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Sample records for apoptosis limits dna

  1. DNA fragmentation in apoptosis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

  2. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  3. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  4. Bacterial DNA delays human eosinophil apoptosis

    OpenAIRE

    Ilmarinen, Pinja; Hasala, Hannele; Sareila, Outi; Moilanen, Eeva; Kankaanranta, Hannu

    2009-01-01

    Bacterial DNA delays human eosinophil apoptosis correspondance: Corresponding author. Tel.: +358 3 3551 6687; fax: +358 3 3551 8082. (Ilmarinen, Pinja) (Ilmarinen, Pinja) The Immunopharmacology Research Group--> , Medical School--> , University of Tampere and Research Unit--> , Tampere University Hospital--> , Tampere--> - FINLAND (Ilmarinen, Pinja) The Immunopharmacology ...

  5. DNA vaccines and apoptosis: to kill or not to kill?

    OpenAIRE

    Leitner, Wolfgang W.; Restifo, Nicholas P

    2003-01-01

    The apoptotic machinery has become the latest target of vaccinologists attempting to improve the efficacy of DNA vaccines. While workers have previously sought to induce apoptotic death in transfected DCs as a means to activate immunity, a new approach (see related article on pages 109–117) instead seeks to delay apoptosis in host DCs after DNA vaccination.

  6. Apoptosis and DNA damage in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Adam J Koppers

    2011-01-01

    DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis,resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

  7. Small DNA pieces in C. elegans are intermediates of DNA fragmentation during apoptosis.

    Directory of Open Access Journals (Sweden)

    P Joseph Aruscavage

    Full Text Available While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, approximately 10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3' phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway.

  8. DNA-damage response network at the crossroads of cell-cycle checkpoints,cellular senescence and apoptosis

    Institute of Scientific and Technical Information of China (English)

    SCHMITT Estelle; PAQUET Claudie; BEAUCHEMIN Myriam; BERTRAND Richard

    2007-01-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation,cellular senescence and cell death.Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities.Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms.Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death.The intimate link between the cell cycle,cellular senescence,apoptosis regulation,cancer development and tumor responses to cancer treatment has become eminently apparent.Extensive research on tumor suppressor genes,oncogenes,the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways,referred to as the DNA-damage response network,are tied to cell proliferation,cell-cycle arrest,cellular senescence and apoptosis.DNA-damage responses are complex,involving "sensor" proteins that sense the damage,and transmit signals to "transducer" proteins,which,in turn,convey the signals to numerous "effector" proteins implicated in specific cellular pathways,including DNA repair mechanisms,cell-cycle checkpoints,cellular senescence and apoptosis.The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation.In addition,several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle,DNA repair/recombination and cellular senescence,effects that are generally distinct from their function in apoptosis.In this review,we report progress in understanding the molecular networks that regulate cell-cycle checkpoints,cellular senescence and apoptosis after DNA damage,and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

  9. FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress.

    Science.gov (United States)

    Jeong, Yeon-Tae; Rossi, Mario; Cermak, Lukas; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Sung, Patrick; Schildkraut, Carl L; Schildkraut, Carl; Pagano, Michele

    2013-01-21

    Proper resolution of stalled replication forks is essential for genome stability. Purification of FBH1, a UvrD DNA helicase, identified a physical interaction with replication protein A (RPA), the major cellular single-stranded DNA (ssDNA)-binding protein complex. Compared with control cells, FBH1-depleted cells responded to replication stress with considerably fewer double-strand breaks (DSBs), a dramatic reduction in the activation of ATM and DNA-PK and phosphorylation of RPA2 and p53, and a significantly increased rate of survival. A minor decrease in ssDNA levels was also observed. All these phenotypes were rescued by wild-type FBH1, but not a FBH1 mutant lacking helicase activity. FBH1 depletion had no effect on other forms of genotoxic stress in which DSBs form by means that do not require ssDNA intermediates. In response to catastrophic genotoxic stress, apoptosis prevents the persistence and propagation of DNA lesions. Our findings show that FBH1 helicase activity is required for the efficient induction of DSBs and apoptosis specifically in response to DNA replication stress. PMID:23319600

  10. Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells.

    Science.gov (United States)

    Chen, Tzu-Hsuan; Pan, Shiow-Lin; Guh, Jih-Hwa; Chen, Chien-Chih; Huang, Yao-Ting; Pai, Hui-Chen; Teng, Che-Ming

    2008-11-01

    Denbinobin is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla. We showed that denbinobin induces apoptosis in human colorectal cancer cells (HCT-116) in a concentration-dependent manner. The addition of a pan-caspase inhibitor (zVAD-fmk) did not suppress the denbinobin-induced apoptotic effect, and denbinobin-induced apoptosis was not accompanied by processing of procaspase-3, -6, -7, -9, and -8. However, denbinobin triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Small interfering RNA targeting of AIF effectively protected HCT-116 cells against denbinobin-induced apoptosis. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors (p21WAF1/CIP1, Bax, PUMA, and NOXA). A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anticancer agent. PMID:18607570

  11. DNA Methylation and Apoptosis Resistance in Cancer Cells

    OpenAIRE

    Pierre-François Cartron; François Marie Vallette; Eric Hervouet; Mathilde Cheray

    2013-01-01

    Apoptosis is a cell death programme primordial to cellular homeostasis efficiency. This normal cell suicide program is the result of the activation of a cascade of events in response to death stimuli. Apoptosis occurs in normal cells to maintain a balance between cell proliferation and cell death. A deregulation of this balance due to modifications in the apoptosic pathway leads to different human diseases including cancers. Apoptosis resistance is one of the most important hallmarks of cance...

  12. Molecular Regulation of DNA Damage-Induced Apoptosis in Neurons of Cerebral Cortex

    OpenAIRE

    Martin, Lee J.; Liu, Zhiping; Pipino, Jacqueline; Chestnut, Barry; Landek, Melissa A.

    2008-01-01

    Cerebral cortical neuron degeneration occurs in brain disorders manifesting throughout life, but the mechanisms are understood poorly. We used cultured embryonic mouse cortical neurons and an in vivo mouse model to study mechanisms of DNA damaged-induced apoptosis in immature and differentiated neurons. p53 drives apoptosis of immature and differentiated cortical neurons through its rapid and prominent activation stimulated by DNA strand breaks induced by topoisomerase-I and -II inhibition. B...

  13. Sophoridinol derivative 05D induces tumor cells apoptosis by topoisomerase1-mediated DNA breakage

    Directory of Open Access Journals (Sweden)

    Zhao W

    2016-05-01

    Full Text Available Wuli Zhao, Caixia Zhang, Chongwen Bi, Cheng Ye, Danqing Song, Xiujun Liu, Rongguang Shao Key Laboratory of Antibiotic Bioengineering, Ministry of Health, Laboratory of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People’s Republic of China Abstract: Sophoridine is a quinolizidine natural product of Sophora alopecuroides and has been applied for treatment of malignant trophoblastic tumors. Although characterized by low toxicity, the limited-spectrum antitumor activity hinders its further applications. 05D, a derivative of sophoridine, exhibits a better anticancer activity on diverse cancer cells, including solid tumors, and hematologic malignancy. It could inhibit topoisomerase 1 (top1 activity by stabilizing DNA–top1 complex and induce mitochondria-mediated apoptosis by promoting DNA single- and double-strand breakage mediated by top1. Also, 05D induced HCT116 cells arrest at G1 phase by inactivating CDK2/CDK4–Rb–E2F and cyclinD1–CDK4–p21 checkpoint signal pathways. 05D suppressed the ataxia telangiectasia mutated (ATM and ATM and Rad3-related (ATR activation and decreased 53BP level, which contributed to DNA damage repair, suggesting that the novel compound 05D might be helpful to improve the antitumor activity of DNA damaging agent by repressing ATM and ATR activation and 53BP level. In addition, the priorities in molecular traits and druggability, such as a simple structure and formulation for oral administration, further prove 05D to be a promising targeting topoisomerase agent. Keywords: topoisomerase inhibitor, topoisomerase 1, DNA breakage, sophoridinol, anticancer, apoptosis, cell cycle

  14. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    Directory of Open Access Journals (Sweden)

    Seok-Jo Kim

    2015-09-01

    Full Text Available Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC programmed cell death (apoptosis that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF and asbestosis (pulmonary fibrosis following asbestos exposure. The mammalian mitochondrial DNA (mtDNA encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1 and mitochondrial aconitase (ACO-2 in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

  15. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis.

