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Sample records for cell lines mitochondria

  1. Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

    Institute of Scientific and Technical Information of China (English)

    Shigang Shan; Kaiyu Liu; Jianxin Peng; Hanchao Yao; Yi Li; Huazhu Hong

    2009-01-01

    Mitochondria are involved in apoptosis of mammalian cells and even single-cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL-ZSU-1). The Western blot results suggested that cytochrome c in the ultraviolet-treated SL-1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time-dependent manner. Flow cytometric analysis of mitochondrial membrane potential (△Ψm) of SL-ZSU-1 cell treated with ultraviolet-C (UV-C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.

  2. Warburg and Crabtree effects in premalignant Barrett's esophagus cell lines with active mitochondria.

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    Martin T Suchorolski

    Full Text Available BACKGROUND: Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. METHODOLOGY/PRINCIPAL FINDINGS: We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.

  3. 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).

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

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    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. Bioenergetic and antiapoptotic properties of mitochondria from cultured human prostate cancer cell lines PC-3, DU145 and LNCaP.

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    Alexander Panov

    Full Text Available The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC, metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ. Unprotected with cyclosporine A (CsA the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

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

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    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.

  7. Stem cell mitochondria during aging.

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    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.

  8. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

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    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.

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

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    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

  10. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

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    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  11. Connecting Mitochondria, Metabolism, and Stem Cell Fate.

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    Wanet, Anaïs; Arnould, Thierry; Najimi, Mustapha; Renard, Patricia

    2015-09-01

    As sites of cellular respiration and energy production, mitochondria play a central role in cell metabolism. Cell differentiation is associated with an increase in mitochondrial content and activity and with a metabolic shift toward increased oxidative phosphorylation activity. The opposite occurs during reprogramming of somatic cells into induced pluripotent stem cells. Studies have provided evidence of mitochondrial and metabolic changes during the differentiation of both embryonic and somatic (or adult) stem cells (SSCs), such as hematopoietic stem cells, mesenchymal stem cells, and tissue-specific progenitor cells. We thus propose to consider those mitochondrial and metabolic changes as hallmarks of differentiation processes. We review how mitochondrial biogenesis, dynamics, and function are directly involved in embryonic and SSC differentiation and how metabolic and sensing pathways connect mitochondria and metabolism with cell fate and pluripotency. Understanding the basis of the crosstalk between mitochondria and cell fate is of critical importance, given the promising application of stem cells in regenerative medicine. In addition to the development of novel strategies to improve the in vitro lineage-directed differentiation of stem cells, understanding the molecular basis of this interplay could lead to the identification of novel targets to improve the treatment of degenerative diseases.

  12. Analysis of mitochondria isolated from single cells.

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    Johnson, Ryan D; Navratil, Marian; Poe, Bobby G; Xiong, Guohua; Olson, Karen J; Ahmadzadeh, Hossein; Andreyev, Dmitry; Duffy, Ciarán F; Arriaga, Edgar A

    2007-01-01

    Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

  13. Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice.

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    Wan, Cuixiang; Li, Shaoqing; Wen, Li; Kong, Jin; Wang, Kun; Zhu, Yingguo

    2007-03-01

    One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.

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

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    Schatten, Heide; Lewis, Marian L.; Chakrabarti, Amitabha

    2001-08-01

    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.

  15. Mitochondria and Energetic Depression in Cell Pathophysiology

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    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.

  16. Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death.

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    Cha, Moon-Yong; Han, Sun-Ho; Son, Sung Min; Hong, Hyun-Seok; Choi, Young-Ju; Byun, Jayoung; Mook-Jung, Inhee

    2012-01-01

    Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads

  17. The cellular energy crisis: mitochondria and cell death.

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    Waterhouse, Nigel J

    2003-01-01

    Exploding nuclear reactors, environmental destruction, and global warming; the danger of energy production is clear. It is quite remarkable that in this modern age, where power usage is at a premium, we find that even on a cellular level, generation of large quantities of power comes at a cost. Mitochondria, which produce the majority of cellular energy in the form of ATP, have recently been shown to play an essential role in the death of a cell by a process known as apoptosis. During apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. This results in activation of caspases, proteases that orchestrate the death of the cell. Cells in which apoptosis is inhibited upstream of mitochondria generally maintain the potential to proliferate, whereas inhibition of caspases downstream of mitochondria generally only delays cell death. Although breaches of the mitochondrial outer membrane result in the release of proteins that are important for respiration, mitochondria appear capable of maintaining at least some of their functions, including ATP production, even after this event. This has important implications both for the mechanism of outer-membrane permeabilization and the mechanism by which the cells eventually die in the absence of caspase activity. The events surrounding the breach of the mitochondrial outer membrane during apoptosis have therefore received much interest over the past few years.

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

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    Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Tak, Hyosun, E-mail: chuberry@naver.com [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Rho, Mina, E-mail: minarho@hanyang.ac.kr [Department of Computer Science, Hanyang University, Seoul 133-791 (Korea, Republic of); Chang, Hae Ryung, E-mail: heyhae@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Yon Hui, E-mail: yhkim@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Kyung Tae, E-mail: bioktkim@ncc.re.kr [Molecular Epidemiology Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Balch, Curt, E-mail: curt.balch@gmail.com [Medical Sciences Program, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN 47405 (United States); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Nam, Seungyoon, E-mail: seungyoon.nam@ncc.re.kr [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of)

    2014-03-28

    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.

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

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

    2015-06-01

    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.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

  20. 碘过量对大鼠甲状腺细胞线粒体的早期影响%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; 李兰英; 李敏芝; 姚小梅

    2014-01-01

    目的 探讨碘过量对Fischer大鼠甲状腺细胞(FRTL)线粒体过氧化损伤的早期影响.方法 分别用含0.0(对照)、0.1 mmol/L碘化钾(KI)的培养基培养FRTL细胞2、4、24 h.利用线粒体超氧化物指示剂(MitoSOX)通过流式细胞术和荧光显微镜检测线粒体超氧化物生成,免疫细胞化学法检测细胞色素C(cyt c)释放,比色法检测细胞培养液上清乳酸脱氢酶(LDH)活力,利用碘化丙啶通过流式细胞术检测死亡细胞百分比,DNA电泳检测凋亡DNA片段.结果 0.1 mmol/L KI处理2、4、24 h时,FRTL细胞线粒体超氧化物生成均较对照组升高,以2h升高最明显;cyt c强阳性细胞率分别为(35.3±3.6)%、(45.8±5.5)%、(30.3±6.1)%,明显高于对照组[(14.8±1.2)%,P均< 0.05];LDH活力分别为(1.85±0.32)、(6.63±0.42)、(8.35±0.34)U/mg,其中4、24 h时明显高于对照组[(0.89±0.04)U/mg,P均<0.05];对照组和0.1 mmol/L KI处理2、4、24 h组死亡细胞百分比分别为4.66%、7.52%、9.29%、13.71%;0.1 mmol/L KI处理24 h时,出现DNA ladder.结论 碘过量处理2h可引起FRTL细胞线粒体过氧化损伤,24 h可引起细胞凋亡.%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(MitoSOX).The cytochrome c (cyt c) released from mitochondria to cytoplasm was detected by immunocytochemistry.The lactate dehydrogenase (LDH) released into supernatant was measured by a LDH kit using colorimetry.The percent of dead cells was assayed by flow cytometry using propidium iodide (PI).DNA with loading buffer was separated in 1% agarose gel.Results Mitochondrial superoxide production in FRTL cells treated

  1. SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells.

    Science.gov (United States)

    Lin, Ye; Hou, Xiaoming; Shen, Wen-Jun; Hanssen, Ruth; Khor, Victor K; Cortez, Yuan; Roseman, Ann N; Azhar, Salman; Kraemer, Fredric B

    2016-02-01

    Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria.

  2. Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells.

    Science.gov (United States)

    Ferrari, Ilaria; Crespi, Arianna; Fornasari, Diego; Pietrini, Grazia

    2016-08-01

    By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology.

  3. Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

    Science.gov (United States)

    Islam, Md Sayeedul; Niwa, Yasuo; Takagi, Shingo

    2009-06-01

    Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

  4. GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum-mitochondria coupling.

    Science.gov (United States)

    Morales, Pablo E; Torres, Gloria; Sotomayor-Flores, Cristian; Peña-Oyarzún, Daniel; Rivera-Mejías, Pablo; Paredes, Felipe; Chiong, Mario

    2014-03-28

    Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)-mitochondria communication, as it allows for a more efficient transfer of Ca(2+) into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER-mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca(2+) transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca(2+) increases in GLP-1 treated cells. Inhibiting both Ca(2+) release from the ER and Ca(2+) entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER-mitochondria communication in VSMC, resulting in higher mitochondrial activity.

  5. Transition metal catalysis in the mitochondria of living cells

    Science.gov (United States)

    Tomás-Gamasa, María; Martínez-Calvo, Miguel; Couceiro, José R.; Mascareñas, José L.

    2016-09-01

    The development of transition metal catalysts capable of promoting non-natural transformations within living cells can open significant new avenues in chemical and cell biology. Unfortunately, the complexity of the cell makes it extremely difficult to translate standard organometallic chemistry to living environments. Therefore, progress in this field has been very slow, and many challenges, including the possibility of localizing active metal catalysts into specific subcellular sites or organelles, remain to be addressed. Herein, we report a designed ruthenium complex that accumulates preferentially inside the mitochondria of mammalian cells, while keeping its ability to react with exogenous substrates in a bioorthogonal way. Importantly, we show that the subcellular catalytic activity can be used for the confined release of fluorophores, and even allows selective functional alterations in the mitochondria by the localized transformation of inert precursors into uncouplers of the membrane potential.

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

    Science.gov (United States)

    Selga, Tūrs; Selga, Maija; Pāvila, Vineta

    2005-12-01

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

  7. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Directory of Open Access Journals (Sweden)

    Jingfei Huang

    Full Text Available Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS generation, activation of mitochondrial permeability transition pores (MPTPs and loss of mitochondrial membrane potential (MMP were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP inhibitor cyclosporin A (CsA, which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  8. New nanocomposites for SERS studies of living cells and mitochondria

    DEFF Research Database (Denmark)

    Sarycheva, A. S.; Brazhe, N. A.; Baizhumanov, A. A.

    2016-01-01

    A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver–silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver......(I) hydroxide as a unique source of plasmonic nanoparticles for SiO2 microspheres. The recorded SERS spectra reveal a set of characteristic peaks at 750, 1127, 1170, 1371, 1565, 1585 and 1638 cm−1, resulting from the normal group vibrations of the pyrrole rings, methine bridges and side radicals in the heme...... molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites...

  9. Oxidation of H2S in mammalian cells and mitochondria.

    Science.gov (United States)

    Abou-Hamdan, Abbas; Guedouari-Bounihi, Hala; Lenoir, Véronique; Andriamihaja, Mireille; Blachier, François; Bouillaud, Frédéric

    2015-01-01

    Hydrogen sulfide (H2S) is the third gasotransmitter described in mammals. These gasotransmitters (H2S, CO, and NO) are small molecules able to diffuse freely across membranes and thus susceptible to reach easily intracellular targets, one of which is the respiratory enzyme cytochrome oxidase subject to complete inhibition by low micromolar concentrations of these gases. However in contrast to NO or CO, H2S can be metabolized by a sulfide quinone reductase feeding the mitochondrial respiratory chain with the hydrogen atoms of sulfide. Sulfide is thus a two-sided molecule: substrate or poison according to the concentration. The aim of this chapter is to present a mean to monitor sulfide oxidation by isolated mitochondria or cells and to summarize how the properties of this amazing couple (mitochondria and sulfide) translate into practical and conceptual consequences.

  10. 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

  11. 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.

  12. 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.

  13. 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

    1995-01-01

    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....

  14. Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.

    Science.gov (United States)

    Wang, Bo; Van Veldhoven, Paul P; Brees, Chantal; Rubio, Noemí; Nordgren, Marcus; Apanasets, Oksana; Kunze, Markus; Baes, Myriam; Agostinis, Patrizia; Fransen, Marc

    2013-12-01

    Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases.

  15. Exendin-4 protects mitochondria from reactive oxygen species induced apoptosis in pancreatic Beta cells.

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    Zhen Li

    Full Text Available OBJECTIVE: Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca(2+-independent phospholipase A2. METHODS: We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder. We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca2-independent phospholipase A2 and Ca(2+-independent phospholipase A2 mRNA. RESULTS: The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05. The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05, and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca(2+-independent phospholipase A2 activity was positively related to Exendin-4 activity. CONCLUSION: Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca(2-independent phospholipase A2.

  16. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  17. A Highly Photostable Hyperbranched Polyglycerol-Based NIR Fluorescence Nanoplatform for Mitochondria-Specific Cell Imaging.

    Science.gov (United States)

    Dong, Chunhong; Liu, Zhongyun; Liu, Junqing; Wu, Changzhu; Neumann, Falko; Wang, Hanjie; Schäfer-Korting, Monika; Kleuser, Burkhard; Chang, Jin; Li, Wenzhong; Ma, Nan; Haag, Rainer

    2016-09-01

    Considering the critical role of mitochondria in the life and death of cells, non-invasive long-term tracking of mitochondria has attracted considerable interest. However, a high-performance mitochondria-specific labeling probe with high photostability is still lacking. Herein a highly photostable hyperbranched polyglycerol (hPG)-based near-infrared (NIR) quantum dots (QDs) nanoplatform is reported for mitochondria-specific cell imaging. Comprising NIR Zn-Cu-In-S/ZnS QDs as extremely photostable fluorescent labels and alkyl chain (C12 )/triphenylphosphonium (TPP)-functionalized hPG derivatives as protective shell, the tailored QDs@hPG-C12 /TPP nanoprobe with a hydrodynamic diameter of about 65 nm exhibits NIR fluorescence, excellent biocompatibility, good stability, and mitochondria-targeted ability. Cell uptake experiments demonstrate that QDs@hPG-C12 /TPP displays a significantly enhanced uptake in HeLa cells compared to nontargeted QDs@hPG-C12 . Further co-localization study indicates that the probe selectively targets mitochondria. Importantly, compared with commercial deep-red mitochondria dyes, QDs@hPG-C12 /TPP possesses superior photostability under continuous laser irradiation, indicating great potential for long-term mitochondria labeling and tracking. Moreover, drug-loaded QDs@hPG-C12 /TPP display an enhanced tumor cell killing efficacy compared to nontargeted drugs. This work could open the door to the construction of organelle-targeted multifunctional nanoplatforms for precise diagnosis and high-efficient tumor therapy.

  18. Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate.

    Science.gov (United States)

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

    2009-05-01

    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 showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient rho(0) cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.

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

    Science.gov (United States)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    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 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 slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by 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 mitochondria by autophagy.

  20. Tanshinone IIA Induces Apoptosis in Human Oral Cancer KB Cells through a Mitochondria-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Pao-Yu Tseng

    2014-01-01

    Full Text Available Tanshinone IIA (Tan IIA, an active phytochemical in the dried root of Salvia miltiorrhiza Bunge, has shown an antiproliferative activity on various human cancer cell lines including nasopharyngeal carcinoma cells. However, the effects of Tan IIA on human oral cancer cells are still unknown. This study aimed to investigate the antiproliferative effects of Tan IIA on human oral cancer KB cells and explored the possible underlying mechanism. Treatment of KB cells with Tan IIA suppressed cell proliferation/viability and induced cell death in a dose-dependent manner through sulforhodamine B colorimetric assay. Observation of cell morphology revealed the involvement of apoptosis in the Tan IIA-induced growth inhibition on KB cells. Cell cycle analysis showed a cell cycle arrest in G2/M phase on Tan IIA-treated cells. The dissipation of mitochondrial membrane potential observed by flow cytometry and the expression of activated caspases with the cleaved poly (ADP-ribose polymerase under immunoblotting analysis indicated that Tan IIA-induced apoptosis in KB cells was mediated through the mitochondria-dependent caspase pathway. These observations suggested that Tan IIA could be a potential anticancer agent for oral cancer.

  1. Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics.

    Science.gov (United States)

    Pancrazi, Laura; Di Benedetto, Giulietta; Colombaioni, Laura; Della Sala, Grazia; Testa, Giovanna; Olimpico, Francesco; Reyes, Aurelio; Zeviani, Massimo; Pozzan, Tullio; Costa, Mario

    2015-11-10

    Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.

  2. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria.

    Science.gov (United States)

    Jain, Aastha; Chugh, Archana

    2016-09-01

    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  3. Morphological and biochemical characterization of mitochondria in Torpedo red blood cells.

    Science.gov (United States)

    Pica, A; Scacco, S; Papa, F; De Nitto, E; Papa, S

    2001-02-01

    A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.

  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

    2016-07-07

    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.

  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

    by 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......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...... 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. Mitochondrial peroxiredoxin-5 as potential modulator of mitochondria-ER crosstalk in MPP+-induced cell death.

    Science.gov (United States)

    De Simoni, Stéphanie; Linard, Dominique; Hermans, Emmanuel; Knoops, Bernard; Goemaere, Julie

    2013-05-01

    Peroxiredoxin-5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH-SY5Y cells to dissect the role of this enzyme in 1-methyl-4-phenylpyridinium (MPP)⁺-induced cell death. Our data show that mitochondria-dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase-9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺-dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2-APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5-inositol-trisphosphate receptors (IP3 R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.

  7. Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria.

    Science.gov (United States)

    Hu, Wan-Ping; Wang, Jeh-Jeng; Yu, Chia-Li; Lan, Cheng-Che E; Chen, Gow-Shing; Yu, Hsin-Su

    2007-08-01

    Previous reports have shown that cellular functions could be influenced by visual light (400-700 nm). Recent evidence indicates that cellular proliferation could be triggered by the interaction of a helium-neon laser (He-Ne laser, 632.8 nm) with the mitochondrial photoacceptor-cytochrome c oxidase. Our previous studies demonstrated that He-Ne irradiation induced an increase in cell proliferation, but not migration, in the melanoma cell line A2058 cell. The aim of this study was to investigate the underlying mechanisms involved in photostimulatory effects induced by an He-Ne laser. Using the A2058 cell as a model for cell proliferation, the photobiologic effects induced by an He-Ne laser were studied. He-Ne irradiation immediately induced an increase in mitochondrial membrane potential (delta psi(mt)), ATP, and cAMP via enhanced cytochrome c oxidase activity and promoted phosphorylation of Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) expressions. He-Ne irradiation-induced A2058 cell proliferation was significantly abrogated by the addition of delta psi(mt) and JNK inhibitors. Moreover, treatment with an He-Ne laser resulted in delayed effects on IL-8 and transforming growth factor-beta1 release from A2058 cells. These results suggest that He-Ne irradiation elicits photostimulatory effects in mitochondria processes, which involve JNK/AP-1 activation and enhanced growth factor release, and ultimately lead to A2058 cell proliferation.

  8. Metabolic pathways in Anopheles stephensi mitochondria.

    Science.gov (United States)

    Giulivi, Cecilia; Ross-Inta, Catherine; Horton, Ashley A; Luckhart, Shirley

    2008-10-15

    No studies have been performed on the mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that have a significant impact on malaria parasite transmission in endemic regions. In the present study, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells [ASE (Anopheles stephensi Mos. 43) cell line] from A. stephensi, a major vector of malaria in India, South-East Asia and parts of the Middle East. ASE cell mitochondria share many features in common with mammalian muscle mitochondria, despite the fact that these cells are of larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays as major a role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate-oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize proline at a rate comparable with that of alpha-glycerophosphate. However, the proline pathway appeared to differ from the currently accepted pathway, in that oxoglutarate could be catabolized completely by the tricarboxylic acid cycle or via transamination, depending on the ATP need.

  9. 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.

  10. MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis.

    Science.gov (United States)

    Yamaoka, Shohei; Nakajima, Masaki; Fujimoto, Masaru; Tsutsumi, Nobuhiro

    2011-02-01

    Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.

  11. Metabolic Pathways in Anopheles stephensi mitochondria

    Science.gov (United States)

    Giulivi, Cecilia; Ross-Inta, Catherine; Horton, Ashley A.; Luckhart, Shirley

    2017-01-01

    No studies have been performed on mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that significantly impact malaria parasite transmission in endemic regions. Here, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells (ASE line) from Anopheles stephensi, a major vector of malaria in India, Southeast Asia and parts of the Middle East. ASE cell mitochondria shared many features in common with mammalian muscle mitochondria, despite the fact that these cells have a larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays a major role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate-oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize Pro at a rate comparable with that of α-glycerophosphate. However, the Pro pathway appeared to differ from the currently accepted pathway, in that ketoglutarate could be catabolyzed completely by the Krebs cycle or via transamination depending on the ATP need. PMID:18588503

  12. 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.

  13. 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

  14. 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.

  15. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability

    Science.gov (United States)

    Gomes, Ligia C.; Di Benedetto, Giulietta; Scorrano, Luca

    2011-01-01

    Summary A plethora of cellular processes, including apoptosis, depend on regulated changes in mitochondrial shape and ultrastructure. Scarce is our understanding of the role of mitochondria and of their morphology during autophagy, a bulk degradation and recycling process of eukaryotic cells’ constituents. Here we show that mitochondrial morphology determines the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in vitro and in vivo. Upon starvation cellular cAMP levels increase and protein kinase A (PKA) becomes activated. PKA in turn phosphorylates the pro-fission dynamin related protein 1 (DRP1) that is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increase dimerization and activity of ATP synthase, and maintain ATP production. When elongation is genetically or pharmacologically blocked, mitochondria conversely consume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology determine the fate of the cell during autophagy. PMID:21478857

  16. Parkin elimination of mitochondria is important for maintenance of lens epithelial cell ROS levels and survival upon oxidative stress exposure.

    Science.gov (United States)

    Brennan, Lisa; Khoury, Josef; Kantorow, Marc

    2017-01-01

    Age-related cataract is associated with oxidative stress and death of lens epithelial cells (LECs) whose survival is dependent on functional mitochondrial populations. Oxidative stress-induced depolarization/damage of LEC mitochondria results in increased reactive oxygen species (ROS) levels and cell death suggesting the need for a LEC mechanism to remove mitochondria depolarized/damaged upon oxidative stress exposure to prevent ROS release and LEC death. To date, a mechanism(s) for removal of depolarized/damaged LEC mitochondria has yet to be identified and the importance of eliminating oxidative stress-damaged mitochondria to prevent LEC ROS release and death has not been established. Here, we demonstrate that Parkin levels increase in LECs exposed to H2O2-oxidative stress. We establish that Parkin translocates to LEC mitochondria depolarized upon oxidative stress exposure and that Parkin recruits p62/SQSTM1 to depolarized LEC mitochondria. We demonstrate that translocation of Parkin results in the elimination of depolarized/damaged mitochondria and that Parkin clearance of LEC mitochondria is dependent on its ubiquitin ligase activity. Importantly, we demonstrate that Parkin elimination of damaged LEC mitochondria results in reduced ROS levels and increased survival upon oxidative stress exposure. These results establish that Parkin functions to eliminate LEC mitochondria depolarized/damaged upon oxidative stress exposure and that elimination of damaged mitochondria by Parkin is important for LEC homeostasis and survival. The data also suggest that mitochondrial quality control by Parkin could play a role in lens transparency.

