grimm, Amandine; Mensah-Nyagan, Ayikoe Guy; Eckert, Anne

    2016-08-01

    Epidemiological studies revealed that two-thirds of Alzheimer's disease (AD) patients are women and the drop of sex steroid hormones after the menopause has been proposed to be one risk factor in AD. Similarly, the decrease of circulating testosterone levels with aging may also increase the risk of AD in men. Studies attest the neuroprotective effects of sex hormones in animal models of AD, but clinical trial data remain controversial. Here, we discuss the implication of mitochondria in gender differences observed in AD patients and animal models of AD. We summarize the role of mitochondria in aging and AD, pointing to the potential correlation between the loss of sex hormones and changes in the brain redox status. We discuss the protective effects of the sex hormones, estradiol, progesterone and testosterone with a specific focus on mitochondrial dysfunction in AD. The understanding of pathological processes linking the loss of sex hormones with mitochondrial dysfunction and mechanisms that initiate the disease onset may open new avenues for the development of gender-specific therapeutic approaches. PMID:27139022

  9. Evaluation of nitroimidazole hypoxic cell radiosensitizers in a human tumor cell line high in intracellular glutathione

    International Nuclear Information System (INIS)

    Five nitroimidazole hypoxic cell radiosensitizers were evaluated in a human lung adenocarcinoma cell line (A549) whose GSH level was 8-fold higher than Chinese hamster V79 cells. One millimolar concentrations of Misonidazole (MISO), SR-2508, RSU-1164, RSU-1172, and Ro-03-8799 sensitized hypoxic A549 cells to radiation, with Ro-03-8799 giving the highest sensitizer enhancement ration (SER) (2.3). However, MISO, SR-2508 and Ro-03-8799 were less effective in this cell line than in V79 cells, presumably due to higher GSH content of the A549 cells. Increased hypoxic radiosensitization was seen with 0.1 mM Ro-03-8799 after GSH depletion by BSO as compared to 0.1 mM Ro-03-8799 alone (SER-1.8 vs 1.3). The combination of GSH depletion and 0.1 mM Ro-03-8799 was considerably more toxic than 0.1 mM or 1.0 mM Ro-03-8799 alone. This sensitivity was much greater than has been observed for SR-2508. These data show that Ro-03-8799 was the most efficient hypoxic cell radiosensitizer in a human tumor cell line considerably higher in GSH than the rodent cell lines often used in hypoxic radiosensitization studies. Thus, Ro-03-8799 may be a more effective hypoxic cell sensitizer in human tumors that are high in GSH

  10. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B;

    1999-01-01

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  11. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B;

    1999-01-01

    blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC......Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped for the...... gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage of...

  12. Mitochondria change dynamics and morphology during grapevine leaf senescence.

    Directory of Open Access Journals (Sweden)

    Cristina Ruberti

    Full Text Available Leaf senescence is the last stage of development of an organ and is aimed to its ordered disassembly and nutrient reallocation. Whereas chlorophyll gradually degrades during senescence in leaves, mitochondria need to maintain active to sustain the energy demands of senescing cells. Here we analysed the motility and morphology of mitochondria in different stages of senescence in leaves of grapevine (Vitis vinifera, by stably expressing a GFP (green fluorescent protein reporter targeted to these organelles. Results show that mitochondria were less dynamic and markedly changed morphology during senescence, passing from the elongated, branched structures found in mature leaves to enlarged and sparse organelles in senescent leaves. Progression of senescence in leaves was not synchronous, since changes in mitochondria from stomata were delayed. Mitochondrial morphology was also analysed in grapevine cell cultures. Mitochondria from cells at the end of their growth curve resembled those from senescing leaves, suggesting that cell cultures might represent a useful model system for senescence. Additionally, senescence-associated mitochondrial changes were observed in plants treated with high concentrations of cytokinins. Overall, morphology and dynamics of mitochondria might represent a reliable senescence marker for plant cells.