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Williams, David B; Kamp, David W

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

  16. The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway.

    NARCIS (Netherlands)

    X.W. Wang (Xin Wei); W. Vermeulen (Wim); J.D. Coursen; M.K. Gibson (Michael); S.E. Lupold; K. Forrester; G. Xu; L. Elmore; H. Yeh; J.H.J. Hoeijmakers (Jan); C.C. Harris

    1996-01-01

    textabstractThe molecular pathway of p53-dependent apoptosis (programmed cell death) is poorly understood. Because p53 binds to the basal transcription-repair complex TFIIH and modulates its DNA helicase activities, we hypothesized that TFIIH DNA helicases XPB and XPD are members of the p53-mediated

  17. Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    AI-GUO WANG; TAO XIA; QI-LONG CHU; MING ZHANG; FANG LIU; XUE-MIN CHEN; KE-DI YANG

    2004-01-01

    Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

  18. Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

    Directory of Open Access Journals (Sweden)

    Rodriguez-Gallego Carlos

    2010-09-01

    Full Text Available Abstract Background DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp. Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p Conclusions An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

  19. Treg cell resistance to apoptosis in DNA vaccination for experimental autoimmune encephalomyelitis treatment.

    Directory of Open Access Journals (Sweden)

    Youmin Kang

    Full Text Available BACKGROUND: Regulatory T (Treg cells can be induced with DNA vaccinations and protect mice from the development of experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis (MS. Tacrolimus (FK506 has been shown to have functions on inducing immunosuppression and augmenting apoptosis of pathologic T cells in autoimmune disease. Here we examined the therapeutic effect of DNA vaccine in conjunction with FK506 on EAE. METHODOLOGY/PRINCIPAL FINDINGS: After EAE induction, C57BL/6 mice were treated with DNA vaccine in conjunction with FK506. Functional Treg cells were induced in treated EAE mice and suppressed Th1 and Th17 cell responses. Infiltrated CD4 T cells were reduced while Treg cells were induced in spinal cords of treated EAE mice. Remarkably, the activated CD4 T cells augmented apoptosis, but the induced Treg cells resisted apoptosis in treated EAE mice, resulting in alleviation of clinical EAE severity. CONCLUSIONS/SIGNIFICANCE: DNA vaccine in conjunction with FK506 treatment ameliorates EAE by enhancing apoptosis of CD4 T cells and resisting apoptosis of induced Treg cells. Our findings implicate the potential of tolerogenic DNA vaccines for treating MS.

  20. Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage

    DEFF Research Database (Denmark)

    Savorani, Cecilia; Manfé, Valentina; Biskup, Edyta;

    2015-01-01

    (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53(wt/wt)), SeAx ((G245S)p53) and Hut-78 ((R196Stop)p53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored...... the transcriptional activity of (G245S)p53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage....... Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma....

  1. Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

    International Nuclear Information System (INIS)

    DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity

  2. DNA Damage, Apoptosis and Langerhans cells – Activators of UV-induced Immune Tolerance

    OpenAIRE

    Timares, Laura; Katiyar, Santosh; Elmets, Craig A.

    2008-01-01

    Solar ultraviolet radiation (UVR) is highly mutagenic but is only partially absorbed by the outer stratum corneum of the epidermis. UVR can penetrate into the deeper layers of the epidermis, depending on melanin content, where it induces DNA damage and apoptosis in epidermal cells, including those in the germinative basal layer. The cellular decision to initiate either the cellular repair processes or undergo apoptosis has evolved to balance the acute need to maintain skin barrier function wi...

  3. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    OpenAIRE

    Seok-Jo Kim; Paul Cheresh; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidenc...

  4. XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

    OpenAIRE

    Kaushik Tiwari, Meetu; Rogers, Faye A.

    2013-01-01

    DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role i...

  5. Regulation of Ceramide Synthase-Mediated Crypt Epithelium Apoptosis by DNA Damage Repair Enzymes

    Science.gov (United States)

    Rotolo, Jimmy A.; Mesicek, Judith; Maj, Jerzy; Truman, Jean-Philip; Haimovitz-Friedman, Adriana; Kolesnick, Richard; Fuks, Zvi

    2015-01-01

    Acute endothelial cell apoptosis and microvascular compromise couple GI tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via ceramide synthase (CS)-stimulated de novo synthesis of the pro-apoptotic sphingolipid ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ATM deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here we show that inactivation of DNA damage repair pathways signal CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double strand breaks (DSBs). Genetic loss-of-function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11ATLD1/ATLD1 and C57BL/6Prkdc/SCID (SCID) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50S/S mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target re-ordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable. PMID:20086180

  6. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2016-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects. PMID:26703663

  7. G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

    Science.gov (United States)

    Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

    2016-10-01

    The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

  8. Umbelliferone suppresses radiation induced DNA damage and apoptosis in hematopoietic cells of mice

    International Nuclear Information System (INIS)

    Radiotherapy is one of the major modes of treatment for different types of cancers. But the success of radiotherapy is limited by injury to the normal cells. Protection of the normal cells from radiation damage by radioprotectors can increase therapeutic efficiency. These radioprotectors can also be used during nuclear emergency situations. Umbelliferone (UMB) is a wide spread natural product of the coumarin family. It occurs in many plants from the Apiaceae family. In the present study radioprotective effect of UMB was investigated in vitro and in vivo. Anti genotoxic effect of Umbelliferone was tested by treating the splenic lymphocytes with various doses of UMB (6.5 μM - 50 μM) prior to radiation (6Gy) exposure. After the radiation exposure, extent of DNA damage was assessed by comet assay at 5 mm and two hours after radiation exposure. At both the time points, it was observed that the pretreatment of UMB reduced the radiation induced DNA damage to a significant extent in comparison to radiation control. UMB pretreatment also significantly reduced the radiation induced apoptosis enumerated by propidium iodide staining assay. Results of clonogenic survival assay using intestinal cell line showed that pretreatment with UMB significantly protected against radiation induced loss of colony forming units. To assess the anti genotoxic role of umbelliferone in vivo two different doses of UMB (20 mg/Kg and 40 mg/Kg of body weight) were injected into Swiss mice or with vehicle and exposed to radiation. Thirty minutes after the radiation comet assay was performed in peripheral leukocytes. Frequency of micro nucleated erythrocytes was scored in bone marrow cells. It was observed that UMB alone did not cause any significant increase in DNA damage in comparison to control. Animals which are exposed to radiation alone showed significant increase in DNA damage and micronuclei frequency. But animals treated with UMB prior to the radiation exposure showed significant decrease

  9. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2016-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

  10. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    Directory of Open Access Journals (Sweden)

    Eun Ah Song

    2016-08-01

    Full Text Available The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA, shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

  11. The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma.

    Science.gov (United States)

    Maes, Ken; De Smedt, Eva; Lemaire, Miguel; De Raeve, Hendrik; Menu, Eline; Van Valckenborgh, Els; McClue, Steve; Vanderkerken, Karin; De Bruyne, Elke

    2014-05-30

    DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.

  12. Mitochondrial DNA depletion causes decreased ROS production and resistance to apoptosis.

    Science.gov (United States)

    Chen, Hulin; Wang, Junling; Liu, Zhongrong; Yang, Huilan; Zhu, Yingjie; Zhao, Minling; Liu, Yan; Yan, Miaomiao

    2016-10-01

    Mitochondrial DNA (mtDNA) depletion occurs frequently in many diseases including cancer. The present study was designed in order to examine the hypothesis that mtDNA‑depleted cells are resistant to apoptosis and to explore the possible mechanisms responsible for this effect. Parental human osteosarcoma 143B cells and mtDNA‑deficient (Rho˚ or ρ˚) 206 cells (derived from 143B cells) were exposed to different doses of solar-simulated ultraviolet (UV) radiation. The effects of solar irradiation on cell morphology were observed under both light and fluorescence microscopes. Furthermore, apoptosis, mitochondrial membrane potential (MMP) disruption and reactive oxygen species (ROS) production were detected and measured by flow cytometry. In both cell lines, apoptosis and ROS production were clearly increased, whereas MMP was slightly decreased. However, apoptosis and ROS production were reduced in the Rho˚206 cells compared with the 143B cells. We also performed western blot analysis and demonstrated the increased release of cytosolic Cyt c from mitochondria in the 143B cells compared with that in the Rho˚206 cells. Thus, we concluded that Rho˚206 cells exhibit more resistance to solar‑simulated UV radiation‑induced apoptosis at certain doses than 143B cells and this is possibly due to decreased ROS production. PMID:27499009

  13. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup;

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... independently of DNA damage signaling pathways during the very early stages of apoptosis....