  17. 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.

  18. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    Science.gov (United States)

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  19. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  20. 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...

  1. Mitochondria and cell death pathways in plants: Actions speak louder than words

    OpenAIRE

    Scott, Iain; Logan, David C

    2008-01-01

    The mitochondrion has a central role during programmed cell death (PCD) in animals, acting as both a sensor of death signals, and as an initiator of the biochemical processes which lead to the controlled destruction of the cell. In contrast to our extensive knowledge of animal cell death, the part played by mitochondria in the death of plant cells has received relatively little attention. Using a combination of whole-organism and cell-based models, we recently demonstrated that changes in mit...

  2. The ER-mitochondria interface: the social network of cell death.

    Science.gov (United States)

    Grimm, Stefan

    2012-02-01

    When cellular organelles communicate bad things can happen. Recent findings uncovered that the junction between the endoplasmic reticulum (ER) and the mitochondria holds a crucial role for cell death regulation. Not only does this locale connect the two best-known organelles in apoptosis, numerous regulators of cell death are concentrated at this spot, providing a terrain for intense signal transfers. Ca2+ is the most prominent signalling factor that is released from the ER and, at high concentration, mediates the transfer of an apoptosis signal to mitochondria as the executioner organelle for cell death. An elaborate array of checks and balances is fine-tuning this process including Bcl-2 family members. Moreover, MAMs, "mitochondria-associated membranes", are distinct membrane sections at the ER that are in close contact with mitochondria and have been found to exchange lipids and lipid-derived molecules such as ceramide for apoptosis induction. Recent work has also described a reverse transfer of apoptosis signals, from mitochondria to the ER, via cytochrome c release and prolonged IP3R opening or through the mitochondrial fission factor Fis1 and Bap31 at the ER, which form the ARCosome, a novel caspase-activation complex.

  3. HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study.

    Directory of Open Access Journals (Sweden)

    Sophie Perrin

    Full Text Available BACKGROUND: The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence. METHODS: 49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999. In more than 88% of treated patients, the viral load was 500/mm(3. ROS (reactive oxygen species production and ΔΨm (inner membrane potential were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters. Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively were analysed by western blotting. RESULTS: Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm or morphological (network volume density, fragmentation and branching parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria. CONCLUSIONS: In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1

  4. Ca2+ transport by mitochondria from L1210 mouse ascites tumor cells.

    Science.gov (United States)

    Reynafarje, B; Lehninger, A L

    1973-06-01

    Mitochondria isolated from the ascites form of L1210 mouse leukemia cells readily accumulate Ca(2+) from the suspending medium and eject H(+) during oxidation of succinate in the presence of phosphate and Mg(2+), with normal stoichiometry between Ca(2+) uptake and electron transport. Ca(2+) loads up to 1600 ng-atoms per mg of protein are attained. As is the case in mitochondria from normal tissues, Ca(2+) uptake takes precedence over oxidative phosphorylation. However, Ca(2+) transport by the L-1210 mitochondria is unusual in other respects, which may possibly have general significance in tumor cells. The apparent affinity of the L1210 mitochondria for Ca(2+) in stimulation of oxygen uptake is about 3-fold greater than in normal liver mitochondria; moreover, the maximal rate of Ca(2+) transport is also considerably higher. Furthermore, when Ca(2+) pulses are added to L1210 mitochondria in the absence of phosphate or other permeant anions, much larger amounts of Ca(2+) are bound and H(+) ejected per atom of oxygen consumed than in the presence of phosphate; up to 7 Ca(2+) ions are bound per pair of electrons passing each energy-conserving site of the electron-transport chain. Such "superstoichiometry" of Ca(2+) uptake can be accounted for by two distinct types of respiration-dependent interaction of Ca(2+) with the L1210 mitochondria. One is the stimulation of oxygen consumption, which is achieved by relatively low concentrations of Ca(2+) (K(m) congruent with 8 muM) and is accompanied by binding of Ca(2+) up to 40 ng-atoms per mg of protein. The second process, also dependent on electron transport, is the binding of further Ca(2+) from the medium in exchange with previously stored membrane-bound protons, in which the affinity for Ca(2+) is much lower (K(m) congruent with 120 muM).

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

    Energy Technology Data Exchange (ETDEWEB)

    Han, Bin [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Izumi, Hiroto; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Fujimoto, Naohiro; Matsumoto, Tetsuro [Department of Urology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Wu, Bin [Department of Urology, Shengjing Hospital of China Medical University, Shenyang (China); Tanimoto, Akihide [Department of Pathology, Kagoshima University Graduate School of Medical and Dental Science, Kagoshima (Japan); Sasaguri, Yasuyuki [Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan); Kohno, Kimitoshi, E-mail: k-kohno@med.uoeh-u.ac.jp [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu (Japan)

    2011-04-29

    Highlights: {yields} Mitochondrial transcription factor A (mtTFA) localizes in nuclei and binds tightly to the nuclear chromatin. {yields} mtTFA contains two putative nuclear localization signals (NLS) in the HMG-boxes. {yields} 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). {yields} 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.

  6. Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells

    Science.gov (United States)

    Nieminen, Anna-Liisa; Azizuddin, Kashif; Zhang, Ping; Kenney, Malcolm E.; Pediaditakis, Peter; Lemasters, John J.; Oleinick, Nancy L.

    2008-02-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote

  7. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    Science.gov (United States)

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  8. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    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.

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of); Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Cho, Dong-Hyung, E-mail: dhcho@khu.ac.kr [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)

    2011-05-13

    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} 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.

  12. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

    Science.gov (United States)

    VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

    2015-01-01

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

  13. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    Science.gov (United States)

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

  14. 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.

  15. Characterization of mitochondria in cisplatin-resistant human ovarian carcinoma cells.

    Science.gov (United States)

    Hirama, Masanori; Isonishi, Seiji; Yasuda, Makoto; Ishikawa, Hiroshi

    2006-11-01

    One of the mechanisms of cisplatin cell cytotoxicity is the mitochondria-associated induction of apoptosis. The morphological or functional change of mitochondria in cisplatin-resistant cells has already been reported. Herein we present additional data describing the mitochondrial genomic and functional changes in cisplatin- resistant cells. Cisplatin increased the level of apoptotic cells in cisplatin-sensitive human ovarian carcinoma OV 2008 and C13 cells by 3.90+/-1.01 (SD; N=3) (pmitochondrial downstream event was functioning well in both the C13 cells and in OV 2008 cells. Mitochondrial transmembrane potential (DeltaPsim) determined by flow cytometry using DiOC6-stained cells revealed a significant depolarization of C13 cells as compared to OV 2008 cells. Treatment of these cells with cisplatin or hydrogen peroxide induces complete mitochondrial DNA damage in OV 2008 cells, while only partial DNA-destruction is observed in C13 cells, strongly suggesting that mitochondria are resistant to cisplatin and oxidative stress response. Continuous oxygen consumption of these cells monitored by a multi-channel dissolved oxygen meter is 1.70-fold higher in OV 2008 cells than C13 cells and the oxygen consumption was decreased by 30% in C13 cells, suggesting mitochondrial respiratory malfunction in these cells. The hypothesis generated here is that mitochondrial DNA resistance to cisplatin and oxidative stress response might be one of the main characteristics concerning the lower level of apoptosis induced by cisplatin. However, the mechanism by which the mitochondrial DNA encoded molecule is involved in cisplatin resistance remains to be determined.

  16. Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples.

    Science.gov (United States)

    Kappler, Lisa; Li, Jia; Häring, Hans-Ulrich; Weigert, Cora; Lehmann, Rainer; Xu, Guowang; Hoene, Miriam

    2016-02-19

    Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 μg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics.

  17. In beta-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion.

    Science.gov (United States)

    Maechler, Pierre; Carobbio, Stefania; Rubi, Blanca

    2006-01-01

    Pancreatic beta-cells are unique neuroendocrine cells displaying the peculiar feature of responding to nutrients, principally glucose, as primary stimulus. This requires translation of a metabolic substrate into intracellular messengers recognized by the exocytotic machinery. Central to this signal transduction mechanism, mitochondria integrate and generate metabolic signals, thereby coupling glucose recognition to insulin secretion. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin exocytosis. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. In particular, importance of cataplerotic and anaplerotic processes is discussed, with special attention to the mitochondrial enzyme glutamate dehydrogenase. Mitochondrial defects, such as mutations and reactive oxygen species production, are presented in the context of beta-cell failure in the course of type 2 diabetes.

  18. Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells.

    Science.gov (United States)

    Ortega-Camarillo, C; Guzmán-Grenfell, A M; García-Macedo, R; Rosales-Torres, A M; Avalos-Rodríguez, A; Durán-Reyes, G; Medina-Navarro, R; Cruz, M; Díaz-Flores, M; Kumate, J

    2006-01-01

    The mechanisms related to hyperglycemia-induced pancreatic beta-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Delta psi (m)), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Delta psi (m), lead to an important pancreatic beta-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.

  19. 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.

  20. Efficient repopulation of genetically derived rho zero cells with exogenous mitochondria.

    Directory of Open Access Journals (Sweden)

    Sandra Heller

    Full Text Available Mitochondria are involved in a variety of cellular biochemical pathways among which the ATP production by oxidative phosphorylation (OXPHOS represents the most important function of the organelle. Since mitochondria contain their own genome encoding subunits of the OXPHOS apparatus, mtDNA mutations can cause different mitochondrial diseases. The impact of these mutations can be characterized by the trans-mitochondrial cybrid technique based on mtDNA-depleted cells (ρ(0 as acceptors of exogenous mitochondria. The aim of the present work was to compare ρ(0 cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation. Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0 acceptor cells. Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.

  1. Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria.

    Science.gov (United States)

    Alonso, Elba; Cano-Abad, María F; Moreno-Ortega, Ana J; Novalbos, Jesús; Milla, Juan; García, Antonio G; Ruiz-Nuño, Ana

    2013-11-01

    The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.

  2. The involvement of PUMP from mitochondria of Araucaria angustifolia embryogenic cells in response to cold stress.

    Science.gov (United States)

    Valente, Caroline; Pasqualim, Patrícia; Jacomasso, Thiago; Maurer, Juliana Bello Baron; Souza, Emanuel Maltempi de; Martinez, Glaucia Regina; Rocha, Maria Eliane Merlin; Carnieri, Eva Gunilla Skare; Cadena, Sílvia Maria Suter Correia

    2012-12-01

    In this study, the responses of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX) in mitochondria from embryogenic cells of A. angustifolia subjected to cold stress (4°C for 24 h or 48 h) is reported. In the mitochondria of stressed cells, PUMP activity increased by approximately 45% (at 24h and 48 h), which was determined by measuring the oxygen consumption after the addition of linoleic acid and the inhibition by BSA and ATP. PUMP activation was confirmed using transmembrane electrical potential (Δψ) assays. Immunoblot assays showed an increase of PUMP expression by 40% and 150% after 24h and 48 h of cold stress, respectively. AOX activity, measured under conditions similar to those of the PUMP assays, was only slightly increased in the mitochondria from stressed cells (at 24h and 48 h), as demonstrated by oxygen consumption experiments. Cell viability was unaffected by cold stress, indicating that the effects on PUMP and AOX were not caused by cell death. These results show that the main response of this gymnosperm to cold stress is the activation of PUMP, which suggests that this protein may be involved in the control of reactive oxygen species generation, which has been previously associated with this condition.

  3. Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

    Science.gov (United States)

    Madreiter-Sokolowski, Corina T.; Gottschalk, Benjamin; Parichatikanond, Warisara; Eroglu, Emrah; Klec, Christiane; Waldeck-Weiermair, Markus; Malli, Roland; Graier, Wolfgang F.

    2017-01-01

    Background/Aims Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death. PMID:27606689

  4. Akt is translocated to the mitochondria during etoposide-induced apoptosis of HeLa cells.

    Science.gov (United States)

    Park, Byoungduck; Je, Young-Tae; Chun, Kwang-Hoon

    2015-11-01

    Akt, or protein kinase B, is a key serine-threonine kinase, which exerts anti-apoptotic effects and promotes cell proliferation in response to various stimuli. Recently, however, it was demonstrated that Akt exhibits a proapoptotic role in certain contexts. During etoposide‑induced apoptosis of HeLa cells, Akt enhances the interaction of second mitochondria‑derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) and X‑linked inhibitor of apoptosis protein by phosphorylating Smac at serine 67, and thus promotes apoptosis. However, the detailed mechanisms underlying Akt regulation in etoposide‑mediated apoptosis remain to be determined. The present study investigated whether etoposide triggers the translocation of Akt into the mitochondria. It was found that Akt activity was increased and sustained during apoptosis triggered by etoposide in HeLa cells. During apoptosis, Akt was translocated from the cytoplasm into the mitochondria in a phosphoinositide 3‑kinase-dependent manner at the early and late stages of apoptosis. Concomitantly, the depletion of Akt in the nuclear fraction was observed after etoposide treatment from analysis of confocal microscopy. The results suggest that etoposide‑stimulated Akt is translocated into the mitochondria, thereby possibly enhancing its interaction with Smac and promoting apoptosis in HeLa cells. These results indicate that Akt may be a promising candidate for a pro-apoptotic approach in cancer treatment.

  5. Mitochondria play an important role in the cell proliferation suppressing activity of berberine

    Science.gov (United States)

    Yan, Xiao-Jin; Yu, Xuan; Wang, Xin-Pei; Jiang, Jing-Fei; Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Xing, Dong-Ming

    2017-01-01

    After being studied for approximately a century, berberine (BBR) has been found to act on various targets and pathways. A great challenge in the pharmacological analysis of BBR at present is to identify which target(s) plays a decisive role. In the study described herein, a rescue experiment was designed to show the important role of mitochondria in BBR activity. A toxic dose of BBR was applied to inhibit cell proliferation and mitochondrial activity, then α-ketobutyrate (AKB), an analogue of pyruvate that serves only as an electron receptor of NADH, was proven to partially restore cell proliferation. However, mitochondrial morphology damage and TCA cycle suppression were not recovered by AKB. As the AKB just help to regenerate NAD+, which is make up for part function of mitochondrial, the recovered cell proliferation stands for the contribution of mitochondria to the activity of BBR. Our results also indicate that BBR suppresses tumour growth and reduces energy charge and mitochondrial DNA (mtDNA) copy number in a HepG2 xenograft model. In summary, our study suggests that mitochondria play an important role in BBR activity regarding tumour cell proliferation and metabolism. PMID:28181523

  6. Puerarin induces mitochondria-dependent apoptosis in hypoxic human pulmonary arterial smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Chan Chen

    Full Text Available BACKGROUND: Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs. Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs, and to determine the possible underlying mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: HPASMCs were cultured for 24 h in normoxia or hypoxia (5% O₂ conditions with and without puerarin. Cell number and viability were determined with a hemacytometer or a cell counting kit. Apoptosis was detected with a TUNEL test, rhodamine-123 (R-123 fluorescence, a colorimetric assay, western blots, immunohistochemical staining and RT-PCR. Hypoxia inhibited mitochondria-dependent apoptosis and promoted HPASMC growth. In contrast, after puerarin (50 µM or more intervention, cell growth was inhibited and apoptosis was observed. Puerarin-induced apoptosis in hypoxic HPASMCs was accompanied by reduced mitochondrial membrane potential, cytochrome c release from the mitochondria, caspase-9 activation, and Bcl-2 down-regulation with concurrent Bax up-regulation. CONCLUSIONS/SIGNIFICANCE: Puerarin promoted apoptosis in hypoxic HPASMCs by acting on the mitochondria-dependent pathway. These results suggest a new mechanism of puerarin relevant to the management of clinical hypoxic pulmonary hypertension.

  7. Puerarin Induces Mitochondria-Dependent Apoptosis in Hypoxic Human Pulmonary Arterial Smooth Muscle Cells

    Science.gov (United States)

    Chen, Chan; Chen, Chun; Wang, Zhiyi; Wang, Liangxing; Yang, Lehe; Ding, Minjiao; Ding, Cheng; Sun, Yu; Lin, Quan; Huang, Xiaoying; Du, Xiaohong; Zhao, Xiaowei; Wang, Chuangyi

    2012-01-01

    Background Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs), and to determine the possible underlying mechanisms. Methodology/Principal Findings HPASMCs were cultured for 24 h in normoxia or hypoxia (5% O2) conditions with and without puerarin. Cell number and viability were determined with a hemacytometer or a cell counting kit. Apoptosis was detected with a TUNEL test, rhodamine-123 (R-123) fluorescence, a colorimetric assay, western blots, immunohistochemical staining and RT-PCR. Hypoxia inhibited mitochondria-dependent apoptosis and promoted HPASMC growth. In contrast, after puerarin (50 µM or more) intervention, cell growth was inhibited and apoptosis was observed. Puerarin-induced apoptosis in hypoxic HPASMCs was accompanied by reduced mitochondrial membrane potential, cytochrome c release from the mitochondria, caspase-9 activation, and Bcl-2 down-regulation with concurrent Bax up-regulation. Conclusions/Significance Puerarin promoted apoptosis in hypoxic HPASMCs by acting on the mitochondria-dependent pathway. These results suggest a new mechanism of puerarin relevant to the management of clinical hypoxic pulmonary hypertension. PMID:22457823

  8. Metabolic shift in lung alveolar cell mitochondria following acrolein exposure.

    Science.gov (United States)

    Agarwal, Amit R; Yin, Fei; Cadenas, Enrique

    2013-11-15

    Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

  9. 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

    rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral....... Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From...... the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course...

  10. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    Directory of Open Access Journals (Sweden)

    Singleton Charles K

    2002-04-01

    Full Text Available Abstract Background Thiamine diphosphate (ThDP is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 μM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. Conclusions The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.

  11. Ultrasensitive fluorescent ratio imaging probe for the detection of glutathione ultratrace change in mitochondria of cancer cells.

    Science.gov (United States)

    Zhang, Hua; Wang, Caixia; Wang, Kui; Xuan, Xiaopeng; Lv, Qingzhang; Jiang, Kai

    2016-11-15

    Glutathione (GSH) ultratrace change in mitochondria of cancer cells can mildly and effectively induce cancer cells apoptosis in early stage. Thus, if GSH ultratrace change in mitochondria of cancer cells could be recognized and imaged, it will be beneficial for fundamental research of cancer therapy. There have reported a lot of fluorescent probes for GSH, but the fluorescent probe with ultrasensitivity and high selectivity for the ratio imaging of GSH ultratrace changes in mitochondria of cancer cells is scarce. Herein, based on different reaction mechanism of sulfonamide under different pH, a sulfonamide-based reactive ratiometric fluorescent probe (IQDC-M) was reported for the recognizing and imaging of GSH ultratrace change in mitochondria of cancer cells. The detection limit of IQDC-M for GSH ultratrace change is low to 2.02nM, which is far less than 1.0‰ of endogenic GSH in living cells. And during the recognition process, IQDC-M can emit different fluorescent signals at 520nm and 592nm, which results in it recognizing GSH ultratrace change on ratio mode. More importantly, IQDC-M recognizing GSH ultratrace change specifically occurs in mitochondria of cancer cells because of appropriate water/oil amphipathy (log P) of IQDC-M. So, these make IQDC-M possible to image and monitor GSH ultratrace change in mitochondria during cancer cells apoptosis for the first time.

  12. Low infra red laser light irradiation on cultured neural cells: effects on mitochondria and cell viability after oxidative stress

    Directory of Open Access Journals (Sweden)

    Giardino Luciana

    2009-04-01

    Full Text Available Abstract Background Considerable interest has been aroused in recent years by the well-known notion that biological systems are sensitive to visible light. With clinical applications of visible radiation in the far-red to near-infrared region of the spectrum in mind, we explored the effect of coherent red light irradiation with extremely low energy transfer on a neural cell line derived from rat pheochromocytoma. We focused on the effect of pulsed light laser irradiation vis-à-vis two distinct biological effects: neurite elongation under NGF stimulus on laminin-collagen substrate and cell viability during oxidative stress. Methods We used a 670 nm laser, with extremely low peak power output (3 mW/cm2 and at an extremely low dose (0.45 mJ/cm2. Neurite elongation was measured over three days in culture. The effect of coherent red light irradiation on cell reaction to oxidative stress was evaluated through live-recording of mitochondria membrane potential (MMP using JC1 vital dye and laser-confocal microscopy, in the absence (photo bleaching and in the presence (oxidative stress of H2O2, and by means of the MTT cell viability assay. Results We found that laser irradiation stimulates NGF-induced neurite elongation on a laminin-collagen coated substrate and protects PC12 cells against oxidative stress. Conclusion These data suggest that red light radiation protects the viability of cell culture in case of oxidative stress, as indicated by MMP measurement and MTT assay. It also stimulates neurite outgrowth, and this effect could also have positive implications for axonal protection.

  13. Beet yellow stunt virus in cells of Sonchus oleraceus L. and its relation to host mitochondria.

    Science.gov (United States)

    Esau, K

    1979-10-15

    In Sonchus oleraceus L. (Asteraceae) infected with the beet yellow stunt virus (BYSV) the virions are found in phloem cells, including the sieve elements. In parenchymatous phloem cells, the virus is present mainly in the cytoplasm. In some parenchymatous cells, containing massive accumulations of virus, the flexuous rodlike virus particles are found partly inserted into mitochondrial cristae. The mitochondrial envelope is absent where virus is present in the cristae. A similar relation between virus and host mitochondria apparently has not been recorded for any other plant virus.

  14. 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.

  15. A sensitive and specific Raman probe based on bisarylbutadiyne for live cell imaging of mitochondria.

    Science.gov (United States)

    Yamakoshi, Hiroyuki; Palonpon, Almar; Dodo, Kosuke; Ando, Jun; Kawata, Satoshi; Fujita, Katsumasa; Sodeoka, Mikiko

    2015-02-01

    We previously showed that bisarylbutadiyne (BADY), which has a conjugated diyne structure, exhibits an intense peak in the cellular Raman-silent region. Here, we synthesized a mitochondria-selective Raman probe by linking bisphenylbutadiyne with triphenylphosphonium, a well-known mitochondrial targeting moiety. This probe, named MitoBADY, has a Raman peak 27 times more intense than that of 5-ethynyl-2'-deoxyuridine. Raman microscopy using submicromolar extracellular probe concentrations successfully visualized mitochondria in living cells. A full Raman spectrum is acquired at each pixel of the scanned sample, and we showed that simultaneous Raman imaging of MitoBADY and endogenous cellular biomolecules can be achieved in a single scan. MitoBADY should be useful for the study of mitochondrial dynamics.