  13. The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS.

    Science.gov (United States)

    Rajanayake, Krishani K; Taylor, William R; Isailovic, Dragan

    2016-08-01

    Glycosphingolipids (GSLs) are important biomolecules, which are linked to many diseases such as GSL storage disorders and cancer. Consequently, the expression of GSLs may be altered in ovarian cancer cell lines in comparison to apparently healthy cell lines. Here, differential expressions of GSLs in an epithelial ovarian cancer cell line SKOV3 and a nontumorigenic epithelial ovarian cell line T29 were studied using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. The isolation of GSLs from SKOV3 and T29 cell lines was carried out using Folch partition. GSLs were successfully detected by MALDI-MS, and structurally assigned by a comparison of their MALDI-MS/MS fragmentation patterns with MS/MS data found in SimLipid database. Additionally, LIPID MAPS was used to assign GSL ion masses in MALDI-MS spectra. Seventeen neutral GSLs were identified in Folch partition lower (chloroform/methanol) phases originating from both cell lines, while five globo series neutral GSLs were identified only in the Folch partition lower phase of SKOV3 cell line. Several different sialylated GSLs were detected in Folch partition upper (water/methanol) phases of SKOV3 and T29 cell lines. Overall, this study demonstrates the alteration and increased glycosylation of GSLs in an epithelial ovarian cancer cell line in comparison to a nontumorigenic epithelial ovarian cell line. PMID:27267063

  14. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

  15. CD3 receptor modulation in Jurkat leukemic cell line.

    Directory of Open Access Journals (Sweden)

    Jacek M Witkowski

    2004-03-01

    Full Text Available CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low (217.6, CD3+(217.9 or CD3(low (217.7. The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.

  16. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  17. Interactions of Streptococcus iniae with phagocytic cell line.

    Science.gov (United States)

    El Aamri, Fatima; Remuzgo-Martínez, S; Acosta, Félix; Real, Fernando; Ramos-Vivas, José; Icardo, José M; Padilla, Daniel

    2015-04-01

    Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection. PMID:24956597

  18. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  19. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    International Nuclear Information System (INIS)

    Research highlights: → C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. → The expression of C/EBPβ, α and PPARγ were increased under hypoxia. → Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) β, α and peroxisome proliferator activating receptor (PPAR) γ were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  20. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation

    OpenAIRE

    Wong, Yvette C.; Holzbaur, Erika L.F.

    2014-01-01

    In mitophagy, damaged mitochondria recruit parkin to ubiquitinate proteins on the outer mitochondrial membrane, targeting mitochondria for autophagosome engulfment and degradation. However, the proteins involved in mediating autophagosome formation to degrade damaged and ubiquitinated mitochondria remain unknown. We used live cell imaging to demonstrate that optineurin is actively recruited to parkin-labeled ubiquitinated mitochondria and is stabilized by its ubiquitin binding domain. Optineu...

  1. Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae Possess Functional Mitochondria.

    Directory of Open Access Journals (Sweden)

    Giuseppina Di Giacomo

    Full Text Available Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm-sensitive probe MitoTracker Red indicated that mitochondria i widely occur in erythrocytes, ii are polarized, and iii seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

  2. Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae) Possess Functional Mitochondria.

    Science.gov (United States)

    Di Giacomo, Giuseppina; Campello, Silvia; Corrado, Mauro; Di Giambattista, Livia; Cirotti, Claudia; Filomeni, Giuseppe; Gentile, Gabriele

    2015-01-01

    Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation. PMID:26367118

  3. Radiosensitivity of brain cancer stem cells from malignant glicoma cell line U251 in vitro

    International Nuclear Information System (INIS)

    Objective: To investigate the radiosensitivity of brain cancer stem cells of different conditions isolated from malignant glioma cell line U251 irt vitro. Methods: The brain cancer stem cells in U251 or the brain cancer stem cells isolated from U251 were irradiated by 60Co γ-rays. TUNEL and Annexin-FITC were employed to detect the apoptosis. The brain cancer stem cells were subcutaneously transplanted to nude mouse. Flow cytometry was used to detect cell cycle. Results: The brain cancer stem cells isolated from malignant glioma cell line U251 were in active cell cycle and sensitive to 60Co γ-rays. Thed apoptotic cells were increased obviously after irradiation. After subcutaneously transplanted to unde mouse, there was no tumor appear. However; the brain cancer stem cells existed in U251 were in G0-G1 and resisted to 60Co γ-rays. They differentiated into the parent glioma type after traqnsplantation. Conclusions: The brain cancer stem cells existed in the malignant glioma cell line is resisted to irradiation, and this phenomenon may explain the glioma relapse irt situ after radiation therapy. (authors)

  4. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Hwang, Meeyul [Research Center for Biomedical Resource of Oriental Medicine, Daegu Haany University (Korea, Republic of); Kim, Ji-Hyun [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Han, Bok-Ghee, E-mail: bokghee@nih.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Jeon, Jae-Pil, E-mail: jpjeon@cdc.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  5. High radiosensitivity of the MOLT-4 leukaemic cell line

    International Nuclear Information System (INIS)

    Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0.8% methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D0=0.49+-0.02 Gy and extrapolation number n=0.92+-0.09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesticulation. (author)

  6. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    Directory of Open Access Journals (Sweden)

    Andreas Krieg

    Full Text Available Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN according to their proliferation index into G1- or G2-neuroendocrine tumors (NET and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC. Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1 or lymph node metastases (NEC-DUE2 from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

  7. Targeting microbiota-mitochondria inter-talk: Microbiota control mitochondria metabolism.

    Science.gov (United States)

    Saint-Georges-Chaumet, Y; Attaf, D; Pelletier, E; Edeas, M

    2015-01-01

    Our aim is to highlight the subtle relationship that exists between microbiota and mitochondria. Microbiota targets mitochondria by modulating the Reactive Oxygen Species (ROS) production and the mitochondrial activity through interactions with toxins, proteins or other metabolites released by gut microbiota. The intriguing relationship that exists between mitochondria and microbiota is strengthened by the probable prokaryotic origin of mitochondria. Emerging data implicates a role for ROS, nitric oxide, Short Chain Fatty Acids and hydrogen sulfide in the cross-talk between microbiota - mitochondria and REDOX signaling. Several studies have shown that microbiota act and modulate mitochondrial activity, and use it as a relay to strengthen host-microbiotal interaction. This modulation depends on the gut bacterial strain quality and diversity to increase its pathogenic versus beneficial effects. Furthermore, based on conclusions from new studies, it is possible that microbiota can directly interact with the host cell gene expression by favoring bacterial and mitochondrial DNA insertion in the nuclear genome. The emerging knowledge of mitochondria-microbiota interaction may be of great importance to better understand the mechanism of mitochondrial and metabolic diseases, and the syndromes associated with change in quality and quantity of microbiotal species. We suggest that microbiota via mitochondrial modulation influence cell homeostasis and metabolism. The challenge will be to find strategies to modulate the quality and diversity of microbiota rather than acting on microbiota metabolites and microbiota related factors. The medicine of tomorrow will be completely personalized. Firstly there will be a test to show the quality, quantity and diversity of microbiota, and secondly a preventive or therapeutic strategy will be administrated (probiotics, diet, prodrug or fecal transplantation). The era of digital medicine is here. PMID:26429302

  8. Hyperoxia decreases glycolytic capacity, glycolytic reserve and oxidative phosphorylation in MLE-12 cells and inhibits complex I and II function, but not complex IV in isolated mouse lung mitochondria.

    Directory of Open Access Journals (Sweden)

    Kumuda C Das

    Full Text Available High levels of oxygen (hyperoxia are frequently used in critical care units and in conditions of respiratory insufficiencies in adults, as well as in infants. However, hyperoxia has been implicated in a number of pulmonary disorders including bronchopulmonary dysplasia (BPD and adult respiratory distress syndrome (ARDS. Hyperoxia increases the generation of reactive oxygen species (ROS in the mitochondria that could impair the function of the mitochondrial electron transport chain. We analyzed lung mitochondrial function in hyperoxia using the XF24 analyzer (extracellular flux and optimized the assay for lung epithelial cells and mitochondria isolated from lungs of mice. Our data show that hyperoxia decreases basal oxygen consumption rate (OCR, spare respiratory capacity, maximal respiration and ATP turnover in MLE-12 cells. There was significant decrease in glycolytic capacity and glycolytic reserve in MLE-12 cells exposed to hyperoxia. Using mitochondria isolated from lungs of mice exposed to hyperoxia or normoxia we have shown that hyperoxia decreased the basal, state 3 and state3 μ (respiration in an uncoupled state respirations. Further, using substrate or inhibitor of a specific complex we show that the OCR via complex I and II, but not complex IV was decreased, demonstrating that complexes I and II are specific targets of hyperoxia. Further, the activities of complex I (NADH dehydrogenase, NADH-DH and complex II (succinate dehydrogenase, SDH were decreased in hyperoxia, but the activity of complex IV (cytochrome oxidase, COX remains unchanged. Taken together, our study show that hyperoxia impairs glycolytic and mitochondrial energy metabolism in in tact cells, as well as in lungs of mice by selectively inactivating components of electron transport system.