  14. Arsenic Trioxide Modulates DNA Synthesis and Apoptosis in Lung Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Kenneth Ndebele

    2010-04-01

    Full Text Available Arsenic trioxide, the trade name Trisenox, is a drug used to treat acute promyleocytic leukemia (APL. Studies have demonstrated that arsenic trioxide slows cancer cells growth. Although arsenic influences numerous signal-transduction pathways, cell-cycle progression, and/or apoptosis, its apoptotic mechanisms are complex and not entirely delineated. The primary objective of this research was to evaluate the effects of arsenic trioxide on DNA synthesis and to determine whether arsenic-induced apoptosis is mediated via caspase activation, p38 mitogen–activated protein kinase (MAPK, and cell cycle arrest. To achieve this goal, lung cancer cells (A549 were exposed to various concentrations (0, 2, 4, 6, 8, and 10 µg/mL of arsenic trioxide for 48 h. The effect of arsenic trioxide on DNA synthesis was determined by the [3H]thymidine incorporation assay. Apoptosis was determined by the caspase-3 fluorescein isothiocyanate (FITC assay, p38 MAP kinase activity was determined by an immunoblot assay, and cell-cycle analysis was evaluated by the propidium iodide assay. The [3H]thymidine-incorporation assay revealed a dose-related cytotoxic response at high levels of exposure. Furthermore, arsenic trioxide modulated caspase 3 activity and induced p38 MAP kinase activation in A549 cells. However, cell-cycle studies showed no statistically significant differences in DNA content at subG1 check point between control and arsenic trioxide treated cells.

  15. DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos

    Science.gov (United States)

    Montero, Juan A.; Sanchez-Fernandez, Cristina; Lorda-Diez, Carlos I.; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2016-01-01

    DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of γH2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells γH2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration. PMID:27752097

  16. DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

    Science.gov (United States)

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-11-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  17. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance12

    Science.gov (United States)

    Stronach, Euan A; Chen, Michelle; Maginn, Elaina N; Agarwal, Roshan; Mills, Gordon B; Wasan, Harpreet; Gabra, Hani

    2011-01-01

    Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors. PMID:22131882

  18. DNA-PK Mediates AKT Activation and Apoptosis Inhibition in Clinically Acquired Platinum Resistance

    Directory of Open Access Journals (Sweden)

    Euan A. Stronach

    2011-11-01

    Full Text Available Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinumresistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK, and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Re-sensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage–mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors.

  19. DNA apoptosis and stability in B-cell chronic lymphoid leukaemia: implication of the DNA double-strand breaks repair system by non homologous recombination

    International Nuclear Information System (INIS)

    After an introduction presenting the diagnosis and treatment of chronic lymphoid leukaemia, its molecular and genetic characteristics, and its cellular origin and clonal evolution, this research thesis describes the apoptosis (definition and characteristics, cancer and chemotherapy, apoptotic ways induced by gamma irradiation), the genotoxic stresses, the different repair mechanisms for different damages, and the DNA repair processes. It reports how human chronic lymphocytic leukaemia B cells can escape DNA damage-induced apoptosis through the non-homologous end-joining DNA repair pathway, and presents non-homologous end-joining DNA repair as a potent mutagenic process in human chronic lymphocytic leukaemia B cells

  20. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Clement G. Yedjou

    2015-12-01

    Full Text Available In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO32] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60 cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO32 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05 increase of necrotic cell death in Pb(NO32-treated cells, indicative of membrane rupture by Pb(NO32 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05 in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO32 exposure significantly (p < 0.05 increased the proportion of caspase-3 positive cells (apoptotic cells compared to the control. The flow cytometry assessment also indicated Pb(NO32 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO32 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO32 exposure and its associated adverse

  1. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

    Science.gov (United States)

    de Renty, Christelle; DePamphilis, Melvin L; Ullah, Zakir

    2014-01-01

    Proliferating trophoblast stem cells (TSCs) can differentiate into nonproliferating but viable trophoblast giant cells (TGCs) that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

  2. Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Christelle de Renty

    Full Text Available Proliferating trophoblast stem cells (TSCs can differentiate into nonproliferating but viable trophoblast giant cells (TGCs that are resistant to DNA damage induced apoptosis. Differentiation is associated with selective up-regulation of the Cip/Kip cyclin-dependent kinase inhibitors p57 and p21; expression of p27 remains constant. Previous studies showed that p57 localizes to the nucleus in TGCs where it is essential for endoreplication. Here we show that p27 also remains localized to the nucleus during TSC differentiation where it complements the role of p57. Unexpectedly, p21 localized to the cytoplasm where it was maintained throughout both the G- and S-phases of endocycles, and where it prevented DNA damage induced apoptosis. This unusual status for a Cip/Kip protein was dependent on site-specific phosphorylation of p21 by the Akt1 kinase that is also up-regulated in TGCs. Although cytoplasmic p21 is widespread among cancer cells, among normal cells it has been observed only in monocytes. The fact that it also occurs in TGCs reveals that p57 and p21 serve nonredundant functions, and suggests that the role of p21 in suppressing apoptosis is restricted to terminally differentiated cells.

  3. 10-Acetylirciformonin B, A Sponge Furanoterpenoid, Induces DNA Damage and Apoptosis in Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Fu-Wen Kuo

    2012-10-01

    Full Text Available 10-Acetylirciformonin B, a furanoterpenoid derived from irciformonin B found in a marine sponge, has been reported to possess potent cytotoxic activity against several cancer cell lines. However, the mechanism of its apoptotic activity against human leukemia cells has never been reported. The purpose of this study was to investigate the cytotoxic effects of 10-acetylirciformonin B and its possible mechanism of action against leukemia HL 60 cells. We found that 10-acetylirciformonin B decreased cell viability through the inhibition of cell growth as well as the induction of DNA damage and apoptosis in a dose-dependent manner. The induction of DNA damage was mediated by the increase of p-CHK2 and γ-H2A.X, which was suggested from the increase of tail movement in the neutral Comet assay. Induction of apoptosis was mediated with the increase in caspases 8, 9 and 3 activation as well as PARP cleavage. In summary, our resultsindicate that 10-acetylirciformonin B treatment causes apoptosis in leukaemia cells; probably through a caspase-dependent regulatory pathway.

  4. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    OpenAIRE

    Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

    2015-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)...

  5. Novel insights into the apoptosis mechanism of DNA topoisomerase I inhibitor isoliquiritigenin on HCC tumor cell

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ze-xin; Li, Jian; Li, Yan; You, Kun; Xu, Hongwei; Wang, Jianguo, E-mail: wangjianguoxx@163.com

    2015-08-21

    The inhibitory effect of DNA topoisomerase (Top I) by isoliquiritigenin(ISO) were investigated and their interaction mechanism was evaluated using methods including UV–vis absorption, fluorescence, coupled with molecular simulation, and using the MTT method of inhibition rate of HCC tumor cell SNU475 proliferation assay, finally, the interaction of ISO with calf thymus DNA was investigated by melting measurements and molecular docking studies. It was found that isoliquiritigenin reversibly inhibited DNA Top I in a competitive manner with the concentrations of ISO resulting in 50% activity lost (IC{sub 50}) were estimated to be 0.178 ± 0.12 mM. Isoliquiritigenin exhibited a strong ability to quench the intrinsic fluorescence of Top I through a static quenching procedure. The positive values of enthalpy change and entropy change suggested that the binding of isoliquiritigenin to Top I was driven mainly by hydrophobic interactions. The molecular docking results revealed isoliquiritigenin actually interacted with the primary amino acid residues on the active site of Top I, and the detection results of fluorescence staining and the inhibitory effect on the growth of HCC SUN475 showed that isoliquiritigenin induced the apoptosis cells increased gradually. The interaction of ISO with DNA can cause the denaturation temperature to be increased, which indicated that the stabilization of the DNA helix was increased in the presence of ISO, which indicated that the results provide strong evidence for intercalative binding of ISO with DNA. - Highlights: • ISO reversibly inhibits TOP I activity in an A dose dependent manner. • Hydrophobic interactions play a major role in ISO–TOP I interaction. • ISO has a high affinity close to the active site pocket of TOP I. • The binding of ISO to DNA induces the stability of the structure of DNA.