  16. 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.

  17. Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

    Science.gov (United States)

    Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

    2017-02-09

    Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca(2+) , mitochondria membrane potential (ΔΨm ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca(2+) production, and decreased the level of ΔΨm and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways.

  18. Role of mitochondria in programmed cell death mediated by arachidonic acid-derived eicosanoids.

    Science.gov (United States)

    Yin, Huiyong; Zhou, Yunhua; Zhu, Mingjiang; Hou, Sarina; Li, Zi; Zhong, Huiqin; Lu, Jianhong; Meng, Tao; Wang, Junhong; Xia, Lin; Xu, Yue; Wu, Yuncheng

    2013-05-01

    Arachidonic acid-derived eicosanoids from cyclooxygenases, lipoxygenases, and cytochrome P450 are important lipid mediators involved in numerous homeostatic and pathophysiological processes. Most eicosanoids act primarily on their respective cell surface G-protein coupled receptors to elicit downstream signaling in an autocrine and paracrine fashion. Emerging evidence indicates that these hormones are also critical in apoptosis in a cell/tissue specific manner. In this review, we summarize the formation of eicosanoids and their roles as mediators in apoptosis, specifically on the roles of mitochondria in mediating these events and the signaling pathways involved. The biological relevance of eicosanoid-mediated apoptosis is also discussed.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

    Science.gov (United States)

    Garg, Sriram G; Martin, William F

    2016-07-02

    Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically

  4. Energy transfer in "parasitic" cancer metabolism: mitochondria are the powerhouse and Achilles' heel of tumor cells.

    Science.gov (United States)

    Martinez-Outschoorn, Ubaldo E; Pestell, Richard G; Howell, Anthony; Tykocinski, Mark L; Nagajyothi, Fnu; Machado, Fabiana S; Tanowitz, Herbert B; Sotgia, Federica; Lisanti, Michael P

    2011-12-15

    It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, "parasitic" energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a "parasitic organism." Hence, we propose the term "Parasitic Cancer Metabolism" to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can

  5. 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.

  6. Trichostatin A Induced Bcl-2 Protein Level Decrease Mediated A549/CDDP Cells Apoptosis by Mitochondria Pathway

    Directory of Open Access Journals (Sweden)

    Jun WU

    2009-11-01

    Full Text Available Background and objective The use of platinum-based combination chemotherpy remains the standard treatment for non-small cell lung cancer. However, the resistance to platinum limits further treatment clinically. Trichostatin A (TSA is one of histone deacetylase (HDAC inhibitors. It inhibits tumor cell proliferation and acts as a chemosensitizer. The aim of this study is to investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Cytotoxicity and cell viability was assayed by Neutral Red method. Morphologic assessment of apoptosis was determined by fluorescence microscope; cell cycle and mitochondrial membrane potential were detected by flow cytometry. In addition, A549/CDDP cells were transfected with Bcl-2 expression Vector and siRNA-bcl-2. Results A549/CDDP cells treated with TSA showed apparently cytotoxicity, IC50 of TSA was (446.59±27.32 nmol/L. The growth curve showed the ratio of growth decreased with the increase of concentration of TSA. The apoptosis appeared 24 hours after treated by (125-500 nmol/L TSA, morphologic changes including nuclear chromatin condensation. Fluorescence strength was observed with fluorescence microscope. Treated by TSA, mitochondrial membrane potential was decreased and cells were arrested at S phase. Western blotting analyses showed that the levels of Bcl-2 decreased, while expression of Bax increased. Simultaneously caspase-3 was activated. Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis, while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA. Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.

  7. Solanine Induces Mitochondria-Mediated Apoptosis in Human Pancreatic Cancer Cells

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    Hongwei Sun

    2014-01-01

    Full Text Available Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer.

  8. Solanine induces mitochondria-mediated apoptosis in human pancreatic cancer cells.

    Science.gov (United States)

    Sun, Hongwei; Lv, Chongqing; Yang, Longlong; Wang, Yingxiu; Zhang, Qingshun; Yu, Suhui; Kong, Hongru; Wang, Meng; Xie, Jianming; Zhang, Chunwu; Zhou, Mengtao

    2014-01-01

    Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP) by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer.

  9. Cancer Cell Mitochondria Targeting by Pancratistatin Analogs is Dependent on Functional Complex II and III

    Science.gov (United States)

    Ma, Dennis; Pignanelli, Christopher; Tarade, Daniel; Gilbert, Tyler; Noel, Megan; Mansour, Fadi; Adams, Scott; Dowhayko, Alexander; Stokes, Kyle; Vshyvenko, Sergey; Hudlicky, Tomas; McNulty, James; Pandey, Siyaram

    2017-01-01

    Enhanced mitochondrial stability and decreased dependence on oxidative phosphorylation confer an acquired resistance to apoptosis in cancer cells, but may present opportunities for therapeutic intervention. The compound pancratistatin (PST) has been shown to selectively induce apoptosis in cancer cells. However, its low availability in nature has hindered its clinical advancement. We synthesized PST analogs and a medium-throughput screen was completed. Analogs SVTH-7, -6, and -5 demonstrated potent anti-cancer activity greater than PST and several standard chemotherapeutics. They disrupted mitochondrial function, activated the intrinsic apoptotic pathway, and reduced growth of tumor xenografts in vivo. Interestingly, the pro-apoptotic effects of SVTH-7 on cancer cells and mitochondria were abrogated with the inhibition of mitochondrial complex II and III, suggesting mitochondrial or metabolic vulnerabilities may be exploited by this analog. This work provides a scaffold for characterizing distinct mitochondrial and metabolic features of cancer cells and reveals several lead compounds with high therapeutic potential. PMID:28220885

  10. 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

  11. Mechanisms of mitochondria and autophagy crosstalk.

    Science.gov (United States)

    Rambold, Angelika S; Lippincott-Schwartz, Jennifer

    2011-12-01

    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 neuronal loss in Parkinson's disease. In addition to autophagy's significance in mitochondrial integrity, several lines of evidence suggest that mitochondria can also substantially influence the autophagic process. The mitochondria's ability to influence and be influenced by autophagy places both elements (mitochondria and autophagy) in a unique position where defects in one or the other system could increase the risk to various metabolic and autophagic related diseases.

  12. 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.

  13. HBsAg inhibits the translocation of JTB into mitochondria in HepG2 cells and potentially plays a role in HCC progression.

    Directory of Open Access Journals (Sweden)

    Yun-Peng Liu

    Full Text Available BACKGROUND AND AIMS: The expression of the jumping translocation breakpoint (JTB gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. METHODS: We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg in vitro were determined by assessing cell apoptosis and motility. RESULTS: Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis, which was significantly enhanced by HBs expression. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells, whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased advantages in cell motility and anti-apoptosis. CONCLUSION: HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development.

  14. Caffeic acid phenethyl ester induces mitochondria-mediated apoptosis in human myeloid leukemia U937 cells.

    Science.gov (United States)

    Jin, Un-Ho; Song, Kwon-Ho; Motomura, Muneo; Suzuki, Ikukatsu; Gu, Yeun-Hwa; Kang, Yun-Jeong; Moon, Tae-Chul; Kim, Cheorl-Ho

    2008-03-01

    Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

  15. 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.

  16. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.

    Science.gov (United States)

    Arnandis, T; Ferrer-Vicens, I; García-Trevijano, E R; Miralles, V J; García, C; Torres, L; Viña, J R; Zaragozá, R

    2012-09-01

    Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b(2) of the v-type H(+) ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.

  17. Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria.

    Science.gov (United States)

    Gupta, Shelly; Prasad, G V R Krishna; Mukhopadhaya, Arunika

    2015-12-25

    Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

  18. Transcellular degradation of axonal mitochondria.

    Science.gov (United States)

    Davis, Chung-ha O; Kim, Keun-Young; Bushong, Eric A; Mills, Elizabeth A; Boassa, Daniela; Shih, Tiffany; Kinebuchi, Mira; Phan, Sebastien; Zhou, Yi; Bihlmeyer, Nathan A; Nguyen, Judy V; Jin, Yunju; Ellisman, Mark H; Marsh-Armstrong, Nicholas

    2014-07-01

    It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.

  19. 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.

  20. Caffeine increases mitochondrial function and blocks melatonin signaling to mitochondria in Alzheimer's mice and cells.

    Science.gov (United States)

    Dragicevic, Natasa; Delic, Vedad; Cao, Chuanhai; Copes, Neil; Lin, Xiaoyang; Mamcarz, Maggie; Wang, Li; Arendash, Gary W; Bradshaw, Patrick C

    2012-12-01

    Caffeine and melatonin have been shown to protect the Swedish mutant amyloid precursor protein (APP(sw)) transgenic mouse model of Alzheimer's disease from cognitive dysfunction. But their mechanisms of action remain incompletely understood. These Alzheimer's mice have extensive mitochondrial dysfunction, which likely contributes to their cognitive decline. To further explore the mechanism through which caffeine and melatonin protect cognitive function in these mice, we monitored the function of isolated mitochondria from APP(sw) mice treated with caffeine, melatonin, or both in their drinking water for one month. Melatonin treatment yielded a near complete restoration of mitochondrial function in assays of respiratory rate, membrane potential, reactive oxygen species production, and ATP levels. Caffeine treatment by itself yielded a small increase in mitochondrial function. However, caffeine largely blocked the large enhancement of mitochondrial function provided by melatonin. Studies with N2a neuroblastoma cells stably expressing APP(sw) showed that specific inhibition of cAMP-dependent phosphodiesterase (PDE) 4 or cGMP-dependent PDE5 also blocked melatonin protection of mitochondrial function, but A(2a) and A₁ adenosine receptor antagonists were without effect. Melatonin or caffeine at the concentrations used to modulate mitochondrial function in the cells had no effect on cAMP-dependent PDE activity or cellular cAMP or cGMP levels. Therefore, caffeine and increased cyclic nucleotide levels likely block melatonin signaling to mitochondria by independent mechanisms that do not involve adenosine receptor antagonism. The results of this study indicate that melatonin restores mitochondrial function much more potently than caffeine in APP(sw) transgenic mouse and cell models of Alzheimer's disease.

  1. Etoposide Induces Mitochondria-Associated Apoptotic Cell Death in Human Gastric Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    LI Jing-hua; CHEN Yue; WANG Jia-si; KONG Wei; JIN Ying-hua

    2008-01-01

    Recent observations indicate that the resistance of apoptosis is an important process of tumor metastasis and metastases are the cause of 90% of human cancer death.Etoposide,a semisynthetic derivative of the podophyllotoxins,is a clinically used anti-cancer reagent,but the effects of it on metastatic gastric carcinoma cells are totally unknown.In this study,etoposide induced apoptotic cell death in human gastric adenocareinoma cell line SGC-7901,derived from metastatic lymph nodes,as evidenced by the analysis of DNA fragmentation,apoptotic body formation,caspase activation,and apoptosis specific changes in cell morphology is demonstrated.The depolarization of mitochondrial membrane and the release of cytochrome c were most early events in etoposide treated SGC-7901 cells,and were followed by caspase-3 activation and PARP cleavage.Caspase-8 activation was not detected under the same condition.Thus,it was proposed that etoposide induces caspase-associated apoptotic cell death in human metastatic gastric carcinoma,which is initiated by mitochondrial cytochrome c release.

  2. Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): formation of mitoptotic bodies and extrusion of mitochondrial material from the cell.

    Science.gov (United States)

    Lyamzaev, Konstantin G; Nepryakhina, Olga K; Saprunova, Valeria B; Bakeeva, Lora E; Pletjushkina, Olga Yu; Chernyak, Boris V; Skulachev, Vladimir P

    2008-01-01

    Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.

  3. 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

    2016-08-01

    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.

  4. Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines

    OpenAIRE

    Silva, Diana F.; Selfridge, J. Eva; Lu, Jianghua; E, Lezi; Roy, Nairita; Hutfles, Lewis; Burns, Jeffrey M.; Michaelis, Elias K.; Yan, Shidu; Cardoso, Sandra M.; Swerdlow, Russell H.

    2013-01-01

    Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD an...

  5. 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.

  6. Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance.

    Science.gov (United States)

    Wang, Huaimin; Feng, Zhaoqianqi; Wang, Youzhi; Zhou, Rong; Yang, Zhimou; Xu, Bing

    2016-12-14

    Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.

  7. Acute effects of ACTH on dissociated adrenocortical cells: quantitative changes in mitochondria and lipid droplets.

    Science.gov (United States)

    Zoller, L C; Malamed, S

    1975-08-01

    To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

  8. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  9. 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.

  10. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  11. Validation of a Strategy for Cancer Therapy: Delivering Aminoglycoside Drugs to Mitochondria in HeLa Cells.

    Science.gov (United States)

    Abe, Jiro; Yamada, Yuma; Harashima, Hideyoshi

    2016-02-01

    Mitochondria in human cancer cells have been implicated in cancer cell proliferation, invasion, metastasis, and even drug-resistance mechanisms, making them a potential target organelle for the treatment of human malignancies. Gentamicin (GM), an aminoglycoside drug (AG), is a small molecule that functions as an antibiotic and has ototoxic and nephrotoxic characteristics. Thus, the delivery of GM to mitochondria in cancer cells would be an innovative anticancer therapeutic strategy. In this study, we attempted mitochondrial delivery of GM in HeLa cells derived from a human cervical cancer. For the mitochondrial delivery, we used MITO-Porter, a liposomal nanocarrier for mitochondrial delivery via membrane fusion. We first encapsulated GM in the aqueous phase of the carrier to construct GM-MITO-Porter. Flow cytometry analysis and fluorescent microscopy observations permitted us to confirm that the GM-MITO-Porter was efficiently taken up by HeLa cells and accumulated in mitochondria, whereas naked GM was not taken up by the cells. Moreover, cell viability assays using HeLa cells showed that the GM-MITO-Porter induced strong cytotoxic effects related to mitochondrial disorder. This finding is the first report of the mitochondrial delivery of an AG to cancer cells for cancer therapeutic strategy.

  12. Multiple enzymatic defects in mitochondria in hematological cells of patients with primary sideroblastic anemia.

    Science.gov (United States)

    Aoki, Y

    1980-07-01

    Activities of mitochondrial enzymes in blood cells from 69 patients with primary sideroblastic anemia were determined to elucidate the pathogenesis of the disease. In erythroblasts of patients with primary acquired type the activities of both delta-aminolevulinic acid synthetase and mitochondrial serine protease were inevitably decreased. The susceptibility to the protease of apo-delta-aminolevulinic acid synthetase prepared from erythroblasts of patients with this type was within the normal range, in contrast to that of pyridoxine-responsive anemia. The activities of mitochondrial enzymes such as cytochrome oxidase, serine protease, and oligomycin-sensitive ATPase, except citrate synthetase, were usually decreased in mature granulocytes of the patients. Patients with hereditary sideroblastic anemia also had decreased delta-aminolevulinic acid synthetase activity in erythroblasts, and decreased serine protease activity in both erythroblasts and mature granulocytes. Mature granulocytes obtained from patients with pyridoxine-responsive anemia before therapy had decreased cytochrome oxidase activity, however, the activity increased to a normal level when the patients were in remission. The activities of other mitochondrial enzymes in mature granulocytes were within normal range in these patients before pyridoxine therapy. The activities of these mitochondrial enzymes in lymphocytes were within normal range in all groups of patients with primary sideroblastic anemia. We suggest that patients with primary acquired, and possibly also those with hereditary sideroblastic anemia have impaired mitochondrial function in both erythroblasts and granulocytes. That only anemia is observed in these patients is because a functional abnormality of mitochondria in erythroblasts is most important because of the role of mitochondria in the formation of heme in erythrocyte development. In contrast to these two types of sideroblastic anemia, only delta-aminolevulinic acid synthetase in

  13. l-Lactate metabolism in HEP G2 cell mitochondria due to the l-lactate dehydrogenase determines the occurrence of the lactate/pyruvate shuttle and the appearance of oxaloacetate, malate and citrate outside mitochondria.

    Science.gov (United States)

    Pizzuto, Roberto; Paventi, Gianluca; Porcile, Carola; Sarnataro, Daniela; Daniele, Aurora; Passarella, Salvatore

    2012-09-01

    As part of an ongoing study of l-lactate metabolism both in normal and in cancer cells, we investigated whether and how l-lactate metabolism occurs in mitochondria of human hepatocellular carcinoma (Hep G2) cells. We found that Hep G2 cell mitochondria (Hep G2-M) possess an l-lactate dehydrogenase (ml-LDH) restricted to the inner mitochondrial compartments as shown by immunological analysis, confocal microscopy and by assaying ml-LDH activity in solubilized mitochondria. Cytosolic and mitochondrial l-LDHs were found to differ from one another in their saturation kinetics. Having shown that l-lactate itself can enter Hep G2 cells, we found that Hep G2-M swell in ammonium l-lactate, but not in ammonium pyruvate solutions, in a manner inhibited by mersalyl, this showing the occurrence of a carrier-mediated l-lactate transport in these mitochondria. Occurrence of the l-lactate/pyruvate shuttle and the appearance outside mitochondria of oxaloacetate, malate and citrate arising from l-lactate uptake and metabolism together with the low oxygen consumption and membrane potential generation are in favor of an anaplerotic role for l-LAC in Hep G2-M.

  14. 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.

  15. Environmental regulation of mitochondria-rich cells in Chalcalburnus tarichi (Pallas, 1811) during reproductive migration.

    Science.gov (United States)

    Oğuz, Ahmet R

    2013-03-01

    Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l⁻¹) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl⁻]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.

  16. Skeletal muscle satellite cells, mitochondria and microRNAs: their involvement in the pathogenesis of ALS

    Directory of Open Access Journals (Sweden)

    Stavroula Tsitkanou

    2016-09-01

    Full Text Available Amyotrophic lateral sclerosis (ALS, also known as motor neurone disease (MND, is a fatal motor neurone disorder. It results in progressive degeneration and death of upper and lower motor neurones, protein aggregation, severe muscle atrophy and respiratory insufficiency. Median survival with ALS is between two to five years from the onset of symptoms. ALS manifests as either familial ALS (FALS (~10% of cases or sporadic ALS (SALS, (~90% of cases. Mutations in the copper/zinc (CuZn superoxide dismutase (SOD1 gene account for ~20% of FALS cases and the mutant SOD1 mouse model has been used extensively to help understand the ALS pathology. As the precise mechanisms causing ALS are not well understood there is presently no cure. Recent evidence suggests that motor neuron degradation may involve a cell non-autonomous phenomenon involving numerous cell types within various tissues. Skeletal muscle is now considered as an important tissue involved in the pathogenesis of ALS by activating a retrograde signalling cascade that degrades motor neurons. Skeletal muscle heath and function are regulated by numerous factors including satellite cells, mitochondria and microRNAs. Studies demonstrate that in ALS these factors show various levels of dysregulation within the skeletal muscle. This review provides an overview of their dysregulation in various ALS models as well as how they may contribute individually and/or synergistically to the ALS pathogenesis.

  17. Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria.

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    Heather A Feaga

    2016-03-01

    Full Text Available Bacteria use trans-translation and the alternative rescue factors ArfA (P36675 and ArfB (Q9A8Y3 to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197 is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans.

  18. NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells.

    Science.gov (United States)

    Tejedo, J; Bernabé, J C; Ramírez, R; Sobrino, F; Bedoya, F J

    1999-10-08

    Exposure of RINm5F cells to interleukin-1beta and to several chemical NO donors such as sodium nitroprusside (SNP), SIN-1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl-2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO-induced apoptosis in the pancreatic beta-cell line is independent of acute activation of the cGMP pathway.

  19. Steroidogenesis in MA-10 Mouse Leydig Cells Is Altered via Fatty Acid Import into the Mitochondria1

    Science.gov (United States)

    Rone, Malena B.; Midzak, Andrew S.; Martinez-Arguelles, Daniel B.; Fan, Jinjiang; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    ABSTRACT Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation. PMID:25210128

  20. KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells.

    Science.gov (United States)

    Tanaka, Kousuke; Sugiura, Yoshimi; Ichishita, Ryohei; Mihara, Katsuyoshi; Oka, Toshihiko

    2011-07-15

    Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

  1. 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.

  2. 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.

  3. 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

  4. Ganoderma atrum Polysaccharide Ameliorates Hyperglycemia-Induced Endothelial Cell Death via a Mitochondria-ROS Pathway.

    Science.gov (United States)

    Li, Wen-Juan; Nie, Shao-Ping; Yao, Yu-Fei; Liu, Xiao-Zhen; Shao, Deng-Yin; Gong, De-Ming; Cui, Steve W; Phillips, Glyn O; He, Ming; Xie, Ming-Yong

    2015-09-23

    The aim of the present study was to examine the role of Ganoderma atrum polysaccharide (PSG-1) in reactive oxygen species (ROS) generation and mitochondrial function in hyperglycemia-induced angiopathy. In this work, ROS scavenger, oxidizing agent tert-butylhydroperoxide (tBH), mitochondrial permeability transition pore (mPTP) blockers, and caspase inhibition are used to investigate whether PSG-1 may promote survival of human umbilical vein cells (HUVECs) through preventing the overproduction of ROS and mitochondrial dysfunction. Experimental results show that exposure of HUVECs to 35.5 mmol/L glucose increases the proportion of cells undergoing apoptosis. PSG-1, mPTP blocker, or caspase inhibition can reduce apoptosis and ROS generation. PSG-1 also increases mitochondrial Bcl-2 protein formation and mitochondrial membrane potential (ΔΨm) but inhibits Bax translocation, cytochrome c release, and caspase activation. In summary, vascular protection of PSG-1 can be mediated by a mitochondria-ROS pathway. ROS generation and mPTP induction are critical for high glucose-mediated apoptosis. PSG-1 ameliorates endothelial dysfunction by inhibiting oxidative stress and subsequent mitochondrial dysfunction.