  9. Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

    2009-01-01

    Objective:To identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity. Methods: CD133+ and CD133- cells were isolated from MHCC97H cell line by magnetic bead cell sorting(MACS), and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by soft agar cloning and tumor formation following nude mice inoculation. Results:CD133+ cells represent a minority(0.5-2.0%) of the tumor cell population with a greater colony-forming efficiency and greater tumor production ability. The colony-forming efficiency of CD133+ cells in soft agar was significantly higher than CD133- cells(36.8±1.4 vs 12.9±0.8, P<0.05).After 6 weeks, 3/5 mice inoculated with 1 × 103 CD133+ cells, 4/5 with 1 × 104 CD133+ cells and 5/5 with 1 × 105 CD133+ cells developed detectable tumors at the injection site, while only one tumor was found in mice treated with same numbers of CD133- cells. Conclusion: CD133 may be a hallmark of liver cancer stem cells (CSC) in human hepatocellular carcinoma(HCC), because the CD133+ cells identified and isolated with anti-CD133 labeled magnetic beads from MHCC97H cell line exhibit high potentials for clonogenicity and tumorigenicity. These CD133+ cells might contribute to hepatocarcinogenesis, as well as the growth and recurrence of human HCC, and therefore may be a useful target for anti-cancer therapy.

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

    International Nuclear Information System (INIS)

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/106 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]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',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition 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 Ni to inhibit adenylate cyclase

  11. Breast cancer cell lines carry cell line-specific genomic alterations that are distinct from aberrations in breast cancer tissues: Comparison of the CGH profiles between cancer cell lines and primary cancer tissues

    International Nuclear Information System (INIS)

    Cell lines are commonly used in various kinds of biomedical research in the world. However, it remains uncertain whether genomic alterations existing in primary tumor tissues are represented in cell lines and whether cell lines carry cell line-specific genomic alterations. This study was performed to answer these questions. Array-based comparative genomic hybridization (CGH) was employed with 4030 bacterial artificial chromosomes (BACs) that cover the genome at 1.0 megabase resolution to analyze DNA copy number aberrations (DCNAs) in 35 primary breast tumors and 24 breast cancer cell lines. DCNAs were compared between these two groups. A tissue microdissection technique was applied to primary tumor tissues to reduce the contamination of samples by normal tissue components. The average number of BAC clones with DCNAs was 1832 (45.3% of spotted clones) and 971 (24.9%) for cell lines and primary tumor tissues, respectively. Gains of 1q and 8q and losses of 8p, 11q, 16q and 17p were detected in >50% of primary cancer tissues. These aberrations were also frequently detected in cell lines. In addition to these alterations, the cell lines showed recurrent genomic alterations including gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 that were barely detected in tumor tissue specimens. These are considered to be cell line-specific DCNAs. The frequency of the HER2 amplification was high in both cell lines and tumor tissues, but it was statistically different between cell lines and primary tumors (P = 0.012); 41.3 ± 29.9% for the cell lines and 15.9 ± 18.6% for the tissue specimens. Established cell lines carry cell lines-specific DCNAs together with recurrent aberrations detected in primary tumor tissues. It must therefore be emphasized that cell lines do not always represent the genotypes of parental tumor tissues

  12. Effects and mechanisms of silibinin on human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth. METHODS: Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined. RESULTS: We demonstrated that silibinin significantly reduced the growth of HUH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly upregulated p21/CDK4 and p27/CDK4 complexes, downregulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and upregulated activated caspase-3 and -9. Silibinin's anti angiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/ PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibininreduced HCC cell proliferation.CONCLUSION: Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and provided a rationale to further test silibinin for HCC chemoprevention.

  13. Chloride channels in the small intestinal cell line IEC-18.

    Science.gov (United States)

    Basavappa, Srisaila; Vulapalli, Sreesatya Raju; Zhang, Hui; Yule, David; Coon, Steven; Sundaram, Uma

    2005-01-01

    Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion. PMID:15389550

  14. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M; Poulsen, H S

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... lung cancer cell lines express the EGF receptor....... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression of...

  15. Actions of steroids in mitochondria.

    Science.gov (United States)

    Gavrilova-Jordan, Larisa P; Price, Thomas M

    2007-05-01

    Investigations of indirect and direct actions of steroids on the mitochondria are relatively new areas of research. In this review we provide brief background information regarding mitochondrial structure and function and then focus upon interactions of glucocorticoid, estrogen, androgen, and progesterone receptors with mitochondria. We evaluate the current evidence for steroid receptor localization in the mitochondria based on techniques of Western blot analysis, immunocytochemistry, electron microscopy, and mass spectrometry. Steroid receptor-dependent interactions with mitochondria may include transcriptional regulation of nuclear DNA-encoded mitochondrial proteins, transcriptional regulation of mitochondrial DNA-encoded proteins, or indirect effects on mitochondria due to interactions with cytoplasmic signaling peptides and non-genomic control of cation fluxes. These interactions may play a role in mitochondrial-dependent processes of oxidative phosphorylation and apoptosis. Physiological examples of these interactions are discussed. PMID:17447205