  6. Novel insights into the apoptosis mechanism of DNA topoisomerase I inhibitor isoliquiritigenin on HCC tumor cell

    International Nuclear Information System (INIS)

    The inhibitory effect of DNA topoisomerase (Top I) by isoliquiritigenin(ISO) were investigated and their interaction mechanism was evaluated using methods including UV–vis absorption, fluorescence, coupled with molecular simulation, and using the MTT method of inhibition rate of HCC tumor cell SNU475 proliferation assay, finally, the interaction of ISO with calf thymus DNA was investigated by melting measurements and molecular docking studies. It was found that isoliquiritigenin reversibly inhibited DNA Top I in a competitive manner with the concentrations of ISO resulting in 50% activity lost (IC50) were estimated to be 0.178 ± 0.12 mM. Isoliquiritigenin exhibited a strong ability to quench the intrinsic fluorescence of Top I through a static quenching procedure. The positive values of enthalpy change and entropy change suggested that the binding of isoliquiritigenin to Top I was driven mainly by hydrophobic interactions. The molecular docking results revealed isoliquiritigenin actually interacted with the primary amino acid residues on the active site of Top I, and the detection results of fluorescence staining and the inhibitory effect on the growth of HCC SUN475 showed that isoliquiritigenin induced the apoptosis cells increased gradually. The interaction of ISO with DNA can cause the denaturation temperature to be increased, which indicated that the stabilization of the DNA helix was increased in the presence of ISO, which indicated that the results provide strong evidence for intercalative binding of ISO with DNA. - Highlights: • ISO reversibly inhibits TOP I activity in an A dose dependent manner. • Hydrophobic interactions play a major role in ISO–TOP I interaction. • ISO has a high affinity close to the active site pocket of TOP I. • The binding of ISO to DNA induces the stability of the structure of DNA

  7. Conformational changes in DNA gyrase revealed by limited proteolysis

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    We have used limited proteolysis to identify conformational changes in DNA gyrase. Gyrase exhibits a proteolytic fingerprint dominated by two fragments, one of approximately 62 kDa, deriving from the A protein, and another of approximately 25 kDa from the B protein. Quinolone binding to the enzym...

  8. LDFF, the large molecular weight DNA fragmentation factor, is responsible for the large molecular weight DNA degradation during apoptosis in Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    Zhi Gang LU; Chuan Mao ZHANG; Zhong He ZHAI

    2004-01-01

    DNA degradation is a biochemical hallmark in apoptosis. It has been demonstrated in many cell types that there are two stages of DNA fragmentation during the apoptotic execution. In the early stage, chromatin DNA is cut into large molecular weight DNA fragments, although the responsible nuclease(s) has not been recognized. In the late stage, the chromatin DNA is cleaved further into short oligonucleosomal fragments by a well-characterized nuclease in apoptosis,the caspase-activated DNase (CAD/DFF40). In this study, we demonstrate that large molecular weight DNA fragmentation also occurs in Xenopus egg extracts in apoptosis. We show that the large molecular weight DNA fragmentation factor (LDFF) is not the Xenopus CAD homolog XCAD. LDFF is activated by caspase-3. The large molecular weight DNA fragmentation activity of LDFF is Mg2+-dependent and Ca2+-independent, can occur in both acidic and neutral pH conditions and can tolerate 45℃ treatment. These results indicate that LDFF in Xenopus egg extracts might be a new DNase (or DNases) responsible for the large DNA fragmentation.

  9. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  10. INTRANUCLEAR MATRIX METALLOPROTEINASES PROMOTE DNA DAMAGE AND APOPTOSIS INDUCED BY OXYGEN–GLUCOSE DEPRIVATION IN NEURONS

    Science.gov (United States)

    HILL, J. W.; PODDAR, R.; THOMPSON, J. F.; ROSENBERG, G. A.; YANG, Y.

    2016-01-01

    Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood–brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24 h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons

  11. Space-charge-limited current in DNA-surfactant complex

    Science.gov (United States)

    Chen, I.-Ching; Lin, Ting-Yu; Hung, Yu-Chueh

    2013-03-01

    In recent years, deoxyribonucleic acid (DNA) biopolymers have attracted much research attention and been considered as a promising material when being employed in many optoelectronic devices. Since performance of many DNA biopolymer-based devices relies on carrier transport, it is crucial to study the carrier mobility of these DNA-surfactant complexes for practical implement. In this work, we present hole mobility characterization of cetyltrimethylammonium (CTMA)-modified DNA biopolymer by using space-charge-limited current (SCLC) method. Devices were fabricated using a sandwich structure with a buffer layer of MoO3 to enhance hole injection and achieve ohmic contact between the anode and the DNA layer. Current-voltage (I-V) curves of the devices were analyzed. A trap-free SCLC behavior can ultimately be achieved and a quadratic dependence in I-V curve was observed. With increasing electric field, a positive field-dependent mobility was demonstrated. The correlation between mobility and temperature was also investigated and a positive relation was found. The characterization results can be further utilized for DNA-based device design and applications.

  12. Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

    Science.gov (United States)

    Matsuyama, Shigemi; Palmer, James; Bates, Adam; Poventud-Fuentes, Izmarie; Wong, Kelvin; Ngo, Justine; Matsuyama, Mieko

    2016-06-01

    Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their

  13. In vitro cytotoxicity, apoptosis, DNA-binding, and antioxidant activity studies of ruthenium (II) complexes.

    Science.gov (United States)

    Huang, Hong-Liang; Liu, Yun-Jun; Zeng, Cheng-Hui; He, Li-Xin; Wu, Fu-Hai

    2010-05-01

    Two new ligands maip (1) (maip = 2-(3-aminophenyl)imizado[4,5-f][1,10]phenanthroline), paip (2) (paip = 2-(4-aminophenyl)imidazo[4,5-f][1,10]phenanthroline), and their ruthenium (II) complexes [Ru(phen)(2)(maip)](ClO(4))(2) (3) and [Ru(phen)(2)(paip)](ClO(4))(2) (4) (phen = 1,10-phenanthroline) have been synthesized and characterized. The cytotoxicity of these compounds was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The apoptosis assay was carried out with acridine orange/ethidium bromide staining methods. The DNA-binding behaviors of complexes 3 and 4 were investigated by viscosity measurements, thermal denaturation, photocleavage, and spectroscopic methods. The results show that the two complexes intercalate into the base pairs of DNA. In the presence of a complex, apoptosis of BEL-7402 cells was observed. Experiments show that these compounds exhibit antioxidant activity against hydroxyl radicals. PMID:20307189

  14. Artesunate Reduces Proliferation, Interferes DNA Replication and Cell Cycle and Enhances Apoptosis in Vascular Smooth Muscle Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded.The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated.DNA replication was evaluated by [3 H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.

  15. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  16. A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest†

    Science.gov (United States)

    Cowling, Victoria H.; Chandriani, Sanjay; Whitfield, Michael L.; Cole, Michael D.

    2006-01-01

    The myc family of oncogenes is well conserved throughout evolution. Here we present the characterization of a domain conserved in c-, N-, and L-Myc from fish to humans, N-Myc317-337, designated Myc box IV (MBIV). A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation. Unlike other Myc mutants, MycΔMBIV is not a simple loss-of-function mutant because it is hyperactive for G2 arrest in primary cells. Microarray analysis of genes regulated by N-MycΔMBIV reveals that it is weakened for transactivation and repression but not nearly as defective as N-MycΔMBII. Although the mutated region is not part of the previously defined DNA binding domain, we find that N-MycΔMBIV has a significantly lower affinity for DNA than the wild-type protein in vitro. Furthermore, chromatin immunoprecipitation shows reduced binding of N-MycΔMBIV to some target genes in vivo, which correlates with the defect in transactivation. Thus, this conserved domain has an unexpected role in Myc DNA binding activity. These data also provide a novel separation of Myc functions linked to the modulation of DNA binding activity. PMID:16705173

  17. Effects of Cadmium on Hepatocellular DNA Damage, Proto-Oncogene Expression and Apoptosis in Rats

    Institute of Scientific and Technical Information of China (English)

    RIAN YU; LIN-GFEI HE; XUE-MIN CHEN

    2007-01-01

    Objective To study the effects of cadmium on hepatocellular DNA damage, expression of proto-oncogenes c-myc, c-fos,and c-jun as well as apoptosis in rats. Methods Cadmium chloride at the doses of 5, 10, and 20 μmol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was measured by single cell gel electrophoresis (or comet assay), while expression of proto-oncogenes c-myc, c-fos, and c-jun in rat hepatocytes were measured by Northern dot hybridization. C-Myc, c-Fos, and c-Jun were detected with immuno-histochemical method. Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP Nick End Labelling) and flow cytometry. Results At the doses of 5, 10, and 20 μmol/kg, cadmium chloride induced DNA damage in rat hepatocytes and the rates of comet cells were 50.20%,88.40%, and 93.80%, respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the dose of cadmium chloride (r=0.9172, P<0.01). Cadmium chloride at the doses of 5, 10, and 20 μmol/kg induced expression of proto-oncogenes c-myc, c-fos, and c-jun. The positive brown-yellow signal for c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in cadmium-treated rat livers. Apoptotic rates (%) of cadmium-treated liver cells at the doses of 5, 10, and 20 μmol/kg were (17.24±2.98), (20.58±1.35), and (24.06±1.77) respectively, being significantly higher than those in the control. The results also displayed an obvious dose-response relationship between apoptotic rates and the dose of cadmium chloride (r=0.8619, P<0.05).Conclusion Cadmium at 5-20 μmol/kg can induce hepatocellular DNA damage, expression of proto-oncogenes c-myc, c-fos,and c-jun as well as apoptosis in rats.