  5. Identification of active elementary flux modes in mitochondria using selectively permeabilized CHO cells.

    Science.gov (United States)

    Nicolae, Averina; Wahrheit, Judith; Nonnenmacher, Yannic; Weyler, Christian; Heinzle, Elmar

    2015-11-01

    Metabolic compartmentation is a key feature of mammalian cells. Mitochondria are the powerhouse of eukaryotic cells, responsible for respiration and the TCA cycle. We accessed the mitochondrial metabolism of the economically important Chinese hamster ovary (CHO) cells using selective permeabilization. We tested key substrates without and with addition of ADP. Based on quantified uptake and production rates, we could determine the contribution of different elementary flux modes to the metabolism of a substrate or substrate combination. ADP stimulated the uptake of most metabolites, directly by serving as substrate for the respiratory chain, thus removing the inhibitory effect of NADH, or as allosteric effector. Addition of ADP favored substrate metabolization to CO2 and did not enhance the production of other metabolites. The controlling effect of ADP was more pronounced when we supplied metabolites to the first part of the TCA cycle: pyruvate, citrate, α-ketoglutarate and glutamine. In the second part of the TCA cycle, the rates were primarily controlled by the concentrations of C4-dicarboxylates. Without ADP addition, the activity of the pyruvate carboxylase-malate dehydrogenase-malic enzyme cycle consumed the ATP produced by oxidative phosphorylation, preventing its accumulation and maintaining metabolic steady state conditions. Aspartate was taken up only in combination with pyruvate, whose uptake also increased, a fact explained by complex regulatory effects. Isocitrate dehydrogenase and α-ketoglutarate dehydrogenase were identified as the key regulators of the TCA cycle, confirming existent knowledge from other cells. We have shown that selectively permeabilized cells combined with elementary mode analysis allow in-depth studying of the mitochondrial metabolism and regulation.

  6. Mitochondria localize to the cleavage furrow in mammalian cytokinesis.

    Science.gov (United States)

    Lawrence, Elizabeth J; Mandato, Craig A

    2013-01-01

    Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.

  7. 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.

  8. Ultrastructure of germ cells, Sertoli cells and mitochondria during spermatogenesis in mature testis of the Chinese Taihang black goats (Capra hircus).

    Science.gov (United States)

    Shi, Liguang; Xun, Wenjuan; Zhou, Hanlin; Hou, Guanyu; Yue, Wenbin; Zhang, Chunxiang; Ren, Youshe; Yang, Rujie

    2013-07-01

    The objective of this study was to describe the ultrastructure of germ cells, Sertoli cells and mitochondria in mature testis of the Chinese Taihang black goat. The characteristics of germ cell nucleus and mitochondria changing during spermatogenesis were investigated by transmission electron microscopy (TEM). The results showed that the spermatogonium was elliptical, and its nucleus was about 4-5 μm. The round mitochondria can be observed throughout the cytoplasm around the nucleus. Small patches of heterochromatin were distributed throughout the nucleus. Spermatocyte was oval-shaped with a nucleus of about 4-4.5 μm in diameter. The heterochromatin began to attach to the inner surface of the nuclear membrane. Spermatid was about 4 μm and oval in shape. Its nucleus was oval or round and approximately 2-3 μm in diameter. The borderline between nucleus membrane and karyoplasm was distinct. During spermiogenesis, spermatid nucleus was condensed and elongated, and chromatin reached the highest condensation in the mature spermatozoon. The mid-piece was surrounded by mitochondria at the neck region. The sperm tail showed the typical "9+2″ structure, contained axoneme and central singlet microtubules. The nuclei of the Sertoli cells were irregular shaped and showed indentations in the membrane. In the mature testes of goat bucks, abundant mitochondria were around the germ cells and Sertoli cells. The scattered mitochondria were aggregated around the base of the flagellum (axoneme) during the spermatid differentiation stage. In conclusion, the present study showed that the spermatogenic process of Taihang black goat followed the pattern of mammals with some specific.

  9. Hitting the Bull’s-Eye in Metastatic Cancers—NSAIDs Elevate ROS in Mitochondria, Inducing Malignant Cell Death

    Directory of Open Access Journals (Sweden)

    Stephen John Ralph

    2015-02-01

    Full Text Available Tumor metastases that impede the function of vital organs are a major cause of cancer related mortality. Mitochondrial oxidative stress induced by hypoxia, low nutrient levels, or other stresses, such as genotoxic events, act as key drivers of the malignant changes in primary tumors to enhance their progression to metastasis. Emerging evidence now indicates that mitochondrial modifications and mutations resulting from oxidative stress, and leading to OxPhos stimulation and/or enhanced reactive oxygen species (ROS production, are essential for promoting and sustaining the highly metastatic phenotype. Moreover, the modified mitochondria in emerging or existing metastatic cancer cells, by their irreversible differences, provide opportunities for selectively targeting their mitochondrial functions with a one-two punch. The first blow would block their anti-oxidative defense, followed by the knockout blow—promoting production of excess ROS, capitulating the terminal stage—activation of the mitochondrial permeability transition pore (mPTP, specifically killing metastatic cancer cells or their precursors. This review links a wide area of research relevant to cellular mechanisms that affect mitochondria activity as a major source of ROS production driving the pro-oxidative state in metastatic cancer cells. Each of the important aspects affecting mitochondrial function are discussed including: hypoxia, HIFs and PGC1 induced metabolic changes, increased ROS production to induce a more pro-oxidative state with reduced antioxidant defenses. It then focuses on how the mitochondria, as a major source of ROS in metastatic cancer cells driving the pro-oxidative state of malignancy enables targeting drugs affecting many of these altered processes and why the NSAIDs are an excellent example of mitochondria-targeted agents that provide a one-two knockout activating the mPTP and their efficacy as selective anticancer metastasis drugs.

  10. Fisetin induces apoptosis in human nonsmall lung cancer cells via a mitochondria-mediated pathway.

    Science.gov (United States)

    Kang, Kyoung Ah; Piao, Mei Jing; Hyun, Jin Won

    2015-03-01

    The present study investigated the apoptotic effects of fisetin, a phenolic compound, against the human nonsmall cell lung cancer cell line, NCI-H460. Fisetin showed dose-dependent cytotoxic activity against NCI-H460 cells, with 50% inhibition of cell viability occurring at a concentration of 75 μg/mL. Fisetin induced both the production of intracellular reactive oxygen species and apoptosis, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the number of sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, fisetin significantly modulated the expression of apoptosis-associated proteins, resulting in reduced expression of B cell lymphoma-2, increased expression of Bcl-2-associated X protein, and activation of caspase-9 and caspase-3. In addition, pretreatment with a caspase inhibitor blocked fisetin-induced cell death.

  11. Mitochondria: a target for bacteria.

    Science.gov (United States)

    Lobet, Elodie; Letesson, Jean-Jacques; Arnould, Thierry

    2015-04-01

    Eukaryotic cells developed strategies to detect and eradicate infections. The innate immune system, which is the first line of defence against invading pathogens, relies on the recognition of molecular patterns conserved among pathogens. Pathogen associated molecular pattern binding to pattern recognition receptor triggers the activation of several signalling pathways leading to the establishment of a pro-inflammatory state required to control the infection. In addition, pathogens evolved to subvert those responses (with passive and active strategies) allowing their entry and persistence in the host cells and tissues. Indeed, several bacteria actively manipulate immune system or interfere with the cell fate for their own benefit. One can imagine that bacterial effectors can potentially manipulate every single organelle in the cell. However, the multiple functions fulfilled by mitochondria especially their involvement in the regulation of innate immune response, make mitochondria a target of choice for bacterial pathogens as they are not only a key component of the central metabolism through ATP production and synthesis of various biomolecules but they also take part to cell signalling through ROS production and control of calcium homeostasis as well as the control of cell survival/programmed cell death. Furthermore, considering that mitochondria derived from an ancestral bacterial endosymbiosis, it is not surprising that a special connection does exist between this organelle and bacteria. In this review, we will discuss different mitochondrial functions that are affected during bacterial infection as well as different strategies developed by bacterial pathogens to subvert functions related to calcium homeostasis, maintenance of redox status and mitochondrial morphology.

  12. In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

    Science.gov (United States)

    Su, Feng; Zhang, Shicui; Li, Hongyan; Guo, Huarong

    2007-04-01

    In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR. MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.

  13. In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid to gill cell line of flounder Paralichthy olivaceus

    Institute of Scientific and Technical Information of China (English)

    SU Feng; ZHANG Shicui; LI Hongyan; GUO Huarong

    2007-01-01

    In vitro acute cytotoxicity of neonicotinoid insecticide imidacloprid (IMI) to the gill cell line of flounder (FG) that collected in the gill of Paralichthys olivaceus, was examined by 3 widely used endpoint bioassays: NR (neutral red), MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and TCP (total cell protein). The result shows that the IMI increased at concentrations ≥0.5 μg/ml. The IC50 value of NR, MTT, and TCP was 41.86, 38.46, and 39.08 μg/ml, respectively. The ultrastructural observation revealed that the mitochondria of the cells exposed to 60 μg/ml IMI for 48 h were severely damaged, swollen or disrupted, while their nuclei and rough endoplasmic reticulum (RER) remained normal. This would suggest that the mitochondria are probably the primary target of IMI.

  14. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Directory of Open Access Journals (Sweden)

    Teruyuki Hayashi

    Full Text Available Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT in combination with fluorescence lifetime imaging microscopy (FLIM. Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  15. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Science.gov (United States)

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  16. Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells

    Directory of Open Access Journals (Sweden)

    Silvia eAlfonso-Loeches

    2014-08-01

    Full Text Available Toll-like receptors (TLRs and Nod-like receptors (NLRs are innate immunity sensors that provide an early/effective response to pathogenic or injury conditions. We have reported that ethanol-induced TLR4 activation triggers signaling inflammatory responses in glial cells, causing neuroinflammation and brain damage. However, it is uncertain if ethanol is able to activate NLRs /inflammasome in astroglial cells, which is the mechanism of activation, and whether there is crosstalk between both immune sensors in glial cells. Here we show that chronic ethanol treatment increases the co-localization of caspase-1 with GFAP+ cells, and up-regulates IL-1β and IL-18 in the frontal medial cortex in WT, but not in TLR4 knock-out mice. We further show that cultured cortical astrocytes expressed several inflammasomes (NLRP3, AIM2, NLRP1 and IPAF, although NLRP3 mRNA is the predominant form. Ethanol, as ATP and LPS treatments, up-regulates NLRP3 expression, and causes caspase-1 cleavage and the release of IL-1β and IL-18 in astrocytes supernatant. Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial (m ROS generation because when using a specific mitochondria ROS scavenger, the mito-TEMPO (500 M or NLRP3 blocking peptide (4g/ml or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 M, abrogates mROS release and reduces the up-regulation of IL-1β and IL-18 induced by ethanol or LPS or ATP. Confocal microscopy studies further confirm that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment within the mitochondria to promote cell death by caspase-1-mediated pyroptosis, which accounts for ≈ 73 % of total cell death (≈22% and the remaining (≈25% die by caspase-3-dependent apoptosis. Suppression of the TLR4 function abrogates most ethanol effects on NLRP3 activation and reduces cell death. These findings suggest that NLRP3 participates, in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4 crosstalk in ethanol

  17. Apoptosis induction and mitochondria alteration in human HeLa tumour cells by photoproducts of Rose Bengal acetate.

    Science.gov (United States)

    Panzarini, Elisa; Tenuzzo, Bernadette; Palazzo, Fabio; Chionna, Alfonsina; Dini, Luciana

    2006-04-01

    The aim of this work was to investigate the apoptosis induction and mitochondria alteration after photodamage exerted by incubation of HeLa cells with Rose Bengal acetate-derivative (RBAc) followed by irradiation for a total dose of 1.6 J/cm2. This treatment was previously demonstrated to reduce cell viability under mild treatment conditions, suggesting the restoration of the photoactive molecule in particularly sensitive cell sites. Indeed, Rose Bengal (RB) is a very efficient photosensitizer, whose photophysical properties are inactivated by addition of the quencher group acetate. The RBAc behaves as a fluorogenic substrate by entering easily the cells where the original, photoactive molecule is restored by specific esterases. Different intracellular sites of photodamage of RB are present. In particular, fluorescence imaging of Rodamine 123 and JC-1 labelled cells showed altered morphology and loss of potential membrane of mitochondria. MTT and NR assays gave indications of alteration of mitochondrial and lysosomal enzyme activities. These damaged sites were likely responsible for triggering apoptosis. Significant amount of apoptotic cell death (about 40%) was induced after light irradiation followed RBAc incubation as revealed by morphological (modification of cell shape and blebs formation), cytochemical (FITC-Annexin-V positive cells) and nuclear fragmentation assays.

  18. 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.

  19. Intracellular position of mitochondria and chloroplasts in bundle sheath and mesophyll cells of C3 grasses in relation to photorespiratory CO2 loss

    Directory of Open Access Journals (Sweden)

    Yuto Hatakeyama

    2016-10-01

    Full Text Available In C3 plants, photosynthetic efficiency is reduced by photorespiration. A part of CO2 fixed during photosynthesis in chloroplasts is lost from mitochondria during photorespiration by decarboxylation of glycine by glycine decarboxylase (GDC. Thus, the intracellular position of mitochondria in photosynthetic cells is critical to the rate of photorespiratory CO2 loss. We investigated the intracellular position of mitochondria in parenchyma sheath (PS and mesophyll cells of 10 C3 grasses from 3 subfamilies (Ehrhartoideae, Panicoideae, and Pooideae by immunostaining for GDC and light and electron microscopic observation. Immunostaining suggested that many mitochondria were located in the inner half of PS cells and on the vacuole side of chloroplasts in mesophyll cells. Organelle quantification showed that 62–75% of PS mitochondria were located in the inner half of cells, and 62–78% of PS chloroplasts were in the outer half. In mesophyll cells, 61–92% of mitochondria were positioned on the vacuole side of chloroplasts and stromules. In PS cells, such location would reduce the loss of photorespiratory CO2 by lengthening the path of CO2 diffusion and allow more efficient fixation of CO2 from intercellular spaces. In mesophyll cells, it would facilitate scavenging by chloroplasts of photorespiratory CO2 released from mitochondria. Our data suggest that the PS cells of C3 grasses have already acquired an initial structure leading to proto-Kranz and further C3–C4 intermediate anatomy. We also found that in the Pooideae, organelle positioning in PS cells on the phloem side resembles that in mesophyll cells.

  20. 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.

  1. The effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca2+]i in the cells

    Science.gov (United States)

    Ji, YuBin; Gao, ShiYong; Ji, ChenFeng; Zou, Xiang

    2008-12-01

    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. HepG2 cells are double stained with and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca2+]i in the cells are observed using LCSM. The results of double staining with TMRE and Fluo-3/AM show that solanine can lower membrane potential and increase the concentration of Ca2+ in the cells 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.

  2. SIRT1 positively regulates autophagy and mitochondria function in embryonic stem cells under oxidative stress.

    Science.gov (United States)

    Ou, Xuan; Lee, Man Ryul; Huang, Xinxin; Messina-Graham, Steven; Broxmeyer, Hal E

    2014-05-01

    SIRT1, an NAD-dependent deacetylase, plays a role in regulation of autophagy. SIRT1 increases mitochondrial function and reduces oxidative stress, and has been linked to age-related reactive oxygen species (ROS) generation, which is highly dependent on mitochondrial metabolism. H2O2 induces oxidative stress and autophagic cell death through interference with Beclin 1 and the mTOR signaling pathways. We evaluated connections between SIRT1 activity and induction of autophagy in murine (m) and human (h) embryonic stem cells (ESCs) upon ROS challenge. Exogenous H2 O2 (1 mM) induced apoptosis and autophagy in wild-type (WT) and Sirt1-/- mESCs. High concentrations of H2O2 (1 mM) induced more apoptosis in Sirt1-/-, than in WT mESCs. However, addition of 3-methyladenine, a widely used autophagy inhibitor, in combination with H2O2 induced more cell death in WT than in Sirt1-/- mESCs. Decreased induction of autophagy in Sirt1-/- mESCs was demonstrated by decreased conversion of LC3-I to LC3-II, lowered expression of Beclin-1, and decreased LC3 punctae and LysoTracker staining. H2O2 induced autophagy with loss of mitochondrial membrane potential and disruption of mitochondrial dynamics in Sirt1-/- mESCs. Increased phosphorylation of P70/85-S6 kinase and ribosomal S6 was noted in Sirt1-/- mESCs, suggesting that SIRT1 regulates the mTOR pathway. Consistent with effects in mESCs, inhibition of SIRT1 using Lentivirus-mediated SIRT1 shRNA in hESCs demonstrated that knockdown of SIRT1 decreased H2O2-induced autophagy. This suggests a role for SIRT1 in regulating autophagy and mitochondria function in ESCs upon oxidative stress, effects mediated at least in part by the class III PI3K/Beclin 1 and mTOR pathways.

  3. Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria and on the viability of hepatoma cells.

    Science.gov (United States)

    Teplova, V V; Kudin, A P; Evtodienko YuV

    1998-01-01

    Effects of cumene hydroperoxide on the Ca(2+)-induced Ca2+ efflux from mitochondria isolated from rat liver and Zaidelja hepatoma were compared. Cumene hydroperoxide at micromolar concentrations (0.3-10 microM) prevented the closing of the permeability transition pore in the inner mitochondrial membrane and, therefore, potentiated the Ca(2+)-induced Ca2+ efflux. This response was 10-100 times greater in hepatoma mitochondria than in rat liver mitochondria. Micromolar concentrations of cumene hydroperoxide induced the death of the hepatoma cells in vitro.

  4. Pancreatic β-cell Na+ channels control global Ca2+ signaling and oxidative metabolism by inducing Na+ and Ca2+ responses that are propagated into mitochondria.

    Science.gov (United States)

    Nita, Iulia I; Hershfinkel, Michal; Kantor, Chase; Rutter, Guy A; Lewis, Eli C; Sekler, Israel

    2014-08-01

    Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in β cells. Although voltage-dependent Na(+) channels are abundantly expressed in β cells and activated by glucose, their role in communicating with mitochondria is unresolved. Here, we combined fluorescent Na(+), Ca(2+), and ATP imaging, electrophysiological analysis with tetrodotoxin (TTX)-dependent block of the Na(+) channel, and molecular manipulation of mitochondrial Ca(2+) transporters to study the communication between Na(+) channels and mitochondria. We show that TTX inhibits glucose-dependent depolarization and blocks cytosolic Na(+) and Ca(2+) responses and their propagation into mitochondria. TTX-sensitive mitochondrial Ca(2+) influx was largely blocked by knockdown of the mitochondrial Ca(2+) uniporter (MCU) expression. Knockdown of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) and Na(+) dose response analysis demonstrated that NCLX mediates the mitochondrial Na(+) influx and is tuned to sense the TTX-sensitive cytosolic Na(+) responses. Finally, TTX blocked glucose-dependent mitochondrial Ca(2+) rise, mitochondrial metabolic activity, and ATP production. Our results show that communication of the Na(+) channels with mitochondria shape both global Ca(2+) and metabolism signals linked to insulin secretion in β cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic β-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

  5. 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.

  6. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

    Science.gov (United States)

    Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

    2013-11-01

    Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles.

  7. Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

    Science.gov (United States)

    Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

    1996-12-01

    Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

  8. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome.

    Science.gov (United States)

    Singh, Bhupendra; Li, Xiurong; Owens, Kjerstin M; Vanniarajan, Ayyasamy; Liang, Ping; Singh, Keshav K

    2015-01-01

    To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.

  9. 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 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.

  10. The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

    Science.gov (United States)

    Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

    2015-11-01

    The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

  11. 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.

  12. Mitochondria and neuroplasticity

    OpenAIRE

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

    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...

  13. 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

  14. Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death.

    Science.gov (United States)

    Kaminski, Marcin; Kiessling, Michael; Süss, Dorothee; Krammer, Peter H; Gülow, Karsten

    2007-05-01

    Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.

  15. 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.

  16. 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.

  17. 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.

  18. Regulation of Mitochondria Function by TRAF3 in B Lymphocytes and B Cell Malignancies

    Science.gov (United States)

    2014-08-01

    Aim 2. To identify novel TRAF3- interacting proteins in mitochondria of B lymphocytes 1 PI: Ping Xie, PhD Considering that TRAF3 does not...mitochondrial proteins. To test this, we propose to identify novel mitochondrial TRAF3- interacting proteins using biochemical affinity purification...mitochondrial TRAF3- interacting proteins : 100% completed. 2c. Mass spectrometry-based sequencing of purified proteins: 100% completed. 2d. Proteomic

  19. On Cellular Darwinism: Mitochondria.

    Science.gov (United States)

    Bull, Larry

    2016-01-01

    The significant role of mitochondria within cells is becoming increasingly clear. This letter uses the NKCS model of coupled fitness landscapes to explore aspects of organelle-nucleus coevolution. The phenomenon of mitochondrial diversity is allowed to emerge under a simple intracellular evolutionary process, including varying the relative rate of evolution by the organelle. It is shown how the conditions for the maintenance of more than one genetic variant of mitochondria are similar to those previously suggested as needed for the original symbiotic origins of the relationship using the NKCS model.

  20. Mitochondria in lung disease.

    Science.gov (United States)

    Cloonan, Suzanne M; Choi, Augustine M K

    2016-03-01

    Mitochondria are a distinguishing feature of eukaryotic cells. Best known for their critical function in energy production via oxidative phosphorylation (OXPHOS), mitochondria are essential for nutrient and oxygen sensing and for the regulation of critical cellular processes, including cell death and inflammation. Such diverse functional roles for organelles that were once thought to be simple may be attributed to their distinct heteroplasmic genome, exclusive maternal lineage of inheritance, and ability to generate signals to communicate with other cellular organelles. Mitochondria are now thought of as one of the cell's most sophisticated and dynamic responsive sensing systems. Specific signatures of mitochondrial dysfunction that are associated with disease pathogenesis and/or progression are becoming increasingly important. In particular, the centrality of mitochondria in the pathological processes and clinical phenotypes associated with a range of lung diseases is emerging. Understanding the molecular mechanisms regulating the mitochondrial processes of lung cells will help to better define phenotypes and clinical manifestations associated with respiratory disease and to identify potential diagnostic and therapeutic targets.

  1. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    Science.gov (United States)

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.

  2. The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells.

    Science.gov (United States)

    Nagai, Masazumi; Vo, Nha H; Shin Ogawa, Luisa; Chimmanamada, Dinesh; Inoue, Takayo; Chu, John; Beaudette-Zlatanova, Britte C; Lu, Rongzhen; Blackman, Ronald K; Barsoum, James; Koya, Keizo; Wada, Yumiko

    2012-05-15

    Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.

  3. 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.

  4. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  5. 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.