  16. Mechanisms of mitochondria and autophagy crosstalk

    OpenAIRE

    Rambold, Angelika S.; Lippincott-Schwartz, Jennifer

    2011-01-01

    Autophagy is a cellular survival pathway that recycles intracellular components to compensate for nutrient depletion and ensures the appropriate degradation of organelles. Mitochondrial number and health are regulated by mitophagy, a process by which excessive or damaged mitochondria are subjected to autophagic degradation. Autophagy is thus a key determinant for mitochondrial health and proper cell function. Mitophagic malfunction has been recently proposed to contribute to progressive neuro...

  17. Dynamic regulation of mitochondrial genome maintenance in germ cells

    OpenAIRE

    Kasashima, Katsumi; Nagao, Yasumitsu; Endo, Hitoshi

    2013-01-01

    Mitochondria play a crucial role in the development and function of germ cells. Mitochondria contain a maternally inherited genome that should be transmitted to offspring without reactive oxygen species-induced damage during germ line development. Germ cells are also involved in the mitochondrial DNA (mtDNA) bottleneck; thus, the appropriate regulation of mtDNA in these cells is very important for this characteristic transmission. In this review, we focused on unique regulation of the mitocho...

  18. Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Reichert Torsten E

    2009-04-01

    Full Text Available Abstract Background The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. Methods The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B and compared with those of an adult keratinocyte cell line (NHEK. Results All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. Conclusion MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.

  19. Chemosensitization and radiosensitization of a lung cancer cell line A549 induced by a composite polymer micelle.

    Science.gov (United States)

    Xu, Jing; Zhang, Bi-Cheng; Li, Xiang-Long; Xu, Wen-Hong; Zhou, Juan; Shen, Li; Wei, Qi-Chun

    2016-08-01

    Multidrug resistance (MDR) to Doxorubicin (DOX) remains a major obstacle to successful cancer treatment. The present study sought to overcome the MDR of lung cancer cells and achieve radiosensitization by developing a composite DOX-loaded micelle (M-DOX). M-DOX containing PEG-PCL/Pluronic P105 was prepared by the solvent evaporation method. Lung cancer cell line A549 was adopted in this study. In vitro cytotoxicity, cellular uptake behavior, subcellular distribution, and radiosensitivity were evaluated by the treatment with M-DOX, and free DOX was used as a control. A549 cells treated with M-DOX as opposed to free DOX showed greater cellular uptake as well as greater cytotoxicity. Furthermore, M-DOX reached the mitochondria and lysosome effectively after cellular uptake, and fluorescence used to track M-DOX was found to be surrounding the nucleus. Finally, colony-forming assays demonstrated that M-DOX treatment improved radiosensitization when compared to free DOX. Based on the increased cytotoxicity and radiosensitization, M-DOX could be considered as a promising drug delivery system to overcome MDR in lung cancer therapy. PMID:27585226

  20. Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas

    International Nuclear Information System (INIS)

    This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV+ lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV+ NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL

  1. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  2. Aging, mitochondria and male reproductive function.

    Science.gov (United States)

    Amaral, Sandra; Ramalho-Santos, João

    2009-12-01

    The rise in life expectancy over the last century, together with higher maternal and paternal ages and have highlighted the issue of reduced fertility with advancing age. Aging of the male reproductive system is incited by multi-factorial changes at molecular, cellular and regulatory levels, and individual characteristics are highly variable, although strongly influenced by lifestyle and environmental factors. Damage accumulated with age leads to progressive deregulation of the hypothalamic-pituitary-gonadal axis and of local auto/paracrine interactions, thereby inducing changes in target organs such as the testis, penis and prostate. Elderly human males produce less testosterone, have fewer motile sperm and a higher incidence of erectile dysfunction and prostate disorders, all of which contribute to lower fertility. Cellular aging can manifest itself at several levels. Aging cells progressively accumulate "waste" products, resulting in a decreased functionally. Changes to mitochondria are among the most remarkable features observed in aging cells and several theories place mitochondria at the hub of cellular events related to aging, namely in terms of the accumulation of oxidative damage to cells and tissues, a process in which these organelles may play a prominent role, although alternative theories have also emerged. Furthermore, mitochondrial energy metabolism is also crucial for male reproductive function and mitochondria may therefore constitute a common link between aging and fertility loss. PMID:20021411

  3. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  4. Dendritic mitochondria reach stable positions during