  18. Electron microscopic observations and DNA chain fragmentation studies on apoptosis in bone tumor cells induced by 153Sm—EDTMP

    Institute of Scientific and Technical Information of China (English)

    ZhuShou-Peng; XiaoDong; 等

    1997-01-01

    The morphological changes observed by electron microscopy indicate that after internal irradiation with 153Sm-ESTMP bone tumor cells displayed feature of apoptosis,such as margination of condensed chromatin,chromatin fragmentation.as well as the membranebouded apoptotic bodies formation.THe quantification analysis of fragmentation DNA for bone tumor cells induced by 153Sm-EDTMP shows that the DNA fragmentation is enhanced with the prolongation of internally irradiated time.These characteristics suggest that 153Sm-EDTMP internal irradiation could induce bone tumor cells to go9 to apoptosis.

  19. Inhibition of DNA topoisomerase I activity and induction of apoptosis by thiazacridine derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Barros, Francisco W.A. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Bezerra, Daniel P., E-mail: danielpbezerra@gmail.com [Department of Physiology, Federal University of Sergipe, São Cristóvão, Sergipe (Brazil); Ferreira, Paulo M.P. [Department of Biological Sciences, Federal University of Piauí, Picos, Piauí (Brazil); Cavalcanti, Bruno C. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Silva, Teresinha G.; Pitta, Marina G.R.; Lima, Maria do C.A. de; Galdino, Suely L.; Pitta, Ivan da R. [Department of Antibiotics, Federal, University of Pernambuco, Recife, Pernembuco (Brazil); Costa-Lotufo, Letícia V.; Moraes, Manoel O. [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil); Burbano, Rommel R. [Institute of Biological Sciences, Federal University of Pará, Belém, Pará (Brazil); Guecheva, Temenouga N.; Henriques, João A.P. [Biotechnology Center, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul (Brazil); Pessoa, Cláudia, E-mail: cpessoa@ufc.br [Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, Ceará (Brazil)

    2013-04-01

    Thiazacridine derivatives (ATZD) are a novel class of cytotoxic agents that combine an acridine and thiazolidine nucleus. In this study, the cytotoxic action of four ATZD were tested in human colon carcinoma HCT-8 cells: (5Z)-5-acridin-9-ylmethylene-3-(4-methylbenzyl)-thiazolidine-2,4-dione — AC-4; (5ZE)-5-acridin-9-ylmethylene-3-(4-bromo-benzyl)-thiazolidine-2,4-dione — AC-7; (5Z)-5-(acridin-9-ylmethylene)-3-(4-chloro-benzyl) -1,3-thiazolidine-2,4-dione — AC-10; and (5ZE)-5-(acridin-9-ylmethylene)-3-(4-fluoro-benzyl)-1,3-thiazolidine-2, 4-dione — AC-23. All of the ATZD tested reduced the proliferation of HCT-8 cells in a concentration- and time-dependent manner. There were significant increases in internucleosomal DNA fragmentation without affecting membrane integrity. For morphological analyses, hematoxylin–eosin and acridine orange/ethidium bromide were used to stain HCT-8 cells treated with ATZD, which presented the typical hallmarks of apoptosis. ATZD also induced mitochondrial depolarisation and phosphatidylserine exposure and increased the activation of caspases 3/7 in HCT-8 cells, suggesting that this apoptotic cell death was caspase-dependent. In an assay using Saccharomyces cerevisiae mutants with defects in DNA topoisomerases 1 and 3, the ATZD showed enhanced activity, suggesting an interaction between ATZD and DNA topoisomerase enzyme activity. In addition, ATZD inhibited DNA topoisomerase I action in a cell-free system. Interestingly, these ATZD did not cause genotoxicity or inhibit the telomerase activity in human lymphocyte cultures at the experimental levels tested. In conclusion, the ATZD inhibited the DNA topoisomerase I activity and induced tumour cell death through apoptotic pathways. - Highlights: ► Thiazacridine derivatives induce mitochondrial-dependent apoptotic cell death. ► Thiazacridine derivatives inhibit DNA topoisomerase I action. ► Thiazacridine derivatives failed to cause genotoxicity on human lymphocytes.

  20. Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-Substrate Complexes of Topoisomerase and DNA

    Directory of Open Access Journals (Sweden)

    Candace L. Minchew

    2011-06-01

    Full Text Available In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific “starting” oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.

  1. Torsional stress in DNA limits collaboration among reverse gyrase molecules.

    Science.gov (United States)

    Ogawa, Taisaku; Sutoh, Kazuo; Kikuchi, Akihiko; Kinosita, Kazuhiko

    2016-04-01

    Reverse gyrase is an enzyme that can overwind (introduce positive supercoils into) DNA using the energy obtained from ATP hydrolysis. The enzyme is found in hyperthermophiles, and the overwinding reaction generally requires a temperature above 70 °C. In a previous study using microscopy, we have shown that 30 consecutive mismatched base pairs (a bubble) in DNA serve as a well-defined substrate site for reverse gyrase, warranting the processive overwinding activity down to 50 °C. Here, we inquire how multiple reverse gyrase molecules may collaborate with each other in overwinding one DNA molecule. We introduced one, two, or four bubbles in a linear DNA that tethered a magnetic bead to a coverslip surface. At 40-71 °C in the presence of reverse gyrase, the bead rotated clockwise as viewed from above, to relax the DNA twisted by reverse gyrase. Dependence on the enzyme concentration indicated that each bubble binds reverse gyrase tightly (dissociation constant 5 min. Rotation with two bubbles was significantly faster compared with one bubble, indicating that overwinding actions are basically additive, but four bubbles did not show further acceleration except at 40 °C where the activity was very low. The apparent saturation is due to the hydrodynamic friction against the rotating bead, as confirmed by increasing the medium viscosity. When torsional stress in the DNA, determined by the friction, approaches ~ 7 pN·nm (at 71 °C), the overwinding activity of reverse gyrase drops sharply. Multiple molecules of reverse gyrase collaborate additively within this limit.

  2. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    Science.gov (United States)

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  3. The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

    Science.gov (United States)

    Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

    2016-08-01

    Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive. PMID:27476333

  4. The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

    Science.gov (United States)

    Castelo Branco, Patrícia Valéria; Soares, Rossy-Eric Pereira; de Jesus, Luís Cláudio Lima; Moreira, Vanessa Ribeiro; Alves, Hugo José; de Castro Belfort, Marta Regina; Silva, Vera Lucia Maciel; Ferreira Pereira, Silma Regina

    2016-08-01

    Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.

  5. The role of intracellular zinc in chromium(VI)-induced oxidative stress, DNA damage and apoptosis.

    Science.gov (United States)

    Rudolf, Emil; Cervinka, Miroslav

    2006-09-25

    Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.

  6. Generation of Reactive Oxygen Species during Apoptosis Induced by DNA-Damaging Agents and/or Histone Deacetylase Inhibitors

    Directory of Open Access Journals (Sweden)

    Barbora Brodská

    2011-01-01

    Full Text Available Reactive oxygen species play an important role in the process of apoptosis in many cell types. In this paper, we analyzed the role of ROS in DNA-damaging agents (actinomycin D or decitabine, which induced apoptosis of leukemia cell line CML-T1 and normal peripheral blood lymphocytes (PBL. The possibility of synergism with histone deacetylase inhibitors butyrate or SAHA is also reported. We found that in cancer cell line, ROS production significantly contributed to apoptosis triggering, while in normal lymphocytes treated by cytostatic or cytotoxic drugs, necrosis as well as apoptosis occurred and large heterogeneity of ROS production was measured. Combined treatment with histone deacetylase inhibitor did not potentiate actinomycin D action, whereas combination of decitabine and SAHA brought synergistic ROS generation and apoptotic features in CML cell line. Appropriate decrease of cell viability indicated promising therapeutic potential of this combination in CML, but side effects on normal PBL should be taken into attention.