  6. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  7. Sinularin Induces Apoptosis through Mitochondria Dysfunction and Inactivation of the pI3K/Akt/mTOR Pathway in Gastric Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Wu

    2016-07-01

    Full Text Available Sinularin is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinularin on two human gastric cancer cell lines, AGS and NCI-N87. Our results demonstrated that sinularin suppressed the proliferation of gastric cancer cells in a dose-dependent manner and induced apoptosis. In addition, the loss of mitochondrial membrane potential, the release of cytochrome C, the activation of Bax, Bad and caspase-3/9, and the suppression of p-Bad, Bcl-xL and Bcl-2 were observed in the cells treated with sinularin. This finding suggests that sinularin-induced apoptosis is associated with mitochondria-mediated apoptosis and occurs through caspase-dependent pathways. Furthermore, sinularin inhibited the phosphoinositol 3-kinase/Akt/mechanistic target of the rapamycin signaling pathway. Taken together, our results show that sinularin-induced apoptosis is mediated by activation of the caspase cascade and mitochondrial dysfunction. Our findings suggest that sinularin merits further evaluation as a chemotherapeutic agent for human gastric cancer.

  8. 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.

  9. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  10. Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

    Science.gov (United States)

    Cho, Young Min; Kim, Ju Han; Kim, Mingoo; Park, Su Jin; Koh, Sang Hyeok; Ahn, Hyo Seop; Kang, Gyeong Hoon; Lee, Jung-Bin; Park, Kyong Soo; Lee, Hong Kyu

    2012-01-01

    It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

  11. Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

    Directory of Open Access Journals (Sweden)

    Young Min Cho

    Full Text Available It has been reported that human mesenchymal stem cells (MSCs can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(- and bromodeoxyuridine [BrdU](+ during the coculture of MSCs (uridine-independent and BrdU-sensitive and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G treatment (uridine-dependent but BrdU-resistant. The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion. Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

  12. 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.

  13. 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.

    2017-01-01

    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, are 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 ErbB2 (HER2) receptor, FAK (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 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 EGF treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of the Hax1 isoform 1 in vitro, and Grb7 expression may slow the 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. PMID:26869103

  14. The Rice Dynamin-Related Protein OsDRP1E Negatively Regulates Programmed Cell Death by Controlling the Release of Cytochrome c from Mitochondria

    Science.gov (United States)

    Zhou, Xueping

    2017-01-01

    Programmed cell death (PCD) mediated by mitochondrial processes has emerged as an important mechanism for plant development and responses to abiotic and biotic stresses. However, the role of translocation of cytochrome c from the mitochondria to the cytosol during PCD remains unclear. Here, we demonstrate that the rice dynamin-related protein 1E (OsDRP1E) negatively regulates PCD by controlling mitochondrial structure and cytochrome c release. We used a map-based cloning strategy to isolate OsDRP1E from the lesion mimic mutant dj-lm and confirmed that the E409V mutation in OsDRP1E causes spontaneous cell death in rice. Pathogen inoculation showed that dj-lm significantly enhances resistance to fungal and bacterial pathogens. Functional analysis of the E409V mutation showed that the mutant protein impairs OsDRP1E self-association and formation of a higher-order complex; this in turn reduces the GTPase activity of OsDRP1E. Furthermore, confocal microscopy showed that the E409V mutation impairs localization of OsDRP1E to the mitochondria. The E409V mutation significantly affects the morphogenesis of cristae in mitochondria and causes the abnormal release of cytochrome c from mitochondria into cytoplasm. Taken together, our results demonstrate that the mitochondria-localized protein OsDRP1E functions as a negative regulator of cytochrome c release and PCD in plants. PMID:28081268

  15. Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

    Institute of Scientific and Technical Information of China (English)

    YU Lei; CUI Rong-tian; MO Ke; WANG Wei; JI Yu-bin; ZOU Xiang

    2008-01-01

    Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L. saponin, CSS)on human hepatocarcinoma cell Line HepG-2. Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method. Morphological observation of the HepG-2 cells was completed by fluorescence microscope. This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining, and to observe if there were typical apoptosis peaks. The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry. The effect of intraceUular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope. Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS, and its IC50 value was 46.16 μg·mL-1. The HepG-2 cells are characteristic apoptosis morphologic changed, and the apoptosis percentage is increased to 66.652 % in the 50 μg·mL-1 dosage group. The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked, and the cellular proportion in G2 period is decreased by the function of CSS for 24 h. The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees. In addition, the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups. Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatoearcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

  16. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  17. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  18. Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera).

    Science.gov (United States)

    Vafopoulou, Xanthe

    2009-12-01

    We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

  19. Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

    Science.gov (United States)

    Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

    2016-01-01

    In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

  20. 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.

  1. ING1 induces apoptosis through direct effects at the mitochondria

    DEFF Research Database (Denmark)

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

    2013-01-01

    The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1,interacts with the proliferating cell nuclear...... 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...

  2. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    OpenAIRE

    Singleton Charles K; Song Qilin

    2002-01-01

    Abstract Background Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine Th...

  3. 地西他滨对同步化的G0/G1期MDS-L细胞系线粒体功能的影响%Role of decitabine in the mitochondria biogenesis of synchronized G0/G1 phase MDS-L cell line

    Institute of Scientific and Technical Information of China (English)

    邱宗建; 杨娟; 宋强

    2014-01-01

    目的:初步探讨地西他滨( DAC)在不整合入基因组DNA的情况下对线粒体生物学功能的影响。方法采用阿非迪霉素( APC)将MDS-L细胞同步化在G0/G1期。 DCFH-DA检测不同浓度DAC(0、5、10、15μmol/L)处理后的同步化G0/G1期细胞系活性氧( ROS)的产生水平。采用qRT-PCR法检测线粒体DNA拷贝数和线粒体编码基因NADH脱氢酶1、6( ND1、ND6) mRNA改变。结果与对照组相比,DAC在低浓度5μmol/L条件下可以通过不依赖DNA损伤的途径增加细胞ROS的产生(P<0.05),同时细胞内线粒体DNA(MtDNA)的拷贝数较对照组明显增加,差异有统计学意义(P<0.05)。但随着DAC浓度增加,DAC在高浓度(15μmol/L)可降低ROS的产生(P<0.05)。此外,在高浓度DAC作用下,ND1和ND6基因表达较对照组明显升高(P<0.05)。结论 DAC通过影响细胞ROS产生,改变线粒体DNA的拷贝数以及调控线粒体基因的表达,产生对线粒体功能的影响,并呈浓度依赖性。%Objective To investigate the effect of decitabin ( DAC) on the mitochondrial biogenesis of synchronized G0/G1 cells.Methods MDS-L cells were treated with aphidicolin ( APC) to synchronize cell cycle at G0/G1 stage. After treatment of decitabine at different concentrations (0, 5, 10, 15μmol/L), reactive oxygen species (ROS) pro-ductions were detected by DCFH-DA.Changes of mitochondrial DNA copy number and expressions of coded genes, NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 6(ND6), were detected with qRT-PCR.Results In com-parison with the control group, decitabine at low concentration (5μmol/L) could promote the production of ROS ( P<0.05) and increase the copy number of mtDNA ( P<0.05); however, as the concentration increased to 15μmol/L, ROS production started to decline to even lower than that of the control (P<0.05).Besides, DAC could significantly change the expressions of ND1 and ND6 at high

  4. Nitric oxide scavenging causes remodeling of the endoplasmic reticulum, Golgi apparatus and mitochondria in pulmonary arterial endothelial cells.

    Science.gov (United States)

    Lee, Jason E; Yuan, Huijuan; Liang, Feng-Xia; Sehgal, Pravin B

    2013-09-01

    The dependence of the structure and function of cytoplasmic organelles in endothelial cells on constitutively produced intracellular nitric oxide (NO) remains largely unexplored. We previously reported fragmentation of the Golgi apparatus in cells exposed to NO scavengers or after siRNA-mediated knockdown of eNOS. Others have reported increased mitochondrial fission in response to an NO donor. Functionally, we previously reported that bovine pulmonary arterial endothelial cells (PAECs) exposed to the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) developed a prosecretory phenotype characterized by prolonged secretion of soluble proteins. In the present study, we investigated whether NO scavenging led to remodeling of the endoplasmic reticulum (ER). Live-cell DAF-2DA imaging confirmed the presence of intracellular NO in association with the BODIPY C5-ceramide-labeled Golgi apparatus. Untreated human PAECs displayed a pattern of peripheral tubulo-reticular ER with a juxtanuclear accumulation of ER sheets. Cells exposed to c-PTIO showed a dramatic increase in ER sheets as assayed using immunofluorescence for the ER structural protein reticulon-4b/Nogo-B and the ER-resident GTPase atlastin-3, live-cell fluorescence assays using RTN4-GFP and KDEL-mCherry, and electron microscopy methods. These ER changes were inhibited by the NO donor diethylamine NONOate, and also produced by L-NAME, but not D-NAME or 8-br-cGMP. This ER remodeling was accompanied by Golgi fragmentation and increased fibrillarity and function of mitochondria (uptake of tetramethyl-rhodamine, TMRE). Despite Golgi fragmentation the functional ER/Golgi trafficking unit was preserved as seen by the accumulation of Sec31A ER exit sites adjacent to the dispersed Golgi elements and a 1.8-fold increase in secretion of soluble cargo. Western blotting and immunopanning data showed that RTN4b was increasingly ubiquitinated following c-PTIO exposure, especially in the

  5. Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

    Science.gov (United States)

    Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

    2005-10-01

    Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

  6. 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...

  7. Conditionally lethal mutations in chinese hamster cells. Characterization of a cell line with a possible defect in the Krebs cycle.

    Science.gov (United States)

    DeFrancesco, L; Werntz, D; Scheffler, I E

    1975-04-01

    A variant Chinese hamster cell line has been isolated from a mutagenized population that has a markedly reduced ability to oxidize a variety of substrates via the Krebs cycle. The production of 14CO2 from 14C-labeled compounds was measured using pyruvate, acetate, beta-hydroxybutyrate, palmitate and glutamate, and in all cases it was neglibible in the mutant. In contrast to this, significant amounts of 14CO2 were produced from 14C-aspartate and 14C-succinate which suggest that some reactions of the Krebs cycle can take place and this conclusion is supported by tracer experiments with labeled compounds. The rate of respiration measured with a Clark oxygen electrode in the mutant was compared to several normal Chinese hamster cell lines and was found to be only 8%. Mitochondria appear to be present in normal numbers and with only minor differences in morphology. The measurement of difference spectra between oxidized and reduced states permits us to conclude that the cytochromes are all present and functional. These results lead us to believe that there may be a defect in the Krebs cycle between alpha-ketoglutarate and succinate. Alternatively a defect in a structural component of the mitochondria or in the electron-transport chain itself may be causing pleiotropic effects in the Krebs cycle and respiration.

  8. The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

    Science.gov (United States)

    Gerő, Domokos; Torregrossa, Roberta; Perry, Alexis; Waters, Alicia; Le-Trionnaire, Sophie; Whatmore, Jacqueline L; Wood, Mark; Whiteman, Matthew

    2016-11-01

    The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

  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. Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

    Science.gov (United States)

    Afriyie, D K; Asare, G A; Bugyei, K; Lin, J; Peng, J; Hong, Z

    2015-01-15

    Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G₀/G₁ sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

  11. Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077.

    Science.gov (United States)

    Modica-Napolitano, J S; Koya, K; Weisberg, E; Brunelli, B T; Li, Y; Chen, L B

    1996-02-01

    We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

  12. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

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

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-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...

  13. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  14. Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death.

    Science.gov (United States)

    Robson, Christine A; Vanlerberghe, Greg C

    2002-08-01

    The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.

  15. THE CYTOTOXIC EFFECTS OF CRUDE BILE ON HUMAN PANCREATIC CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To identify effects of bile acids on pancreatic cancer, The ultrastructure and growth of PANC-1 and MIA PaCa-2 cell lines in crude bile modified medium were studied. Methods The growth of PANC-1 and MIA PaCa-2 cells in RPMI 1640 with or without 1%, 2% and 4% of the purified crude bile (containing total bile acids 10.17mmol/L) was assessed for 2, 4, 6, 8d by using MTT assay to determine inhibitory rate. The cell surface and intracellular ultrastructure of PANC-1 cells was investigated by SEM and TEM at 24h and 48h, respectively. Re sults The proliferation of both cell lines in bile treated medium were greatly retarded (P <0.001). The inhibitory rate of 1%, 2% and 4% bile on Panc-1 cells in 4d were 38%, 60% and 66%, respectively (P <0. 05), on MIA PaCa-2 cells at 4d were 28%, 39% and 52%, respectively (P <0. 05). The cells grown in bile for 48h lost their mi crovilli, their mitochondria and other organelles became vacuolated. Conclusion The bile acids in bile has cytotoxicity on PANC-1 and MIAPACA-2 cells, which may inhibit pancreatic cancer progress in patients clinically.

  16. Role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic postconditioning

    Institute of Scientific and Technical Information of China (English)

    Kai Sun; Zhi-Su Liu; Quan Sun

    2004-01-01

    AIM: To investigate the role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic postconditioning (IPC).METHODS: A rat model of acute hepatic ischemia-reperfusion was established, 24 healthy male Wistar rats were randomly divided into sham-operated group, ischemia-reperfusion group (IR) and IPC group. IPC was achieved by several brief pre-reperfusions followed by a persistent reperfusion.Concentration of malondialdehyde (MDA) and activity of several antioxidant enzymes in hepatic tissue were measured respectively. Apoptotic cells were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and expression of Bcl-2 protein was measured by immunohistochemical techniques. Moreover, mitochondrial ultrastructure and parameters of morphology of the above groups were observed by electron microscope.RESULTS: Compared with IR group, the concentration of MDA and the hepatocellular apoptotic index in IPC group was significantly reduced (P<0.05), while the activity of antioxidant enzymes and OD value of Bcl-2 protein were markedly enhanced (P<0.05). Moreover, the injury of mitochondrial ultrastructure in IPC group was also obviously relieved.CONCLUSION: IPC can depress the synthesis of oxygen free radicals to protect the mitochondrial ultrastructure and increase the expression of Bcl-2 protein that lies across the mitochondrial membrane. Consequently, IPC can reduce hepatocellular apoptosis after reperfusion and has a protective effect on hepatic ischemia-reperfusion injury.

  17. Diversity in host clone performance within a Chinese hamster ovary cell line.

    Science.gov (United States)

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics.

  18. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Genetics Group Web site at http://www.nist.gov/mml/biochemical/genetics/index.cfm . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

  19. How to prevent 'half-bastard' progeny? or An alternative for three-parent babies: two-parent babies through transplantation of sperm mitochondria.

    Science.gov (United States)

    Bongaerts, Ger P A

    2006-01-01

    Body development and activity depend on the level of internal energy generation. Therefore, unaffected, optimally active mitochondria are indispensable in a healthy and vital body. A mutation in the DNA of the semi-autonomous mitochondria (mtDNA) may cause an inheritable insufficiency that is due to decreased energy generation needed for adequate development. Sperm mitochondria will not enter the egg cell during fusion of male and female gametocytes. Since women with mutated mtDNA will increasingly know and realize the effect of such mutation in their own body, they will more often ask for treatment to stop the effect of such inconvenient mutation in their progeny. Thus far, solutions for this problem were thought to be: (i) nucleus transplantation just after fertilization into a nucleus-free egg cell of a second healthy woman and later (ii) transplantation of healthy mitochondria from a second woman into the egg cell before fertilization. Although both transplantations create babies with three, instead of two-parents that have contributed to the genetic content, in case of the newer mitochondria transplantation technique the part of the second woman is somewhat more reduced, but still clearly present. Thus, assisted-reproduction techniques that mix egg cell mitochondria from two women may create not only 'three-parents' babies, but also fears for 'three-parents' babies, since this handling may create non-scientific problems, especially regarding emotional, ethical, religious and juridical aspects of life. Transplantation of healthy sperm mitochondria of the partner into the egg cell with insufficient mitochondria is thought to be the best solution for this problem, since it may create a 'two-parents' instead of a 'three-parents' baby. This only implies that at the moment of (successful) transplantation the biological dogma is broken that mitochondria are maternally inherited: the mitochondria of the maternal line of the woman will have been substituted by the

  20. Energy metabolism characterization of a novel cancer stem cell-like line 3AB-OS.

    Science.gov (United States)

    Palorini, Roberta; Votta, Giuseppina; Balestrieri, Chiara; Monestiroli, Andrea; Olivieri, Sandro; Vento, Renza; Chiaradonna, Ferdinando

    2014-02-01

    Cancer stem cells (CSC) have a central role in driving tumor growth. Since metabolism is becoming an important diagnostic and therapeutic target, characterization of CSC line energetic properties is an emerging need. Embryonic and adult stem cells, compared to differentiated cells, exhibit a reduced mitochondrial activity and a stronger dependence on aerobic glycolysis. Here, we aimed to comparatively analyze bioenergetics features of the human osteosarcoma 3AB-OS CSC-like line, and the parental osteosarcoma MG63 cells, from which 3AB-OS cells have been previously selected. Our results suggest that 3AB-OS cells depend on glycolytic metabolism more strongly than MG63 cells. Indeed, growth in glucose shortage or in presence of galactose or pyruvate (mitochondrial specific substrates) leads to a significant reduction of their proliferation compared to MG63 cells. Accordingly, 3AB-OS cells show an increased expression of lactate dehydrogenase A (LDHA) and a larger accumulation of lactate in the culture medium. In line with these findings 3AB-OS cells as compared to MG63 cells present a reduced mitochondrial respiration, a stronger sensitivity to glucose depletion or glycolysis inhibition and a lessened sensitivity to oxidative phosphorylation inhibitors. Additionally, in contrast to MG63 cells, 3AB-OS display fragmented mitochondria, which become networked as they grow in glucose-rich medium, while almost entirely loose these structures growing in low glucose. Overall, our findings suggest that 3AB-OS CSC energy metabolism is more similar to normal stem cells and to cancer cells characterized by a glycolytic anaerobic metabolism.

  1. Solanine Induces Mitochondria-Mediated Apoptosis in Human Pancreatic Cancer Cells

    OpenAIRE

    Hongwei Sun; Chongqing Lv; Longlong Yang; Yingxiu Wang; Qingshun Zhang; Suhui Yu; Hongru Kong; Meng Wang; Jianming Xie; Chunwu Zhang; Mengtao Zhou

    2014-01-01

    Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptos...

  2. 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...

  3. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  4. 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

  5. Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

    Science.gov (United States)

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Majczak, Anna; Kedzior, Jakub; Slominska, Ewa; Lipinski, Marcin; Wakabayashi, Takashi

    2005-04-01

    Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

  6. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    Science.gov (United States)

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  7. 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...... the H2O2 necessary for NADH oxidation by apoplastic peroxidases, mitochondrial oxygen consumption could be measured in permeabilized cells. Inhibitor-sensitive oxidation of the respiratory substrates succinate, malate and NADH was observed after the addition of the appropriate coenzymes (ATP, NAD...

  8. Glutathione and Mitochondria

    Directory of Open Access Journals (Sweden)

    Vicent eRibas

    2014-07-01

    Full Text Available Glutathione (GSH is the main nonprotein 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 existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease and Alzheimer’s disease.

  9. Fisetin inhibits cellular proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Sabarwal, Akash; Agarwal, Rajesh; Singh, Rana P

    2017-02-01

    The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 μM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased the levels of p53 and its S15 phosphorylation in gastric cancer cells. We also observed that growth suppression and death of non-neoplastic human intestinal FHs74int cells were minimally affected by fisetin. Fisetin strongly increased apoptotic cells and showed mitochondrial membrane depolarization in gastric cancer cells. DNA damage was observed as early as 3 h after fisetin treatment which was accompanied with gamma-H2A.X(S139) phosphorylation and cleavage of PARP. Fisetin-induced apoptosis was observed to be independent of p53. DCFDA and MitoSOX analyses showed an increase in mitochondrial ROS generation in time- and dose-dependent fashion. It also increased cellular nitrite and superoxide generation. Pre-treatment with N-acetyl cysteine (NAC) inhibited ROS generation and also caused protection from fisetin-induced DNA damage. The formation of comets were observed in only fisetin treated cells which was blocked by NAC pre-treatment. Further investigation of the source of ROS, using mitochondrial respiratory chain (MRC) complex inhibitors, suggested that fisetin caused ROS generation specifically through complex I. Collectively, these results for the first time demonstrated that fisetin possesses anticancer potential through ROS production most likely via MRC complex I leading to apoptosis in human gastric carcinoma cells. © 2016 Wiley Periodicals, Inc.

  10. The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease.

    Science.gov (United States)

    Solesio, María E; Prime, Tracy A; Logan, Angela; Murphy, Michael P; Del Mar Arroyo-Jimenez, María; Jordán, Joaquín; Galindo, María F

    2013-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50nM, 30min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.

  11. Colon cancer cell chemosensitisation by fish oil emulsion involves apoptotic mitochondria pathway.

    Science.gov (United States)

    Granci, Virginie; Cai, Fang; Lecumberri, Elena; Clerc, Aurélie; Dupertuis, Yves M; Pichard, Claude

    2013-04-14

    Adjuvant use of safe compounds with anti-tumour properties has been proposed to improve cancer chemotherapy outcome. We aimed to investigate the effects of fish oil emulsion (FOE) rich in n-3 PUFA with the standard chemotherapeutic agents 5-fluorouracil (5-FU), oxaliplatin (OX) or irinotecan (IRI) on two human colorectal adenocarcinoma cells with different genetic backgrounds. The HT-29 (Bax+/+) and LS174T (Bax-/-) cells were co-treated for 24-72 h with 1 μm-5-FU, 1 μm-OX or 10 μm-IRI and/or FOE dilution corresponding to 24 μm-EPA and 20·5 μm-DHA. Soyabean oil emulsion (SOE) was used as isoenergetic and isolipid control. Cell viability, apoptosis and nuclear morphological changes were evaluated by cytotoxic colorimetric assay, flow cytometry analysis with annexin V and 4',6'-diamidino-2-phenylindole staining, respectively. A cationic fluorescent probe was used to evaluate mitochondrial dysfunction, and protein expression involved in mitochondrial apoptosis was determined by Western blot. In contrast to SOE, co-treatment with FOE enhanced significantly the pro-apoptotic and cytotoxic effects of 5-FU, OX or IRI in HT-29 but not in LS174T cells (two-way ANOVA, P <0.01). These results were confirmed by the formation of apoptotic bodies in HT-29 cells. A significant increase in mitochondrial membrane depolarisation was observed after the combination of 5-FU or IRI with FOE in HT-29 but not in LS174T cells (P <0.05). Co-administration of FOE with the standard agents, 5-FU, OX and IRI, could be a good alternative to increase the efficacy of chemotherapeutic protocols through a Bax-dependent mitochondrial pathway.

  12. Effects of a novel porphyrin-based photosensitizer on sensitive and multidrug-resistant human gastric cancer cell lines.

    Science.gov (United States)

    Chen, Jingjing; Mao, Lina; Liu, Shuping; Liang, Yanling; Wang, Sicheng; Wang, Yeyu; Zhao, Qiang; Zhang, Xiaojing; Che, Yanjun; Gao, Lijing; Liu, Tianjun

    2015-10-01

    Photodynamic therapy (PDT) has been considered to be a possible candidate approach in combating multidrug resistance (MDR) phenomenon during the treatment of cancer. To investigate the photocytotoxicity of a novel porphyrin-based photosensitizer, meso-5-[ρ-DTPA-aminophenyl]-10, 15, 20-triphenyl-porhyrin (DTP) (Fig. 1A), on MDR cells, the intracellular DTP uptake, phototoxicity and subcellular DTP localization were studied by using a human gastric cancer MGC803 cell line and its paclitaxel selected subline MGC803/PA expressing MDR phenotype. No significant difference was observed in intracellular DTP accumulation between sensitive and resistant cell lines after exposure to 1.56 μM concentration for 6h. DTP-PDT induced significant photocytotoxicity on both MGC803 and MGC803/PA cell lines and the photokilling was greater in MGC803 cell line in comparison to MGC803/PA. The fluence that caused 50% cell death was 4.42 and 6.29 J/cm(2) in MGC803 and MGC803/PA cell lines, respectively. The presence of Pgp inhibitors verapamil and cyclosporin A could not modify the intracellular DTP level in MGC803/PA cell line and the phototoxic effects. DTP was localized at lysosomes of MGC803 cell line but at lysosomes and mitochondria of MGC803/PA. Our results indicated that DTP-mediated PDT could eradicate gastric cancer cells whether or not they express MDR although the efficacy is slightly reduced in the MDR cells. The photokilling in MDR cells could not be altered by MDR inhibitor verapamil. The slightly different photocytotoxicity between sensitive and resistant cell lines could not explained by classical Pgp MDR and might be attributed to the differential intracellular DTP localization sites.

  13. Early perturbation in mitochondria redox homeostasis in response to environmental stress predicts cell fate in diatoms.

    Science.gov (United States)

    van Creveld, Shiri Graff; Rosenwasser, Shilo; Schatz, Daniella; Koren, Ilan; Vardi, Assaf

    2015-02-01

    Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of global photosynthesis. Nevertheless, little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a redox-sensitive green fluorescent protein sensor targeted to various subcellular organelles in the marine diatom Phaeodactylum tricornutum, to map the spatial and temporal oxidation patterns in response to environmental stresses. Specific organelle oxidation patterns were found in response to various stress conditions such as oxidative stress, nutrient limitation and exposure to diatom-derived infochemicals. We found a strong correlation between the mitochondrial glutathione (GSH) redox potential (EGSH) and subsequent induction of cell death in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), and a volatile halocarbon (BrCN) that mediate trophic-level interactions in marine diatoms. Induction of cell death in response to DD was mediated by oxidation of mitochondrial EGSH and was reversible by application of GSH only within a narrow time frame. We found that cell fate can be accurately predicted by a distinct life-death threshold of mitochondrial EGSH (-335 mV). We propose that compartmentalized redox-based signaling can integrate the input of diverse environmental cues and will determine cell fate decisions as part of algal acclimation to stress conditions.

  14. Crosstalk between mitochondria and peroxisomes

    Institute of Scientific and Technical Information of China (English)

    Jean; Demarquoy; Fran?oise; Le; Borgne

    2015-01-01

    Mitochondria and peroxisomes are small ubiquitous organelles. They both play major roles in cell metabolism,especially in terms of fatty acid metabolism,reactive oxygen species(ROS) production,and ROS scavenging,and it is now clear that they metabolically interact with each other. These two organelles share some properties,such as great plasticity and high potency to adapt their form and number according to cell requirements. Their functions are connected,and any alteration in the function of mitochondria may induce changes inperoxisomal physiology. The objective of this paper was to highlight the interconnection and the crosstalk existing between mitochondria and peroxisomes. Special emphasis was placed on the best known connections between these organelles:origin,structure,and metabolic interconnections.

  15. Eel osmotic stress transcriptional factor 1 (Ostf1 is highly expressed in gill mitochondria-rich cells, where ERK phosphorylated

    Directory of Open Access Journals (Sweden)

    Tse William KF

    2012-03-01

    Full Text Available Abstract Background Osmotic stress transcriptional factor 1 (Ostf1 was firstly identified in tilapia in 2005. Then numerous studies have investigated its regulation and expression profile in fish gill tissues in related to osmoregulation. Generally, hyperosmotic stress induced ostf1 mRNA expression level, however there is no report studying the cellular localization of Ostf1 expression in any osmoregulatory tissue. In this study immunohistochemical (IHC approach was used to study the cellular localization of Ostf1 in gill cells of Japanese eels. Findings Ostf1 protein was found to be localized in branchial mitochondria-rich/chloride cell (MRC/CC as revealed by Naα5 and CFTR co-localization. The protein was detectable at day 3 after fresh water to seawater transfer and was mainly localized in MRCs. Moreover, elevated levels of extracellular signal regulated kinase (ERK phosphorylation was observed at day 3 of the transfer and was co-localized with MRCs. Conclusions Our data identified Ostf1 expression in gill MRCs. The observation supports the role of Ostf1 in osmosensing and/or osmoregulation in fish gills, particularly its functional relationship with MRCs. The observation of the co-expression of pERK and Ostf1 in MRCs suggests a cross-talk mechanism between the mitogen-activated protein kinases (MAPKs and Ostf1 in response to hyperosmotic challenge. To summarize, this report has addressed the cellular localization of Ostf1 and provides evidence to illustrate the involvement of Ostf1 and ERK on osmosensing and osmoregulatory function of gill MRCs.

  16. Mitochondria-targeted antioxidant enzyme activity regulates radioresistance in human pancreatic cancer cells

    OpenAIRE

    Fisher, Carolyn J.; Goswami, Prabhat C.

    2008-01-01

    In recent years, cellular redox environment gained significant attention as a critical regulator of cellular responses to oxidative stress. Cellular redox environment is a balance between production of reactive oxygen species and their removal by antioxidant enzymes. We investigated the hypothesis that mitochondrial antioxidant enzyme activity regulates radioresistance in human pancreatic cancer cells. Vector-control and manganese superoxide dismutase (MnSOD) overexpressing human pancreatic c...

  17. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  18. 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

  19. The mitochondria-plasma membrane contact site.

    Science.gov (United States)

    Westermann, Benedikt

    2015-08-01

    Mitochondria are dynamic organelles that are highly motile and frequently fuse and divide. It has recently become clear that their complex behavior is governed to a large extent by interactions with other cellular structures. This review will focus on a mitochondria-plasma membrane tethering complex that was recently discovered and molecularly analyzed in budding yeast, the Num1/Mdm36 complex. This complex attaches mitochondria to the cell cortex and ensures that a portion of the organelles is retained in mother cells during cell division. At the same time, it supports mitochondrial division and integrates mitochondrial dynamics into cellular architecture. Recent evidence suggests that similar mechanisms might exist also in mammalian cells.

  20. 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

  1. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

  2. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  3. 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.

  4. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  5. Ultrastructural evidence for differentiation in a human glioblastoma cell line treated with inhibitors of eicosanoid metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, D.E.; Anderson, K.M. (Rush Presbyterian St. Luke' s Medical Center, Chicago, IL (United States)); Seed, T.M. (Argonne National Lab., IL (United States))

    1990-01-01

    Human glioblastoma cells incubated in the presence of inhibitors of eicosanoid biosynthesis show decreased cellular proliferation without cytotoxicity. The authors studied the ultrastructural morphology of a human glioblastoma cell line cultured with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, or 5,8,11,14-eicosatetraynoic acid, a cyclooxygenase and lipoxygenase inhibitor. When glioblastoma cells were treated for 3 days with antiproliferative concentrations of either agent, they shared many morphological characteristics, including evidence for increased astrocytic differentiation with only limited signs of toxicity. The inhibited glioma cells demonstrated an increase in the number and length of astrocytic processes containing greater numbers of glial filaments, and the NDGA-treated cells also demonstrated extensive lateral pseudopod formation along the processes. The glioblastoma cell shape also become more elongated, losing the usual nuclear lobularity and nuclear inclusions, especially in NDGA-treated cells. Many cytoplasmic organelles packed the cytosol of the inhibited glioma cells, including prominent Golgi apparatus, dilated smooth endoplasmic reticulum evolving into dilated vesicles, cytoplasmic vacuoles, and numerous concentric laminations. There was limited evidence for toxicity, however, as the mitochondria were more pleomorphic with some mitochondrial distension and disruption of the cristae along with an increase in cytoplasmic vacuolization. The authors conclude that the inhibitors of eicosanoid biosynthesis. NDGA and 5,8,11,14-eicosatetraynoic acid, not only suppress glioblastoma cell proliferation, but also include increased astrocytic differentiation.

  6. 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

  7. Dual mechanisms of sHA 14-1 in inducing cell death through endoplasmic reticulum and mitochondria.

    Science.gov (United States)

    Hermanson, David; Addo, Sadiya N; Bajer, Anna A; Marchant, Jonathan S; Das, Sonia Goutam Kumar; Srinivasan, Balasubramanian; Al-Mousa, Fawaz; Michelangeli, Francesco; Thomas, David D; Lebien, Tucker W; Xing, Chengguo

    2009-09-01

    HA 14-1 is a small-molecule Bcl-2 antagonist that promotes apoptosis in malignant cells, but its mechanism of action is not well defined. We recently reported that HA 14-1 has a half-life of only 15 min in vitro, which led us to develop a stable analog of HA 14-1 (sHA 14-1). The current study characterizes its mode of action. Because of the antiapoptotic function of Bcl-2 family proteins on the endoplasmic reticulum (ER) and mitochondria, the effect of sHA 14-1 on both organelles was evaluated. sHA 14-1 induced ER calcium release in human leukemic cells within 1 min, followed by induction of the ER stress-inducible transcription factor ATF4. Similar kinetics and stronger intensity of ER calcium release were induced by the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, accompanied by similar kinetics and intensity of ATF4 induction. sHA 14-1 directly inhibited SERCA enzymatic activity but had no effect on the inositol triphosphate receptor. Evaluation of the mitochondrial pathway showed that sHA 14-1 triggered a loss of mitochondrial transmembrane potential (Delta psi m) and weak caspase-9 activation, whereas thapsigargin had no effect. (R)-4-(3-Dimethylamino-1-phenylsulfanylmethyl-propylamino)-N-{4-[4-(4'-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-3-nitrobenzenesulfonamide (ABT-737), a well established small-molecule Bcl-2 antagonist, rapidly induced loss of Delta psi m and caspase-9 activation but had no effect on the ER. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone had some protective effect on sHA 14-1-induced cell death. These collective results suggest a unique dual targeting mechanism of sHA 14-1 on the apoptotic resistance machinery of tumor cells that includes antiapoptotic Bcl-2 family proteins and SERCA proteins.

  8. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  9. Sulfide detoxification in plant mitochondria.

    Science.gov (United States)

    Birke, Hannah; Hildebrandt, Tatjana M; Wirtz, Markus; Hell, Rüdiger

    2015-01-01

    In contrast to animals, which release the signal molecule sulfide in small amounts from cysteine and its derivates, phototrophic eukaryotes generate sulfide as an essential intermediate of the sulfur assimilation pathway. Additionally, iron-sulfur cluster turnover and cyanide detoxification might contribute to the release of sulfide in mitochondria. However, sulfide is a potent inhibitor of cytochrome c oxidase in mitochondria. Thus, efficient sulfide detoxification mechanisms are required in mitochondria to ensure adequate energy production and consequently survival of the plant cell. Two enzymes have been recently described to catalyze sulfide detoxification in mitochondria of Arabidopsis thaliana, O-acetylserine(thiol)lyase C (OAS-TL C), and the sulfur dioxygenase (SDO) ethylmalonic encephalopathy protein 1 (ETHE1). Biochemical characterization of sulfide producing and consuming enzymes in mitochondria of plants is fundamental to understand the regulatory network that enables mitochondrial sulfide homeostasis under nonstressed and stressed conditions. In this chapter, we provide established protocols to determine the activity of the sulfide releasing enzyme β-cyanoalanine synthase as well as sulfide-consuming enzymes OAS-TL and SDO. Additionally, we describe a reliable and efficient method to purify OAS-TL proteins from plant material.

  10. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  11. Role of Mitochondria in Parvovirus Pathology

    OpenAIRE

    Jonna Nykky; Matti Vuento; Leona Gilbert

    2014-01-01

    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. ...

  12. 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.

  13. 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.

  14. Atypical cell populations associated with acquired resistance to cytostatics and cancer stem cell features: the role of mitochondria in nuclear encapsulation.

    Science.gov (United States)

    Díaz-Carballo, David; Gustmann, Sebastian; Jastrow, Holger; Acikelli, Ali Haydar; Dammann, Philip; Klein, Jacqueline; Dembinski, Ulrike; Bardenheuer, Walter; Malak, Sascha; Araúzo-Bravo, Marcos J; Schultheis, Beate; Aldinger, Constanze; Strumberg, Dirk

    2014-11-01

    Until recently, acquired resistance to cytostatics had mostly been attributed to biochemical mechanisms such as decreased intake and/or increased efflux of therapeutics, enhanced DNA repair, and altered activity or deregulation of target proteins. Although these mechanisms have been widely investigated, little is known about membrane barriers responsible for the chemical imperviousness of cell compartments and cellular segregation in cytostatic-treated tumors. In highly heterogeneous cross-resistant and radiorefractory cell populations selected by exposure to anticancer agents, we found a number of atypical recurrent cell types in (1) tumor cell cultures of different embryonic origins, (2) mouse xenografts, and (3) paraffin sections from patient tumors. Alongside morphologic peculiarities, these populations presented cancer stem cell markers, aberrant signaling pathways, and a set of deregulated miRNAs known to confer both stem-cell phenotypes and highly aggressive tumor behavior. The first type, named spiral cells, is marked by a spiral arrangement of nuclei. The second type, monastery cells, is characterized by prominent walls inside which daughter cells can be seen maturing amid a rich mitochondrial environment. The third type, called pregnant cells, is a giant cell with a syncytium-like morphology, a main nucleus, and many endoreplicative functional progeny cells. A rare fourth cell type identified in leukemia was christened shepherd cells, as it was always associated with clusters of smaller cells. Furthermore, a portion of resistant tumor cells displayed nuclear encapsulation via mitochondrial aggregation in the nuclear perimeter in response to cytostatic insults, probably conferring imperviousness to drugs and long periods of dormancy until nuclear eclosion takes place. This phenomenon was correlated with an increase in both intracellular and intercellular mitochondrial traffic as well as with the uptake of free extracellular mitochondria. All these cellular

  15. Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis.

    Science.gov (United States)

    Karnewar, Santosh; Vasamsetti, Sathish Babu; Gopoju, Raja; Kanugula, Anantha Koteswararao; Ganji, Sai Krishna; Prabhakar, Sripadi; Rangaraj, Nandini; Tupperwar, Nitin; Kumar, Jerald Mahesh; Kotamraju, Srigiridhar

    2016-04-11

    Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE(-/-) mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE(-/-) mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.

  16. Finite-particle tracking reveals submicroscopic-size changes of mitochondria during transport in mitral cell dendrites

    Science.gov (United States)

    Gennerich, Arne; Schild, Detlev

    2006-03-01

    The mechanisms of molecular motor regulation during bidirectional organelle transport are still uncertain. There is, for instance, the unsettled question of whether opposing motor proteins can be engaged in a tug-of-war. Clearly, any non-synchronous activation of the molecular motors of one cargo can principally lead to changes in the cargo's shape and size; the cargo's size and shape parameters would certainly be observables of such changes. We therefore set out to measure position, shape and size parameters of fluorescent mitochondria (during their transport) in dendrites of cultured neurons using a finite-particle tracking algorithm. Our data clearly show transport-related submicroscopic-size changes of mitochondria. The observed displacements of the mitochondrial front and rear ends are consistent with a model in which microtubule plus- and minus-end-directed motor proteins or motors of the same type but moving along anti-parallel microtubules are often out-of-phase and occasionally engaged in a tug-of-war. Mostly the leading and trailing ends of mitochondria undergo similar characteristic movements but with a substantial time delay between the displacements of both ends, a feature reminiscent of an inchworm-like motility mechanism. More generally, we demonstrate that observing the position, shape and size of actively transported finite objects such as mitochondria can yield information on organelle transport that is generally not accessible by tracking the organelles' centroid alone.

  17. Safrole induces cell death in human tongue squamous cancer SCC-4 cells through mitochondria-dependent caspase activation cascade apoptotic signaling pathways.

    Science.gov (United States)

    Yu, Fu-Shun; Huang, An-Cheng; Yang, Jai-Sing; Yu, Chun-Shu; Lu, Chi-Cheng; Chiang, Jo-Hua; Chiu, Chang-Fang; Chung, Jing-Gung

    2012-07-01

    Safrole is one of important food-borne phytotoxin that exhibits in many natural products such as oil of sassafras and spices such as anise, basil, nutmeg, and pepper. This study was performed to elucidate safrole-induced apoptosis in human tongue squamous carcinoma SCC-4 cells. The effect of safrole on apoptosis was measured by flow cytometry and DAPI staining and its regulatory molecules were studied by Western blotting analysis. Safrole-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and Bid and down-regulation of the protein levels of Bcl-2 (up-regulation of the ratio of Bax/Bcl-2), resulting in cytochrome c release, promoted Apaf-1 level and sequential activation of caspase-9 and caspase-3 in a time-dependent manner. We also used real-time PCR to show safrole promoted the mRNA expressions of caspase-3, -8, and -9 in SCC-4 cells. These findings indicate that safrole has a cytotoxic effect in human tongue squamous carcinoma SCC-4 cells by inducing apoptosis. The induction of apoptosis of SCC-4 cells by safrole is involved in mitochondria- and caspase-dependent signal pathways.

  18. 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.

  19. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  20. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

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

    2011-01-01

    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...... except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein......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...

  1. Protection of normal brain cells from γ-irradiation-induced apoptosis by a mitochondria-targeted triphenyl-phosphonium-nitroxide: a possible utility in glioblastoma therapy.

    Science.gov (United States)

    Huang, Zhentai; Jiang, Jianfei; Belikova, Natalia A; Stoyanovsky, Detcho A; Kagan, Valerian E; Mintz, Arlan H

    2010-10-01

    Glioblastoma multiforme is the most frequent and aggressive primary brain tumor. A strong rationale to identify innovative approaches to treat these tumors is required since treatment failures result in local recurrences and median survivals range from 9 to 12 months. Glioma cells are reported to have less mitochondrial content compared to adjacent normal brain cells. Based on this difference, we suggest a new strategy, utilizing protection of normal brain cells by mitochondria-targeted electron scavengers and antioxidants-nitroxides-thus allowing for the escalation of the radiation doses. In this paper, we report that a conjugate of nitroxide with a hydrophobic cation, triphenyl-phosphonium (TPEY-Tempo), significantly protected brain endothelial cells from γ-irradiation-induced apoptosis while radiosensitizing brain tumor cells. Thus, TPEY-Tempo may be a promising adjunct in the treatment of glioblastoma with the potential to not only prolong survival but also to maintain quality of life and reduce treatment toxicity.

  2. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

    Full Text Available Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus. To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

  3. Mitochondria and redox homoeostasis as chemotherapeutic targets of Araucaria angustifolia (Bert.) O. Kuntze in human larynx HEp-2 cancer cells.

    Science.gov (United States)

    Branco, Cátia dos Santos; de Lima, Émilin Dreher; Rodrigues, Tiago Selau; Scheffel, Thamiris Becker; Scola, Gustavo; Laurino, Claudia Cilene Fernandes Correia; Moura, Sidnei; Salvador, Mirian

    2015-04-25

    Natural products are among one of the most promising fields in finding new molecular targets in cancer therapy. Laryngeal carcinoma is one of the most common cancers affecting the head and neck regions, and is associated with high morbidity rate if left untreated. The aim of this study was to examine the antiproliferative effect of Araucaria angustifolia on laryngeal carcinoma HEp-2 cells. The results showed that A. angustifolia extract (AAE) induced a significant cytotoxicity in HEp-2 cells compared to the non-tumor human epithelial (HEK-293) cells, indicating a selective activity of AAE for the cancer cells. A. angustifolia extract was able to increase oxidative damage to lipids and proteins, and the production of nitric oxide, along with the depletion of enzymatic antioxidant defenses (superoxide dismutase and catalase) in the tumor cell line. Moreover, AAE was able to induce DNA damage, nuclear fragmentation and chromatin condensation. A significant increase in the Apoptosis Inducing Factor (AIF), Bax, poly-(ADP-ribose) polymerase (PARP) and caspase-3 cleavage expression were also found. These effects could be related to the ability of AAE to increase the production of reactive oxygen species through inhibition of the mitochondrial electron transport chain complex I activity and ATP production by the tumor cells. The phytochemical analysis of A. angustifolia, performed using High Resolution Mass Spectrometry (HRMS) in MS and MS/MS mode, showed the presence of dodecanoic and hexadecanoic acids, and phenolic compounds, which may be associated with the chemotherapeutic effect observed in this study.

  4. Photodynamic damage study of HeLa cell line using ALA

    Science.gov (United States)

    AlSalhi, M. S.; Atif, M.; AlObiadi, A. A.; Aldwayyan, A. S.

    2011-04-01

    The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425-525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.

  5. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

    Directory of Open Access Journals (Sweden)

    William Dott

    2014-01-01

    Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

  6. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

  7. 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

  8. Lipids of mitochondria.

    Science.gov (United States)

    Horvath, Susanne E; Daum, Günther

    2013-10-01

    A unique organelle for studying membrane biochemistry is the mitochondrion whose functionality depends on a coordinated supply of proteins and lipids. Mitochondria are capable of synthesizing several lipids autonomously such as phosphatidylglycerol, cardiolipin and in part phosphatidylethanolamine, phosphatidic acid and CDP-diacylglycerol. Other mitochondrial membrane lipids such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sterols and sphingolipids have to be imported. The mitochondrial lipid composition, the biosynthesis and the import of mitochondrial lipids as well as the regulation of these processes will be main issues of this review article. Furthermore, interactions of lipids and mitochondrial proteins which are highly important for various mitochondrial processes will be discussed. Malfunction or loss of enzymes involved in mitochondrial phospholipid biosynthesis lead to dysfunction of cell respiration, affect the assembly and stability of the mitochondrial protein import machinery and cause abnormal mitochondrial morphology or even lethality. Molecular aspects of these processes as well as diseases related to defects in the formation of mitochondrial membranes will be described.