  7. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo.

    Science.gov (United States)

    Phesse, T J; Myant, K B; Cole, A M; Ridgway, R A; Pearson, H; Muncan, V; van den Brink, G R; Vousden, K H; Sears, R; Vassilev, L T; Clarke, A R; Sansom, O J

    2014-06-01

    Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes to DNA damage. Remarkably, c-Myc deletion completely abrogated the immediate wave of apoptosis following both ionizing irradiation and cisplatin treatment, recapitulating the phenotype of p53 deficiency in the intestine. Consistent with this, c-Myc-deficient intestinal enterocytes did not upregulate p53. Mechanistically, this was linked to an upregulation of the E3 Ubiquitin ligase Mdm2, which targets p53 for degradation in c-Myc-deficient intestinal enterocytes. Further, low level overexpression of c-Myc, which does not impact on basal levels of apoptosis, elicited sustained apoptosis in response to DNA damage, suggesting c-Myc activity acts as a crucial cell survival rheostat following DNA damage. We also identify the importance of MYC during DNA damage-induced apoptosis in several other tissues, including the thymus and spleen, using systemic deletion of c-Myc throughout the adult mouse. Together, we have elucidated for the first time in vivo an essential role for endogenous c-Myc in signalling DNA damage-induced apoptosis through the control of the p53 tumour suppressor protein. PMID:24583641

  8. NUC-1, a caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis.

    Science.gov (United States)

    Wu, Y C; Stanfield, G M; Horvitz, H R

    2000-03-01

    One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and discovered that TUNEL labels apoptotic cells only during a transient intermediate stage. Mutations in nuc-1 allowed the generation of TUNEL-reactive DNA but blocked the conversion of TUNEL-reactive DNA to a subsequent TUNEL-unreactive state. Completion of DNA degradation did not occur in the absence of cell-corpse engulfment. Our data suggest that the process of degradation of the DNA of a cell corpse occurs in at least three distinct steps and requires activities provided by both the dying and the engulfing cell.

  9. Characterization of HIFU ablation using DNA fragmentation labeling as apoptosis stain

    Science.gov (United States)

    Anquez, Jeremie; Corréas, Jean-Michel; Pau, Bernard; Lacoste, François; Yon, Sylvain

    2012-11-01

    The goal of this work was to compare modalities to precisely quantify the extent of thermally induced lesions: gross pathology vs. histopathology vs. devascularization. Liver areas of 14 rabbits were targeted with HIFU and RF ablations in an acute study. Contrast enhanced computorized tomography (CE-CT) scan images were acquired two hours after HIFU and RF treatment to obtain the devascularized volumes of the livers. The animals were then euthanized and deep frozen. The livers were sliced and each slice was photographed and stacked yielding a volume of gross pathology. The volume VGP of the HIFU lesions were derived. The area AGP of the lesions were computed on a particular slice. The lesions were segmented as hypo intense (devascularized) regions on CE-CT images and their volumes VC were computed. The ratios VC/VGP were computed for all the HIFU lesions on all the 14 subjects with a mean value of 1.2. Histology was performed on the livers using Hematoxyline Eosine Staining (HES) and DNA Fragmentation labeling (TUNEL® technology) which characterizes apoptosis. Apoptotic regions of area AT were segmented on the images stained by TUNEL®. No necrosis was identified on the HES data. While TUNEL® did not mark the cores of the RF lesions as apoptotic, the periphery of HIFU and RF lesions was always recognized with TUNEL® as apoptotic. The ratio AGP/AT was computed. The mean value was 0.95 and 0.25 for HIFU and RF lesions respectively. These findings show that the devascularized territory seen on CE-CT scan coincide with the coagulated territories seen with gross pathology. Those actually correspond to cells in apoptosis. It is confirmed that HES stain does not show necrosis 2 hours after thermal ablation. TUNEL® technology for DNA fragmentation labeling appears as a useful marker for thermally induced acute lesions in the liver.

  10. Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis.

    Science.gov (United States)

    Srivastava, Shikha; Somasagara, Ranganatha R; Hegde, Mahesh; Nishana, Mayilaadumveettil; Tadi, Satish Kumar; Srivastava, Mrinal; Choudhary, Bibha; Raghavan, Sathees C

    2016-01-01

    Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy. PMID:27068577

  11. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    Science.gov (United States)

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-01

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  12. Histone H1 Limits DNA Methylation in Neurospora crassa.

    Science.gov (United States)

    Seymour, Michael; Ji, Lexiang; Santos, Alex M; Kamei, Masayuki; Sasaki, Takahiko; Basenko, Evelina Y; Schmitz, Robert J; Zhang, Xiaoyu; Lewis, Zachary A

    2016-01-01

    Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the [Formula: see text]hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the [Formula: see text]hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa. PMID:27172195

  13. Protection from Palmitate-Induced Mitochondrial DNA Damage Prevents from Mitochondrial Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and Impaired Insulin Signaling in Rat L6 Skeletal Muscle Cells

    OpenAIRE

    Yuzefovych, Larysa V.; Solodushko, Viktoriya A.; Wilson, Glenn L.; Rachek, Lyudmila I.

    2011-01-01

    Saturated free fatty acids have been implicated in the increase of oxidative stress, mitochondrial dysfunction, apoptosis, and insulin resistance seen in type 2 diabetes. The purpose of this study was to determine whether palmitate-induced mitochondrial DNA (mtDNA) damage contributed to increased oxidative stress, mitochondrial dysfunction, apoptosis, impaired insulin signaling, and reduced glucose uptake in skeletal muscle cells. Adenoviral vectors were used to deliver the DNA repair enzyme ...

  14. Mechanisms of a novel anticancer therapeutic strategy involving atmospheric pressure plasma-mediated apoptosis and DNA strand break formation.

    Science.gov (United States)

    Chung, Woo-Hyun

    2016-01-01

    Atmospheric pressure plasma has been developed for a variety of biomedical applications due to its chemically reactive components. Recently, the plasma has emerged as a promising novel cancer therapy based on its ability to selectively ablate cancer cells while leaving normal cells essentially unaffected. The therapeutic effect of plasma is attributed to intracellular generation of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondria-mediated apoptosis and to activation of the DNA damage checkpoint signaling pathway via severe DNA strand break formation. However, the biochemical mechanisms responsible for appropriate activation of these physiological events and which pathway is more crucial for plasma-mediated cytotoxicity have not been clarified. Understanding the molecular link between ROS/RNS-mediated apoptosis and DNA damage-involved chromosome instability is critical for the development of more efficacious therapeutic strategies for selective killing of diverse cancer cells.

  15. The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Highlights: ► Zebularine inhibited cell growth of gastric cancer in a time- and dose-dependent manner. ► Chromatin condensation and nuclear fragmentation were induced. ► Zebularine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by zebularine. -- Abstract: DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

  16. The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Wei, E-mail: polo5352877@163.com [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China); Zhou, Wei; Yu, Hong-gang; Luo, He-Sheng; Shen, Lei [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan (China)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Zebularine inhibited cell growth of gastric cancer in a time- and dose-dependent manner. Black-Right-Pointing-Pointer Chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Zebularine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by zebularine. -- Abstract: DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 {mu}M accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

  17. The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

    International Nuclear Information System (INIS)

    Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

  18. RESCUE OF HIPPO CO-ACTIVATOR YAP1 TRIGGERS DNA DAMAGE-INDUCED APOPTOSIS IN HEMATOLOGICAL CANCERS

    Science.gov (United States)

    Cottini, Francesca; Hideshima, Teru; Xu, Chunxiao; Sattler, Martin; Dori, Martina; Agnelli, Luca; Hacken, Elisa ten; Bertilaccio, Maria Teresa; Antonini, Elena; Neri, Antonino; Ponzoni, Maurilio; Marcatti, Magda; Richardson, Paul G.; Carrasco, Ruben; Kimmelman, Alec C.; Wong, Kwok-Kin; Caligaris-Cappio, Federico; Blandino, Giovanni; Kuehl, W. Michael; Anderson, Kenneth C.; Tonon, Giovanni

    2014-01-01

    Oncogene–induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53–independent, pro-apoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway co–activator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1–induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine–threonine kinase, STK4. Importantly, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a novel synthetic–lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels. PMID:24813251

  19. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

    Directory of Open Access Journals (Sweden)

    Duensing Stefan

    2008-07-01

    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

  20. DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingzhen [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Shen, Chunzi [Centers for Disease Control and Prevention, Zibo (China); Yang, Liu; Li, Chunhui; Yi, Anji [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Wang, Zhiping, E-mail: zhipingw@sdu.edu.cn [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China)

    2013-12-01

    Carbon disulfide (CS{sub 2}) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS{sub 2} via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD{sub 50} (157.85 mg/kg), 0.2 LD{sub 50} (315.7 mg/kg) and 0.4 LD{sub 50} (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS{sub 2}. - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss.