  9. 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

  10. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  11. 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.

  12. 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.

  13. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

  14. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  15. 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.

  16. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  17. 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.

  18. 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.

  19. 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.

  20. Further characterization of the first seminoma cell line TCam-2.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2008-03-01

    Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs.

  1. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  2. 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.

  3. 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.

  4. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  5. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  6. 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.

  7. 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.

  8. Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: treating cancer like an infectious disease.

    Science.gov (United States)

    Lamb, Rebecca; Ozsvari, Bela; Lisanti, Camilla L; Tanowitz, Herbert B; Howell, Anthony; Martinez-Outschoorn, Ubaldo E; Sotgia, Federica; Lisanti, Michael P

    2015-03-10

    Here, we propose a new strategy for the treatment of early cancerous lesions and advanced metastatic disease, via the selective targeting of cancer stem cells (CSCs), a.k.a., tumor-initiating cells (TICs). We searched for a global phenotypic characteristic that was highly conserved among cancer stem cells, across multiple tumor types, to provide a mutation-independent approach to cancer therapy. This would allow us to target cancer stem cells, effectively treating cancer as a single disease of "stemness", independently of the tumor tissue type. Using this approach, we identified a conserved phenotypic weak point - a strict dependence on mitochondrial biogenesis for the clonal expansion and survival of cancer stem cells. Interestingly, several classes of FDA-approved antibiotics inhibit mitochondrial biogenesis as a known "side-effect", which could be harnessed instead as a "therapeutic effect". Based on this analysis, we now show that 4-to-5 different classes of FDA-approved drugs can be used to eradicate cancer stem cells, in 12 different cancer cell lines, across 8 different tumor types (breast, DCIS, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma (brain)). These five classes of mitochondrially-targeted antibiotics include: the erythromycins, the tetracyclines, the glycylcyclines, an anti-parasitic drug, and chloramphenicol. Functional data are presented for one antibiotic in each drug class: azithromycin, doxycycline, tigecycline, pyrvinium pamoate, as well as chloramphenicol, as proof-of-concept. Importantly, many of these drugs are non-toxic for normal cells, likely reducing the side effects of anti-cancer therapy. Thus, we now propose to treat cancer like an infectious disease, by repurposing FDA-approved antibiotics for anti-cancer therapy, across multiple tumor types. These drug classes should also be considered for prevention studies, specifically focused on the prevention of tumor recurrence and distant metastasis. Finally, recent

  9. Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway.

    Science.gov (United States)

    Lee, Kyu-Sun; Wu, Zhihao; Song, Yan; Mitra, Siddhartha S; Feroze, Abdullah H; Cheshier, Samuel H; Lu, Bingwei

    2013-12-15

    The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. Here we show that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impaired the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. Our results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling.

  10. The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes.

    Science.gov (United States)

    Kahlert, Stefan; Schönfeld, Peter; Reiser, Georg

    2005-02-01

    The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.

  11. Polo Kinase Phosphorylates Miro to Control ER-Mitochondria Contact Sites and Mitochondrial Ca(2+) Homeostasis in Neural Stem Cell Development.

    Science.gov (United States)

    Lee, Seongsoo; Lee, Kyu-Sun; Huh, Sungun; Liu, Song; Lee, Do-Yeon; Hong, Seung Hyun; Yu, Kweon; Lu, Bingwei

    2016-04-18

    Mitochondria play central roles in buffering intracellular Ca²⁺ transients. While basal mitochondrial Ca²⁺ (Ca²⁺ mito) is needed to maintain organellar physiology, Ca²⁺ mito overload can lead to cell death. How Ca²⁺ mito homeostasis is regulated is not well understood. Here we show that Miro, a known component of the mitochondrial transport machinery, regulates Drosophila neural stem cell (NSC) development through Ca²⁺ mito homeostasis control, independent of its role in mitochondrial transport. Miro interacts with Ca²⁺ transporters at the ER-mitochondria contact site (ERMCS). Its inactivation causes Ca²⁺ mito depletion and metabolic impairment, whereas its overexpression results in Ca²⁺ mito overload, mitochondrial morphology change, and apoptotic response. Both conditions impaired NSC lineage progression. Ca²⁺ mito homeostasis is influenced by Polo-mediated phosphorylation of a conserved residue in Miro, which positively regulates Miro localization to, and the integrity of, ERMCS. Our results elucidate a regulatory mechanism underlying Ca²⁺ mito homeostasis and how its dysregulation may affect NSC metabolism/development and contribute to disease.

  12. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

  13. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  14. Exogenous ether lipids predominantly target mitochondria.

    Directory of Open Access Journals (Sweden)

    Lars Kuerschner

    Full Text Available 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 amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, 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 ether lipid metabolism and intracellular ether lipid trafficking.

  15. Development of a cell line from Echinococcus granulosus germinal layer.

    Science.gov (United States)

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  16. 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.

  17. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  18. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    Science.gov (United States)

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  19. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy.

    Science.gov (United States)

    Yang, Yuhui; Karakhanova, Svetlana; Hartwig, Werner; D'Haese, Jan G; Philippov, Pavel P; Werner, Jens; Bazhin, Alexandr V

    2016-12-01

    Mitochondria are indispensable for energy metabolism, apoptosis regulation, and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression, and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the "vicious cycle" between mitochondria, ROS, genomic instability, and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria-targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria-targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria-targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria-targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. J. Cell. Physiol. 231: 2570-2581, 2016. © 2016 Wiley Periodicals, Inc.

  20. Apoptotic effect of noscapine in breast cancer cell lines.

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  1. Calcium Transport by Corn Mitochondria 1

    Science.gov (United States)

    Silva, Marco Aurelio P.; Carnieri, Eva G. S.; Vercesi, Anibal E.

    1992-01-01

    Mitochondria from some plant tissues possess the ability to take up Ca2+ by a phosphate-dependent mechanism associated with a decrease in membrane potential, H+ extrusion, and increase in the rate of respiration (AE Vercesi, L Pereira da Silva, IS Martins, CF Bernardes, EGS Carnieri, MM Fagian [1989] In G Fiskum, ed, Cell Calcium Metabolism. Plenum Press, New York, pp 103-111). The present study reexamined the nature of the phosphate requirement in this process. The main observations are: (a) Respiration-coupled Ca2+ uptake by isolated corn (Zea mays var Maya Normal) mitochondria or carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced efflux of the cation from such mitochondria are sensitive to mersalyl and cannot be dissociated from the silmultaneous movement of phosphate in the same direction. (b) Ruthenium red-induced efflux is not affected by mersalyl and can occur in the absence of phosphate movement. (c) In Ca2+-loaded corn mitochondria, mersalyl causes net Ca2+ release unrelated to a decrease in membrane potential, probably due to an inhibition of Ca2+ cycling at the level of the influx pathway. It is concluded that corn mitochondria (and probably other plant mitochondria) do possess an electrophoretic influx pathway that appears to be a mersalyl-sensitive Ca2+/inorganic phosphate-symporter and a phosphate-independent efflux pathway possibly similar to the Na2+-independent Ca2+ efflux mechanism of vertebrate mitochondria, because it is not stimulated by Na+. PMID:16668661

  2. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  3. Investigation of the selenium metabolism in cancer cell lines.

    Science.gov (United States)

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

    2011-02-01

    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 μM 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 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 except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  4. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

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

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    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

  6. 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

  7. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  8. 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...... are 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...

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

    Directory of Open Access Journals (Sweden)

    Elena Barbieri

    2016-01-01

    Full Text Available 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.

  10. The apoptotic members CD95, BclxL, and Bcl-2 cooperate to promote cell migration by inducing Ca2+ flux from the endoplasmic reticulum to mitochondria

    Science.gov (United States)

    Fouqué, A; Lepvrier, E; Debure, L; Gouriou, Y; Malleter, M; Delcroix, V; Ovize, M; Ducret, T; Li, C; Hammadi, M; Vacher, P; Legembre, P

    2016-01-01

    Metalloprotease-processed CD95L (cl-CD95L) is a soluble cytokine that implements a PI3K/Ca2+ signaling pathway in triple-negative breast cancer (TNBC) cells. Accordingly, high levels of cl-CD95L in TNBC women correlate with poor prognosis, and administration of this ligand in an orthotopic xenograft mouse model accelerates the metastatic dissemination of TNBC cells. The molecular mechanism underlying CD95-mediated cell migration remains unknown. Here, we present genetic and pharmacologic evidence that the anti-apoptotic molecules BclxL and Bcl-2 and the pro-apoptotic factors BAD and BID cooperate to promote migration of TNBC cells stimulated with cl-CD95L. BclxL was distributed in both endoplasmic reticulum (ER) and mitochondrion membranes. The mitochondrion-localized isoform promoted cell migration by interacting with voltage-dependent anion channel 1 to orchestrate Ca2+ transfer from the ER to mitochondria in a BH3-dependent manner. Mitochondrial Ca2+ uniporter contributed to this flux, which favored ATP production and cell migration. In conclusion, this study reveals a novel molecular mechanism controlled by BclxL to promote cancer cell migration and supports the use of BH3 mimetics as therapeutic options not only to kill tumor cells but also to prevent metastatic dissemination in TNBCs. PMID:27367565

  11. 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.

  12. Uvangoletin induces mitochondria-mediated apoptosis in HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances.

    Science.gov (United States)

    Zheng, Zhuanzhen; Qiao, Zhenhua; Gong, Rong; Wang, Yalin; Zhang, Yiqun; Ma, Yanping; Zhang, Li; Lu, Yujin; Jiang, Bo; Li, Guoxia; Dong, Chunxia; Chen, Wenliang

    2016-02-01

    This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

  13. Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

    Science.gov (United States)

    Rodríguez-Cariño, C; Duffy, C; Sánchez-Chardi, A; McNeilly, F; Allan, G M; Segalés, J

    2011-01-01

    Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.

  14. Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines.

    Science.gov (United States)

    Silva, Diana F; Selfridge, J Eva; Lu, Jianghua; E, Lezi; Roy, Nairita; Hutfles, Lewis; Burns, Jeffrey M; Michaelis, Elias K; Yan, ShiDu; Cardoso, Sandra M; Swerdlow, Russell H

    2013-10-01

    Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD+/NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

  15. Calpains, mitochondria, and apoptosis.

    Science.gov (United States)

    Smith, Matthew A; Schnellmann, Rick G

    2012-10-01

    Mitochondrial activity is critical for efficient function of the cardiovascular system. In response to cardiovascular injury, mitochondrial dysfunction occurs and can lead to apoptosis and necrosis. Calpains are a 15-member family of Ca(2+)-activated cysteine proteases localized to the cytosol and mitochondria, and several have been shown to regulate apoptosis and necrosis. For example, in endothelial cells, Ca(2+) overload causes mitochondrial calpain 1 cleavage of the Na(+)/Ca(2+) exchanger leading to mitochondrial Ca(2+) accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases. Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis. In cardiomyocytes, calpain 1 activates caspase 3 and poly-ADP ribose polymerase during tumour necrosis factor-α-induced apoptosis, and calpain 1 cleaves apoptosis-inducing factor after Ca(2+) overload. Many of these observations have been elucidated with calpain inhibitors, but most calpain inhibitors are not specific for calpains or a specific calpain family member, creating more questions. The following review will discuss how calpains affect mitochondrial function and apoptosis within the cardiovascular system.

  16. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  17. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

  18. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    Science.gov (United States)

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  19. Transfer of mitochondria from astrocytes to neurons after stroke

    Science.gov (United States)

    Hayakawa, Kazuhide; Esposito, Elga; Wang, Xiaohua; Terasaki, Yasukazu; Liu, Yi; Xing, Changhong; Ji, Xunming; Lo, Eng H.

    2016-01-01

    Recently, it was suggested that neurons can release and transfer damaged mitochondria to astrocytes for disposal and recycling 1. This ability to exchange mitochondria may represent a potential mode of cell-cell signaling in the central nervous system (CNS). Here, we show that astrocytes can also release functional mitochondria that enter into neurons. Astrocytic release of extracellular mitochondria particles was mediated by a calcium-dependent mechanism involving CD38/cyclic ADP ribose signaling. Transient focal cerebral ischemia in mice induced astrocytic mitochondria entry to adjacent neurons that amplified cell survival signals. Suppression of CD38 signaling with siRNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke. PMID:27466127

  20. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    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.

  1. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  2. Cyclosporin and mitochondria: a neuroprotective approach

    Directory of Open Access Journals (Sweden)

    Alok Singh

    2013-06-01

    Full Text Available Cyclosporin A (CsA an immunophilin, discovered in 1969 and approved in 1983 to be used as immunosuppressant agent and is widely used in organ transplantation and auto-immune disorders. Its ability to alter mitochondria and apoptotic pathways makes it attractive agent to be employed in variety of diseases including age related neurodegenerative diseases. Mitochondria play pivotal role in cell energetics and reactive oxygen species production and are known to be key regulator of apoptosis hence it is important in a wide range of diseases. The structural and functional properties of mitochondria enable the targeting of drugs supposed to modulate the function of organelle for therapeutic advantage. By targeting mitochondria we can prevent oxidative damage associated with neurodegenerative diseases and ischemia and reperfusion tissue injury. Similarly targeting Bcl-2 can be helpful in cancer by triggering apoptosis. [Int J Basic Clin Pharmacol 2013; 2(3.000: 339-340

  3. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

  4. Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

    Full Text Available Abstract Background Taurolidine (TRD represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining. Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

  5. Exometabolom analysis of breast cancer cell lines: Metabolic signature.

    Science.gov (United States)

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-08-21

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

  6. 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.

  7. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are 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...

  8. Sulindac and its metabolites: sulindac sulfide and sulindac sulfone enhance cytotoxic effects of arsenic trioxide on leukemic cell lines.

    Science.gov (United States)

    Stępnik, Maciej; Ferlińska, Magdalena; Smok-Pieniążek, Anna; Gradecka-Meesters, Dobrosława; Arkusz, Joanna; Stańczyk, Małgorzata

    2011-08-01

    The effects of arsenic trioxide (ATO) in combination with sulindac (SUL), sulindac sulfide (SS) or sulindac sulfone (SF) on human (Jurkat, HL-60, K562 and HPB-ALL) and mouse (EL-4) leukemic cell lines were investigated. The cells showed different sensitivity to sulindacs (2.5-200 μM) with SS being the most cytotoxic (72 h WST-1 reduction test). The cytotoxicity of ATO was enhanced by combination with sulindacs. The combination of ATO (1 μM) with SS or SF at concentrations over 50 μM induced considerable cytotoxicity in all cell lines. Normal human lymphocytes exposed for 48 h to the combinations showed smaller decrease in viability. Measurements of Jurkat, HL-60 and K562 cells exposed to ATO (1 μM) and sulindacs (100 μM or 200 μM for K562 cells) indicated apoptosis as the main cell death mechanism. The mitochondrial membrane potential measurements (JC-1 probe) indicated an active involvement of mitochondria in the process. The results did not indicate involvement of an inhibitory effect of the combinations on NF-κB activity in Jurkat, HL-60 and K562 cells.

  9. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid.

  10. Inhibition of mitochondrial gene transcription suppresses neurotensin secretion in the human carcinoid cell line BON.

    Science.gov (United States)

    Li, Nan; Wang, Qingding; Li, Jing; Wang, Xiaofu; Hellmich, Mark R; Rajaraman, Srinivasan; Greeley, George H; Townsend, Courtney M; Evers, B Mark

    2005-02-01

    Mitochondria, organelles essential for ATP production, play a central role in a number of cellular functions, including the regulation of insulin secretion. Neurotensin (NT), an important regulatory intestinal hormone, has been implicated in fatty acid translocation, gut motility and secretion, and intestinal cell growth; however, mechanisms regulating NT secretion have not been entirely defined. The purpose of this study was to determine the effect of inhibition of mitochondrial gene transcription on NT secretion. BON cells, a novel human carcinoid cell line that produces and secretes NT peptide and expresses the gene encoding NT (designated NT/N), were treated with ethidium bromide (EB; 0.05, 0.1, and 0.4 microg/ml), an inhibitor of DNA and RNA synthesis, or vehicle over a time course (1-4 days). Cells were then stimulated with either ACh (100 microM) or phorbol 12 myristate,13-acetate (PMA, 10 nM) for 30 min. Media and cells were extracted, and NT peptide measured by RIA. Treatment with EB had no effect on BON cell viability or cell cycle distribution over the 4-day course. In contrast, EB treatment produced a dose-dependent reduction of mitochondrial gene expression; however, NT/N gene expression was not altered. Mitochondrial inhibition by EB treatment suppressed NT secretion induced by ACh and PMA, both in a dose-dependent manner. EB-mediated inhibition of NT secretion and mitochondrial gene expression was reversed with removal of EB. Our results demonstrate that inhibition of mitochondrial gene transcription suppresses both ACh- and PMA-stimulated NT release. These findings are the first to demonstrate that mitochondrial function is important for agonist-mediated NT secretion.

  11. 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.

  12. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Gualtieri, Maurizio [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Ovrevik, Johan [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Mollerup, Steen [Section for Toxicology, National Institute of Occupational Health, N-0033 Oslo (Norway); Asare, Nana [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Longhin, Eleonora [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Dahlman, Hans-Jorgen [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Camatini, Marina [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Centre Research POLARIS, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Holme, Jorn A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway)

    2011-08-01

    Highlights: {yields} PM2.5 induces mitotic arrest in BEAS-2B cells. {yields} PM2.5 induces DNA damage and activates DNA damage response. {yields} AhR regulated genes (Cyp1A1, Cyp1B1 and AhRR) are upregulated after PM exposure. {yields} 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.

  14. 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 hydroxylated as required...

  15. Antiapoptotic effect of novel compound from Herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells.

    Science.gov (United States)

    Liu, Xin Hua; Pan, Li Long; Gong, Qi Hai; Zhu, Yi Zhun

    2010-12-01

    Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H₂O₂)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H₂O₂ significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H₂O₂-stimulated cell. Moreover, H9c2 cells stimulated by H₂O₂ was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (ΔΨ(m)) and activation of caspase 3. Furthermore, H₂O₂ also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H₂O₂-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2.

  16. Mitochondria and Organismal Longevity

    OpenAIRE

    2012-01-01

    Mitochondria are essential for various biological processes including cellular energy production. The oxidative stress theory of aging proposes that mitochondria play key roles in aging by generating reactive oxygen species (ROS), which indiscriminately damage macromolecules and lead to an age-dependent decline in biological function. However, recent studies show that increased levels of ROS or inhibition of mitochondrial function can actually delay aging and increase lifespan. The aim of thi...

  17. The Rice Mitochondria Proteome and its Response During Development and to the Environment

    Directory of Open Access Journals (Sweden)

    Shaobai eHuang

    2013-02-01

    Full Text Available Rice (Oryza sativa L. is both a major crop species and the key model grass for molecular and physiological research. Mitochondria are important in rice, as in all crops, as the main source of ATP for cell maintenance and growth. However, the practical significance of understanding the function of mitochondria in rice is increased by the widespread farming practice of using hybrids to boost rice production. This relies on cytoplasmic male-sterile (CMS lines with abortive pollen caused by dysfunctional mitochondria. We provide an overview of what is known about the mitochondrial proteome of rice seedlings. To date, more than 320 proteins have been identified in purified rice mitochondria using mass spectrometry. The insights from this work include a broad understanding of the major subunits of mitochondrial respiratory complexes and TCA cycle enzymes, carbon and nitrogen metabolism enzymes as well as details of the supporting machinery for biogenesis and the subset of stress-responsive mitochondrial proteins. Many proteins with unknown functions have also been found in rice mitochondria. Proteomic analysis has also revealed the features of rice mitochondrial protein presequences required for mitochondrial targeting, as well as cleavage site features for processing of precursors after import. Changes in the abundance of rice mitochondrial proteins in response to different stresses, especially anoxia and light, are summarized. Future research on quantitative analysis of the rice mitochondrial proteomes at the spatial and developmental level, its response to environmental stresses and recent advances in understanding of basis of rice CMS systems are highlighted.

  18. Extensive fusion of mitochondria in spinal cord motor neurons.

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    Geoffrey C Owens

    Full Text Available The relative roles played by trafficking, fission and fusion in the dynamics of mitochondria in neurons have not been fully elucidated. In the present study, a slow widespread redistribution of mitochondria within cultured spinal cord motor neurons was observed as a result of extensive organelle fusion. Mitochondria were labeled with a photoconvertible fluorescent protein (mitoKaede that is red-shifted following brief irradiation with blue light. The behavior of these selectively labeled mitochondria was followed by live fluorescence imaging. Marking mitochondria within the cell soma revealed a complete mixing, within 18 hours, of these organelles with mitochondria coming from the surrounding neurites. Fusion of juxtaposed mitochondria was directly observed in neuritic processes at least 200 microns from the cell body. Within 24 hours, photoconverted mitoKaede was dispersed to all of the mitochondria in the portion of neurite under observation. When time lapse imaging over minutes was combined with long-term observation of marked mitochondria, moving organelles that traversed the field of view did not initially contain photoconverted protein, but after several hours organelles in motion contained both fluorescent proteins, coincident with widespread fusion of all of the mitochondria within the length of neurite under observation. These observations suggest that there is a widespread exchange of mitochondrial components throughout a neuron as a result of organelle fusion.

  19. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  20. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 > 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.

  2. Bufalin Induces Apoptosis of Human Osteosarcoma U-2 OS Cells through Endoplasmic Reticulum Stress, Caspase- and Mitochondria-Dependent Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Ching-Hsiao Lee

    2017-03-01

    Full Text Available Bone cancer is one of the cancer-related diseases, and there are increased numbers of patients with bone cancer worldwide. Therefore the efficacy of treatment of bone cancer is considered extremely vital. Bufalin has been showed to have biological activities including anticancer activities in vitro and in vivo. However, the exact associated mechanisms for bufalin induced apoptosis in human bone cancer cells are still unclear. In the present study, we investigated the effect of bufalin on the cytotoxic effects in U-2 OS human osteosarcoma cells. For examining apoptotic cell deaths, we used flow cytometry assay, Annexin V/PI double staining, and TUNNEL assay. Reactive oxygen species (ROS, Ca2+, mitochondrial membrane potential (ΔΨm, and caspase-8, -9 and -3 activities were measured by flow cytometry assay. Furthermore, western blotting and a confocal laser microscopy examination were used for measuring the alterations of apoptotic associated protein expression and translocation, respectively. The results indicated that bufalin induced cell morphological changes, decreased the viable cell number, induced apoptotic cell death, and increased the apoptotic cell number, and affected apoptotic associated protein expression in U-2 OS cells. Bufalin increased apoptotic proteins such as Bak, and decreased anti-apoptotic proteins such as Bcl-2 and Bcl-x in U-2 OS cells. Furthermore, bufalin increased the protein levels of cytochrome c (Cyto c, AIF (Apoptosis inducing factor and Endo G (Endonuclease G in cytoplasm that were also confirmed by confocal microscopy examination. Based on those findings, bufalin induced apoptotic cell death in U-2 OS cells may be via endoplasmic reticulum (ER stress, caspase-, and mitochondria-dependent pathways; thus, we may suggest that bufalin could be used as an anti-cancer agent for the treatment of osteosarcoma in the future, and further in vivo studies are needed.

  3. Derivation of human embryonic stem cell line Genea019

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    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.

  4. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  5. 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.

  6. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  7. Differential production of superoxide by neuronal mitochondria

    Directory of Open Access Journals (Sweden)

    Levin Leonard A

    2008-01-01

    Full Text Available Abstract Background Mitochondrial DNA (mtDNA mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs, a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells. Results In the presence of the complex I substrate glutamate/malate or the complex II substrate succinate, the rate of superoxide production in RGC-5 cells was significantly lower than cerebral or neuroblastoma cells. Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A. Immunoblotting and real-time quantitative PCR of METC components demonstrated different patterns of expression among the three different sources of neuronal mitochondria. Conclusion RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components. Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

  8. Reconstruction of endometrium from human endometrial side population cell lines.

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    Irene Cervelló

    Full Text Available Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC population recently identified by several groups using the side population (SP technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP cell lines (ICE 1-7: four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3 and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN. Phenotype analysis corroborated their epithelial (CD9+ or stromal (vimentin+ cell origin and mesenchymal (CD90+, CD73+ and CD45⁻ attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα or progesterone receptor (PR. The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis.

  9. 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.

  10. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

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

    Science.gov (United States)

    Gualtieri, Maurizio; Ovrevik, Johan; Mollerup, Steen; Asare, Nana; Longhin, Eleonora; Dahlman, Hans-Jørgen; Camatini, Marina; Holme, Jørn A

    2011-08-01

    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.

  12. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  13. Do we age because we have mitochondria?

    Science.gov (United States)

    Bereiter-Hahn, Jürgen

    2014-01-01

    The process of aging remains a great riddle. Production of reactive oxygen species (ROS) by mitochondria is an inevitable by-product of respiration, which has led to a hypothesis proposing the oxidative impairment of mitochondrial components (e.g., mtDNA, proteins, lipids) that initiates a vicious cycle of dysfunctional respiratory complexes producing more ROS, which again impairs function. This does not exclude other processes acting in parallel or targets for ROS action in other organelles than mitochondria. Given that aging is defined as the process leading to death, the role of mitochondria-based impairments in those organ systems responsible for human death (e.g., the cardiovascular system, cerebral dysfunction, and cancer) is described within the context of "garbage" accumulation and increasing insulin resistance, type 2 diabetes, and glycation of proteins. Mitochondrial mass, fusion, and fission are important factors in coping with impaired function. Both biogenesis of mitochondria and their degradation are important regulatory mechanisms stimulated by physical exercise and contribute to healthy aging. The hypothesis of mitochondria-related aging should be revised to account for the limitations of the degradative capacity of the lysosomal system. The processes involved in mitochondria-based impairments are very similar across a large range of organisms. Therefore, studies on model organisms from yeast, fungi, nematodes, flies to vertebrates, and from cells to organisms also add considerably to the understanding of human aging.

  14. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members o...

  15. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  16. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    Science.gov (United States)

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

  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. 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.

  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. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  1. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  2. Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells.

    Science.gov (United States)

    Song, Xinxin; Kim, Han-Cheon; Kim, Seog-Young; Basse, Per; Park, Bae-Hang; Lee, Byeong-Chel; Lee, Yong J

    2012-05-01

    Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.

  3. Mechanism(s of Toxic Action of Zn2+ and Selenite: A Study on AS-30D Hepatoma Cells and Isolated Mitochondria

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    Elena A. Belyaeva

    2011-01-01

    Full Text Available Mitochondria of AS-30D rat ascites hepatoma cells are found to be the main target for Zn2+ and sodium selenite (Na2SeO3. High [mu]M concentrations of Zn2+ or selenite were strongly cytotoxic, killing the AS-30D cells by both apoptotic and necrotic ways. Both Zn2+ and selenite produced strong changes in intracellular generation of reactive oxygen species (ROS and the mitochondrial dysfunction via the mitochondrial electron transport chain (mtETC disturbance, the membrane potential dissipation, and the mitochondrial permeability transition pore opening. The significant distinctions in toxic action of Zn2+ and selenite on AS-30D cells were found. Selenite induced a much higher intracellular ROS level (the early event compared to Zn2+ but a lower membrane potential loss and a lower decrease of the uncoupled respiration rate of the cells, whereas the mtETC disturbance was the early and critical event in the mechanism of Zn2+ cytotoxicity. Sequences of events manifested in the mitochondrial dysfunction produced by the metal/metalloid under test are compared with those obtained earlier for Cd2+, Hg2+, and Cu2+ on the same model system.

  4. Terpenoids Isolated From the Shoot of Plectranthus hadiensis Induces Apoptosis in Human Colon Cancer Cells Via the Mitochondria-Dependent Pathway.

    Science.gov (United States)

    Menon, Darsan B; Gopalakrishnan, V K

    2015-01-01

    The plant Plectranthus hadiensis is a rich source of many bioactive phytochemicals, especially terpenoids. The terpenoid fraction was isolated and phytochemical characterization was done using GC-MS. The aim of the present study was to find out the antiproliferative activity and the mechanism of cell death induction by the terpenoid fraction on human colon cancer cells (HCT-15). MTT assay was performed with different concentrations of the fraction (10, 20, and 50 µg/mL) to obtain IC50 value for 24 h to induce cell death. The induction of apoptosis were studied by Hoechst staining, acridine orange/ethidium bromide staining, Comet assay, DNA fragmentation, and caspase-3 activity assays. The mechanism of apoptosis induction was studied by expression analysis of antiapoptotic Bcl-2 and proapoptotic Bax using RT-PCR and also by Western blot analysis of proteins involved in the apoptotic pathway. The terpenoid fraction induced significant morphological changes and DNA fragmentation in the cells. Positive Hoechst staining and acridine orange/ethidium bromide staining indicated apoptosis induction by the fraction. DNA fragmentation, which is a characteristic feature of apoptosis, was also observed. Upregulation of caspase-3 activity and proapoptotic Bax, and the downregulation of antiapoptotic Bcl-2 and COX-2 confirmed that the apoptosis induction was via the mitochondria-dependent pathway.

  5. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  6. 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.

  7. 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.

  8. Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC.

    Science.gov (United States)

    Che, Ting-Fang; Lin, Ching-Wen; Wu, Yi-Ying; Chen, Yu-Ju; Han, Chia-Li; Chang, Yih-leong; Wu, Chen-Tu; Hsiao, Tzu-Hung; Hong, Tse-Ming; Yang, Pan-Chyr

    2015-11-10

    Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo. Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

  9. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  10. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  11. 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.

  12. Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi

    2015-05-01

    Full Text Available Background/Aims: Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs, has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT, an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

  13. Different susceptibility of colon cancer DLD-1 and LOVO cell lines to apoptosis induced by DMU-212, a synthetic resveratrol analogue.

    Science.gov (United States)

    Piotrowska, Hanna; Myszkowski, Krzysztof; Amarowicz, Ryszard; Murias, Marek; Kulcenty, Katarzyna; Wierzchowski, Marcin; Jodynis-Liebert, Jadwiga

    2013-12-01

    The cytotoxic activity of DMU-212 has been shown to vary in cell lines derived from the same type of cancer, i.e. ovarian, breast and colorectal ones. However, the molecular mechanism of DMU-212 cytotoxicity has not been clarified in colon cancer cells. This study aims to elucidate the mechanism of antitumor effects of DMU-212 in two human colon cancer cell lines, DLD-1 and LOVO. We showed the stronger cytotoxic activity in DLD-1 cells in which DMU-212 evoked a greater pro-apoptotic effect as compared to that of LOVO cells. The analysis of the expression pattern of 84 apoptosis-related genes indicated transcripts specific to the mitochondria-mediated apoptosis pathway in both colon cancer cell lines used. We found that DMU-212 caused up-regulation of pro-apoptotic Bak1, Bok, Bik, Noxa, Bad, Bax, p53 and Apaf1 transcripts level in DLD-1 cell line, whereas anti-apoptotic Bcl-2, Bcl-xL and Bag1 mRNA expression was decreased. Changes in apoptosis-related genes expression were less pronounced in LOVO cells which did not express CYP1B1 protein and showed lower expression of CYP1A1 protein level than that in DLD-1 cells. Our results suggest that anticancer activity of DMU-212 is closely related to its biotransformation catalysed by these cytochrome P450 isoenzymes.

  14. 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.

  15. 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).

  16. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  17. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies.

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  18. Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

    Science.gov (United States)

    Pessôa, L V F; Bressan, F F; Chiaratti, M R; Pires, P R L; Perecin, F; Smith, L C; Meirelles, F V

    2015-10-30

    Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

  19. Protein trafficking at the crossroads to mitochondria.

    Science.gov (United States)

    Wasilewski, Michal; Chojnacka, Katarzyna; Chacinska, Agnieszka

    2017-01-01

    Mitochondria are central power stations in the cell, which additionally serve as metabolic hubs for a plethora of anabolic and catabolic processes. The sustained function of mitochondria requires the precisely controlled biogenesis and expression coordination of proteins that originate from the nuclear and mitochondrial genomes. Accuracy of targeting, transport and assembly of mitochondrial proteins is also needed to avoid deleterious effects on protein homeostasis in the cell. Checkpoints of mitochondrial protein transport can serve as signals that provide information about the functional status of the organelles. In this review, we summarize recent advances in our understanding of mitochondrial protein transport and discuss examples that involve communication with the nucleus and cytosol.

  20. 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.

  1. Effects of cadmium chloride on some mitochondria-related activity and gene expression of human MDA-MB231 breast tumor cells.

    Science.gov (United States)

    Cannino, Giuseppe; Ferruggia, Elisa; Luparello, Claudio; Rinaldi, Anna Maria

    2008-08-01

    It was reported that cadmium is able to exert a cytotoxic effect on tumor MDA-MB231 cells, which shows signs of "non-classical" apoptosis and is characterized by drastic changes in gene expression pattern. In this study, we have extended our knowledge of metal-breast cancer cell interactions by analyzing some mitochondria-related aspects of the stress response to CdCl(2) at either 5 or 50 microM 24- or 96-h exposure, by cytochemical, conventional PCR and Northern/Western blot techniques. We demonstrated that (i) no modification of the mitochondrial mass was detectable due to CdCl(2) exposure; (ii) the respiration activity appeared to be increased after 96-h exposures, while the production of reactive oxygen species was significantly induced, as well; (iii) hsp60, hsp70, COXII and COXIV expressions were dependent on the duration of Cd exposure; (iv) a different hsp60 protein distribution was observed in mitochondrial and post-mitochondrial extracts and (v) 96-h exposure induced the over-expression of hsc/hsp70 proteins and, conversely, the down-regulation of cytochrome oxidase subunits II and IV. These observations, in addition to providing more information on the cellular and molecular aspects of the interaction between CdCl(2) and MDA-MB231 breast tumor cells, contribute to the comprehension of the intracellular molecular mechanisms implicated in the regulation of some mitochondrial proteins.

  2. 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.

  3. 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.

  4. Mitochondria in biology and medicine--2012

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Rasmussen, Lene Juel

    2014-01-01

    As the understanding of mitochondria and their importance for the cell and organism is developing, increasing evidence is demonstrating the organelle to be intricately involved in an extensive range of pathologies. This range of pathologies include general signs of premature aging, neuro-muscular......As the understanding of mitochondria and their importance for the cell and organism is developing, increasing evidence is demonstrating the organelle to be intricately involved in an extensive range of pathologies. This range of pathologies include general signs of premature aging, neuro...... as biomarkers for the diseases and most important, it opens the possibility of a treatment or a cure for a disease. "Mitochondria in Biology and Medicine" was the title of the second annual conference of Society of Mitochondrial Research and Medicine-India. The conference was organized by Rana P. Singh, Keshav...

  5. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  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. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  8. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  9. 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

    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...

  10. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  11. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  12. 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.

  13. Mitochondria-endoplasmic reticulum choreography: structure and signaling dynamics.

    Science.gov (United States)

    Pizzo, Paola; Pozzan, Tullio

    2007-10-01

    Mitochondria and endoplasmic reticulum (ER) have different roles in living cells but they interact both physically and functionally. A key aspect of the mitochondria-ER relationship is the modulation of Ca(2+) signaling during cell activation, which thus affects a variety of physiological processes. We focus here on the molecular aspects that control the dynamics of the organelle-organelle interaction and their relationship with Ca(2+) signals, also discussing the consequences that these phenomena have, not only for cell physiology but also in the control of cell death.

  14. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  15. 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.

  16. THP-1 cell line: an in vitro cell model for immune-modulation approach : Review

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  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. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 μM; 48 h IC50: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 μM; 48 h IC50: 17 μM). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7-123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

  19. Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

    Science.gov (United States)

    Gao, Shaorong; McGarry, Michelle; Ferrier, Tricia; Pallante, Benedetta; Priddle, Helen; Gasparrini, Bianca; Fletcher, Judy; Harkness, Linda; De Sousa, Paul; McWhir, Jim; Wilmut, Ian

    2003-02-01

    Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

  20. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  1. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  2. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  3. BRITER: a BMP responsive osteoblast reporter cell line.

    Directory of Open Access Journals (Sweden)

    Prem Swaroop Yadav

    Full Text Available BACKGROUND: BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. METHODOLOGY/PRINCIPAL FINDINGS: We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line, responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. CONCLUSION: As the dynamic range of the assay (for BMP responsiveness is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance.

  4. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Goestring, Lovisa [Affibody AB, Bromma (Sweden); Palm, Stig [Sahlgrenska Academy at Goeteborg University, Department of Radiation Physics, Goeteborg (Sweden); Carlsson, Joergen [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Rudbeck Laboratory, Biomedical Radiation Sciences, Uppsala (Sweden)

    2008-06-15

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin {sup registered} treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from {sup 211}At decays using the HER2-binding affibody molecule {sup 211}At-(Z{sub HER2:4}){sub 2} as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of {sup 211}At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from {sup 211}At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  5. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  6. 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.

  7. Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 cell line.

    Directory of Open Access Journals (Sweden)

    Rame Taha

    Full Text Available BACKGROUND: Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. METHODOLOGY/PRINCIPAL FINDINGS: The objective of the present work was to evaluate how iron-ascorbate (FE/ASC-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM (1 increased malondialdehyde levels assessed by HPLC; (2 reduced ATP production noted by luminescence assay; (3 provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4 upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5 affected mitochondrial respiratory chain complexes I, II, III and IV; (6 elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7 lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8 altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2 without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.

  8. Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile.

    Directory of Open Access Journals (Sweden)

    Yin Sim Tor

    Full Text Available Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05. DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05. The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide

  9. 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.

  10. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

  11. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  12. BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa.

    Science.gov (United States)

    Akazawa, Yuko; Matsuda, Katsuya; Isomoto, Hajime; Matsushima, Kayoko; Kido, Yoko; Urabe, Shigetoshi; Yamaghchi, Naoyuki; Ohnita, Ken; Takeshima, Fuminao; Kondo, Hisayoshi; Tsugawa, Hitoshi; Suzuki, Hidekazu; Moss, Joel; Nakao, Kazuhiko; Nakashima, Masahiro

    2015-09-01

    BH3-only protein, Bim, is a pro-apoptotic protein that mediates mitochondria-dependent cell death. However, the role of Bim in Helicobacter pylori-associated gastritis remains unclear. This study aimed to assess the cellular localization of Bim and its possible role in H. pylori-induced gastritis. The study was conducted on biopsy specimens obtained from 80 patients who underwent upper gastrointestinal endoscopy (H. pylori-negative: n=30, positive: n=50). Association between Bim mRNA expression and severity of gastritis was evaluated and the localization of Bim was examined by immunofluorescence. Bim mRNA expression was positively correlated with the degree of gastritis, as defined by the Sydney system. Immunohistochemical analysis confirmed increased Bim expression in H. pylori-infected gastric mucosa compared with uninfected mucosa in both humans and mice. Bim localized in myeloperoxidase- and CD138-positive cells of H. pylori-infected lamina propria and submucosa of the gastric tract, indicating that this protein is predominantly expressed in neutrophils and plasma cells. In contrast, Bim did not localize in CD20-, CD3-, or CD68-positive cells. Bim was expressed in the mitochondria, where it was partially co-localized with activated Bax and cleaved-PARP. In conclusion, Bim is expressed in neutrophils and plasma cells in H. pylori-associated gastritis, where it may participate in the termination of inflammatory response by causing mitochondria-mediated apoptosis in specific leucocytes.

  13. Direct electric current treatment modifies mitochondrial function and lipid body content in the A549 cancer cell line.

    Science.gov (United States)

    Holandino, Carla; Teixeira, Cesar Augusto Antunes; de Oliveira, Felipe Alves Gomes; Barbosa, Gleyce Moreno; Siqueira, Camila Monteiro; Messeder, Douglas Jardim; de Aguiar, Fernanda Silva; da Veiga, Venicio Feo; Girard-Dias, Wendell; Miranda, Kildare; Galina, Antonio; Capella, Marcia Alves Marques; Morales, Marcelo Marcos

    2016-10-01

    Electrochemical therapy (EChT) entails treatment of solid tumors with direct electric current (DC). This work evaluated the specific effects of anodic flow generated by DC on biochemical and metabolic features of the A549 human lung cancer cell line. Apoptosis was evaluated on the basis of caspase-3 activity and mitochondrial transmembrane potential dissipation. Cell morphology was analyzed using transmission electron microscopy, and lipid droplets were studied through morphometric analysis and X-ray qualitative elemental microanalysis. High-resolution respirometry was used to assess mitochondrial respiratory parameters. Results indicated A549 viability decreased in a dose-dependent manner with a prominent drop between 18 and 24h after treatment (p<0.001), together with a two-fold increase in caspase-3 activity. AF-treatment induced a significantly increase (p<0.01) in the cell number with disrupted mitochondrial transmembrane potential. Furthermore, treated cells demonstrated important ultrastructural mitochondria damage and a three-fold increase in the cytoplasmic lipid bodies' number, quantified by morphometrical analyses. Conversely, 24h after treatment, the cells presented a two-fold increase of residual oxygen consumption, accounting for 45.3% of basal oxygen consumption. These results show remarkable alterations promoted by anodic flow on human lung cancer cells which are possibly involved with the antitumoral effects of EChT.

  14. Correlation between Twist expression and multidrug resistance of breast cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Yue-Xi Wang; Xiao-Mei Chen; Jun Yan; Zhi-Ping Li

    2016-01-01

    Objective:To study the correlation between Twist expression and multidrug resistance of breast cancer cell lines. Methods:Human breast cancer cell lines MCF-7, cisplatin-resistant human breast cancer cell lines MCF-7/DDP, doxorubicin-resistant human breast cancer cell lines MCF-7/Adr and taxol-resistant human breast cancer cell lines MCF/PTX were cultured, Twist in human breast cancer cell lines MCF-7 was overexpressed and treated with doxorubicin, and then cell viability and expression levels of EMT marker molecules and related signaling pathway molecules were detected. Results:mRNA contents and protein contents of Twist in drug-resistant breast cancer cell lines MCF-7/DDP, MCF-7/Adr and MCF/PTX were higher than those in MCF-7 cell lines;after doxorubicin treatment, inhibitory rates of cell viability in MCF-7 cell lines were higher than those in MCF-7/Adr and MCF-7/Twist cell lines;E-cadherin expression levels in MCF-7/Adr cell lines and MCF-7/Twist cell lines were lower than those in MCF-7 cell lines, and mRNA contents and protein contents of N-cadherin, Vimentin, TGF-β, Smad, Wnt,β-catenin, TNF-αand NF-kB were higher than those in MCF-7 cell lines. Conclusion:Increased expression of Twist is associated with the occurrence of drug resistance in breast cancer cells.

  15. 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.

  16. "Helicobacter Pylori Attachment To 7 Mamalian Cell Lines "

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard

    2006-06-01

    Full Text Available Background and Aim: Helicobacter pylori is the etiologic agent of chronic –active gastritis, gastroduodenal ulcers in humans, and a co-factor in the occurrence of gastric cancer and mucosa-associated lymphoid tumors, Adhesion of H.pylori to the gastric mucosa is a critical and also initial step in the pathogenesis of the disease. Bacterial adhesion inhibitory agents provide a novel pharmacologic approach to the management of infectious diseases. Materials and Methods: 22 H. pylori strains, isolated from the antral biopsies of 49 patients with dyspepsia, gastritis, gastric ulcer, duodenal ulcer,…were assayed by ELISA (UPRto investigate the diversity of attachment to 7 mamalian cell lines. Results: The concentration of H.pylori and cell suspention ,the condition and temperature, can alter the attachment rate.Best bacterial concentration was equal to 1 Mc farland,and for cell suspension was 5*10 cells/ml.90 minutes in 37C incubation period result in maximum attachment. H.pylori can attach to all 7 cell lines, there are no significant differences between 22 H.pylori strains in attachment to cells. The attachment pattern of H.pylori to the cells showed significant reduction respectly from HepII, HeLa, SW742, AGS,HT29/219, HT29 to Caco-2.Maximum attachment were seen to HepII, HeLa and SW742 cells, and among these HepII was the best cells for this purpose. Conclusion: Our studies suggest that Hep II, HeLa and SW742 cells could serve as a suitable in-vitro model for the study of H.pylori adhesions, attachment, inhibition of attachment and detachment assays and among these Hep II cell is prefer recommended.

  17. 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

    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...

  18. 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.

  19. 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,...

  20. Isolation of Mitochondria from Potato Tubers

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Salvato, Fernanda; Chen, Mingjie;

    2014-01-01

    the in 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...

  1. 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.

  2. 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.

  3. Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Engelholm, S A; Vindeløv, L L; Spang-Thomsen, M

    1985-01-01

    Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with differ......Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...

  4. 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...

  5. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  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. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  8. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  9. Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: differences in mitochondria.

    Science.gov (United States)

    Harada, K; Endo, Y; Warashina, A; Inoue, M

    2015-08-20

    The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized.

  10. 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.

  11. 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.

  12. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    Directory of Open Access Journals (Sweden)

    Edyta Biskup

    2010-05-01

    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

  13. 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