  1. Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Gundala, Sushma Reddy; Yang, Chunhua [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States); Mukkavilli, Rao [Advinus Therapeutics, Karnataka (India); Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States); Reid, Michelle D. [Department of Pathology, Emory University School of Medicine, Atlanta, GA (United States); Aneja, Ritu, E-mail: raneja@gsu.edu [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States)

    2014-10-01

    Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ∼ 72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. - Highlights: • HC perturbs cell-cycle progression by induction of reactive oxygen species (ROS). • HC mediated cytotoxicity by ROS-induced DNA damage leading to

  2. Role of mitochondrial DNA decrease in apoptosis of human bronchial epithelial cells induced by radon and its progeny

    International Nuclear Information System (INIS)

    Human bronchia epithelia with mtDNA decrease (ρ-HBE) cells generated by treatment of ethidium bromide (EB) were exposed to radon gas in a special inhalation chamber. Cell proliferation was determined by cell survival assay Cell apoptosis and membrane potential of mitochondria were analyzed by flow cytometry. The results showed that the survival fraction of ρ-HBE cells significantly increased compared with that of ρ+ HBE cells after irradiation with radon and its progeny. Although the apoptosis rate of p HBE cells was lower than that of the ρ+ HBE cells at early period, the total apoptosis rate was increased, along with the membrane potential decrease of mitochondria in ρ-HBE cells. The results indicate that the increased potential of ρ-HBE proliferation correlates with the total apoptotic rate and mitochondrial membrane potential. (authors)

  3. The ruthenium complex cis-(dichloro)tetrammineruthenium(III) chloride induces apoptosis and damages DNA in murine sarcoma 180 cells

    Indian Academy of Sciences (India)

    Aliny Pereira De Lima; Flávia De Castro Pereira; Cesar Augusto Sam Tiago Vilanova-Costa; Alessandra De Santana Braga Barbosa Ribeiro; Luiz Alfredo Pavanin; Wagner Batista Dos Santos; Elisângela De Paula Silveira-Lacerda

    2010-09-01

    Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. Recently, we reported that the cis-(dichloro)tetrammineruthenium(III) chloride compound has cytotoxic effects on murine sarcoma 180 (S-180) cells. In an effort to understand the mechanism responsible for their cytotoxicity, study we investigated the genotoxicity, cell cycle distribution and induction of apoptosis caused by cis-(dichloro)tetrammineruthenium(III) chloride in S-180 tumour cells. cis-(dichloro)tetrammineruthenium(III) chloride treatment induced significant DNA damage in S-180 cells, as detected by the alkaline comet assay. In the cell cycle analysis, cis-(dichloro)tetrammineruthenium(III) chloride caused an increase in the number of cells in G1 phase, accompanied by a decrease in the S and G2 phases after 24 h of treatment. In contrast, the cell cycle distribution of S-180 cells treated with cis-(dichloro)tetrammineruthenium(III) chloride for 48 h showed a concentration-dependent increase in the sub-G1 phase (indicating apoptosis), with a corresponding decrease in cells in the G1, S and G2 phases. In addition, cis-(dichloro)tetrammineruthenium(III) chloride treatment induced apoptosis in a time-dependent manner, as observed by the increased numbers of annexin V-positive cells. Taken together, these findings strongly demonstrate that DNA damage, cell cycle changes and apoptosis may correlate with the cytotoxic effects of cis-(dichloro)tetrammineruthenium(III) chloride on S-180 cells.

  4. Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres

    Directory of Open Access Journals (Sweden)

    Kamal Ahmed

    2011-04-01

    Full Text Available Abstract Background Breast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event. Results Involvement of pro-apoptotic protein (Bax, active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2 and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres. Conclusions The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

  5. Transient p53 Suppression Increases Reprogramming of Human Fibroblasts without Affecting Apoptosis and DNA Damage

    Directory of Open Access Journals (Sweden)

    Mikkel A. Rasmussen

    2014-09-01

    Full Text Available The discovery of human-induced pluripotent stem cells (iPSCs has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53 gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation.

  6. AZD1775 sensitizes T cell acute lymphoblastic leukemia cells to cytarabine by promoting apoptosis over DNA repair.

    Science.gov (United States)

    Ford, James B; Baturin, Dmitry; Burleson, Tamara M; Van Linden, Annemie A; Kim, Yong-Mi; Porter, Christopher C

    2015-09-29

    While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia. PMID:26334102

  7. Reduced ultraviolet-induced DNA damage and apoptosis in human skin with topical application of a photolyase-containing DNA repair enzyme cream: clues to skin cancer prevention.

    Science.gov (United States)

    Berardesca, Enzo; Bertona, Marco; Altabas, Karmela; Altabas, Velimir; Emanuele, Enzo

    2012-02-01

    The exposure of human skin to ultraviolet radiation (UVR) results in the formation of DNA photolesions that give rise to photoaging, mutations, cell death and the onset of carcinogenic events. Photolyase (EC 4.1.99.3) is a DNA repair enzyme that reverses damage caused by exposure to UVR. We sought to investigate whether addition of photolyase enhances the protection provided by a traditional sunscreen (SS), by reducing the in vivo formation of cyclobutane-type pyrimidine dimers (CPDs) and UVR-induced apoptosis in human skin. Ten volunteers (Fitzpatrick skin type II) were exposed to solar-simulated (ss) UVR at a three times minimal erythema dose for 4 consecutive days. Thirty minutes prior to each exposure, the test materials [vehicle, SS (sun protection factor 50) alone, and SS plus photolyase from Anacystis nidulans] were applied topically to three different sites. One additional site was left untreated and one received ssUVR only. Biopsy specimens were taken 72 h after the last irradiation. The amount of CPDs and the extent of apoptosis were measured by ELISA. Photolyase plus SS was superior to SS alone in reducing both the formation of CPDs and apoptotic cell death (both P<0.001). In conclusion, the addition of photolyase to a traditional SS contributes significantly to the prevention of UVR-induced DNA damage and apoptosis when applied topically to human skin. PMID:22086236

  8. Possible involvement of DNA methylation in 5-azacytidine-induced neuronal cell apoptosis

    OpenAIRE

    Nakayama, Hiroyuki; Kajikawa, S.; Shinozuka, J.; Su, W. P.; Doi, K

    1999-01-01

    Eight chemicals that are cytidine analogues or nucleosides (5-azacytidine (SAzC), 5-azadeoxycytidine, 6-azacytidine, 5-azacytosin, cytidine, 3-deazaadenine, 3-deazauridine and 6-azauridine) were examined for the ability to induce neuronal apoptosis. 5AzC and 5-azadeoxycytidine induced apoptosis in the brain and spinal cord of the fetuses at 24 hr after the injection to dams, while the other chemicals tested failed to induce apoptosis. In the system of PC12 cell...

  9. Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family

    Directory of Open Access Journals (Sweden)

    Rothblatt Jonathan

    2008-07-01

    Full Text Available Abstract Background In contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activator in a primordial p53 pathway that involves CEP-1 activation and the CEP-1 dependent transcriptional induction of the worm BH3 only domain encoding genes egl-1 and ced-13 to induce germ cell apoptosis. EGL-1 and CED-13 proteins inactivate Bcl-2 like CED-9 to trigger CED-4 and CED-3 caspase dependent germ cell apoptosis. To address the function of p53 in global transcriptional regulation we investigate genome-wide transcriptional responses upon DNA damage and cep-1 deficiency. Results Examining C. elegans expression profiles using whole genome Affymetrix GeneChip arrays, we found that 83 genes were induced more than two fold upon ionizing radiation (IR. None of these genes, with exception of an ATP ribosylase homolog, encode for known DNA repair genes. Using two independent cep-1 loss of function alleles we did not find genes regulated by cep-1 in the absence of IR. Among the IR-induced genes only three are dependent on cep-1, namely egl-1, ced-13 and a novel C. elegans specific gene. The majority of IR-induced genes appear to be involved in general stress responses, and qRT-PCR experiments indicate that they are mainly expressed in somatic tissues. Interestingly, we reveal an extensive overlap of gene expression changes occurring in response to DNA damage and in response to bacterial infection. Furthermore, many genes induced by IR are also transcriptionally regulated in longevity mutants suggesting that DNA damage and aging induce an overlapping stress response. Conclusion We performed genome-wide gene expression analyses which indicate that only a surprisingly small number of genes are regulated by CEP-1 and that DNA damage induced apoptosis via the

  10. The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression.

    Science.gov (United States)

    Zhang, Xiaolu; Li, Bingnan; de Jonge, Nick; Björkholm, Magnus; Xu, Dawei

    2015-03-10

    DNA methyltransferase inhibitors (DNMTIs) such as 5-azacytidine (5-AZA) have been used for treatment of acute myeloid leukemia (AML) and other malignancies. Although inhibiting global/gene-specific DNA methylation is widely accepted as a key mechanism behind DNMTI anti-tumor activity, other mechanisms are likely involved in DNMTI's action. Because telomerase reverse transcriptase (TERT) plays key roles in cancer through telomere elongation and telomere lengthening-independent activities, and TERT has been shown to confer chemo- or radio-resistance to cancer cells, we determine whether DNMTIs affect telomere function and whether TERT/telomerase interferes with their anti-cancer efficacy. We showed that 5-AZA induced DNA damage and telomere dysfunction in AML cell lines by demonstrating the presence of 53-BP1 foci and the co-localization of 53-BP1 foci with telomere signals, respectively. Telomere dysfunction was coupled with diminished TERT expression, shorter telomere and apoptosis in 5-AZA-treated cells. However, 5-AZA treatment did not lead to changes in the methylation status of subtelomere regions. Down-regulation of TERT expression similarly occurred in primary leukemic cells derived from AML patients exposed to 5-AZA. TERT over-expression significantly attenuated 5-AZA-mediated DNA damage, telomere dysfunction and apoptosis of AML cells. Collectively, 5-AZA mediates the down-regulation of TERT expression, and induces telomere dysfunction, which consequently exerts an anti-tumor activity. PMID:25682873

  11. Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

    International Nuclear Information System (INIS)

    The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions

  12. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide(As2O3) on the mitochondrial DNA(mtDNA) of acute promyelocytic leukemia(APL) cells.The NB4 cell line was treated with 2.0 μmol/L As2O3 in vitro,and the primary APL cells were treated with 2.0 μmol/L As2O3 in vitro and 0.16 mg kg-1 d-1 As2O3 in vivo.The mitochondrial DNA of all the cells above was amplified by PCR,directly sequenced and analyzed by Sequence Navigatore and Factura software.The apoptosis rates were assayed by flow cytometry.Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use,but the mutation spots were remarkably increased after As2O3 treatment,which was positively correlated to the rates of cellular apoptosis,the correlation coefficient:rNB4-As2O3=0.973818,and rAPL-As2O3=0.934703.The mutation types include transition,transversion,codon insertion or deletion,and the mutation spots in all samples were not constant and regular.It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo.Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.

  13. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    Energy Technology Data Exchange (ETDEWEB)

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  14. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    International Nuclear Information System (INIS)

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest

  15. Detection on emamectin benzoate-induced apoptosis and DNA damage in Spodoptera frugiperda Sf-9 cell line.

    Science.gov (United States)

    Wu, Xiwei; Zhang, Lei; Yang, Chao; Zong, Mimi; Huang, Qingchun; Tao, Liming

    2016-01-01

    Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34μM at 72h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations.

  16. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    Directory of Open Access Journals (Sweden)

    Likui Wang

    2014-09-01

    Full Text Available Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2 called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE, which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

  17. Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells

    International Nuclear Information System (INIS)

    Ochratoxin A (OTA) is a nephrotoxic/-carcinogenic mycotoxin, produced by several Aspergillus- and Penicillium-strains. Humans are exposed to OTA via food contamination, a causal relationship of OTA to human endemic Balkan nephropathy is still under debate. Since DNA-adducts of OTA or its metabolites could not be identified unambiguously, its carcinogenic effectiveness might be related to secondary effects, such as oxidative cell damage or cell proliferation. In this study, OTA mediated induction of (oxidative) DNA damage, cytotoxicity (necrosis, growth inhibition, apoptosis) and modulation of glutathione were investigated in cell lines (V79, CV-1) and primary rat kidney cells. After 24 h incubation, viability of V79 cells was strongly decreased by OTA concentrations >2.5 μmol/L, whereas CV-1 cells were clearly less sensitive. Strong growth inhibition occurred in both cell lines (IC50 ∼2 μmol/L). Apoptosis, detected with an immunochemical test and with flow cytometry, was induced by >1 μmol/L OTA. Oxidative DNA damage, detected by comet assay after additional treatment with repair enzymes, was induced in all cell systems already at five-fold lower concentrations. Glutathione in CV-1 cells was depleted after 1 h incubation (>100 μmol/L). In contrast, an increase was measured after 24 h incubation (>0.5 μmol/L). In conclusion, OTA induces oxidative DNA damage at low, not yet cytotoxic concentrations. Oxidative DNA damage might initiate cell transformation eventually in connection with proliferative response following cytotoxic cell death. Both events might represent pivotal factors in the chain of cellular events leading into nephro-carcinogenicity of OTA

  18. Inhibition of DNA primase and induction of apoptosis by 3,3'-diethyl-9-methylthia-carbocyanine iodide in hepatocellular carcinoma BEL-7402 cells

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ming Li; Zong-Chao Liu; Zhong-Zhen Guan; Xiao-Feng Zhu; Jun-Min Zhou; Bing-Fen Xie; Gong-Kan Feng; Zhen-Yu Zhu; Wen-Qi Jiang

    2004-01-01

    AIM: To evaluate the effects of 3,3'-diethyl-9-methylthia carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.RESULTS: DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09 μmol/L. Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation)and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53,Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase3 might be involved in the induction of apoptosis.

  19. DNA damage, apoptosis and cell cycle changes induced by fluoride in rat oral mucosal cells and hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Ling-Fei He; Jian-Gang Chen

    2006-01-01

    AIM: To study the effect of fluoride on oxidativestress,DNA damage and apoptosis as well as cell cycle of rat oral mucosal cells and hepatocytes.METHODS: Ten male SD rats weighing 80~120 g were randomly divided into control group and fluoride group,5 animals each group. The animals in fluoride group had free access to deionized water containing 150 mg/L sodium fluoride (NaF). The animals in control group were given distilled water. Four weeks later, the animals were killed. Reactive oxygen species (ROS) in oral mucosa and liver were measured by Fenton reaction, lipid peroxidation product, malondialdehyde (MDA), was detected by thiobarbituric acid (TBA) reaction, reduced glutathione (GSH) was assayed by dithionitrobenzoic acid (DTNB)reaction. DNA damage in oral mucosal cells and hepatocytes was determined by single cell gel (SCG) electrophoresis or comet assay. Apoptosis and cell cycle in oral mucosal cells and hepatocytes were detected by flow cytometry.RESULTS: The contents of ROS and MDA in oral mucosa and liver tissue of fluoride group were significantly higher than those of control group (P<0.01), but the level of GSH was markedly decreased (P<0.01). The contents of ROS, MDA and GSH were (134.73±12.63) U/mg protein,(1.48±0.13) mmol/mg protein and (76.38±6.71) mmol/mg protein in oral mucosa respectively, and (143.45±11.76) U/mg protein, (1.44±0.12) mmol/mg protein and (78.83±7.72) mmol/mg protein in liver tissue respectively. The DNA damage rate in fluoride group was 50.20%in oral mucosal cells and 44.80% in hepatocytes, higher than those in the control group (P<0.01). The apoptosis rate in oral mucosal cells was (13.63±1.81) % in fluoride group, and (12.76±1.67) % in hepatocytes,higher than those in control group. Excess fluoride could differently lower the number of oral mucosal cells and hepatocytes at G0/G1 and S G2/M phases (P<0.05).CONCLUSION: Excess fluoride can induce oxidative stress and DNA damage and lead to apoptosis and cell cycle

  20. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis*

    OpenAIRE

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-01-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycl...

  1. Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases.

    Science.gov (United States)

    Clark-Ganheart, Cecily A; Iqbal, Sara N; Brown, Donna L; Black, Susan; Fries, Melissa H

    2014-05-01

    Circulating cell free fetal DNA (cffDNA) is an effective screening modality for fetal aneuploidy. We report two cases of false positive results. The first case involves a female, with self-reported Down syndrome. CffDNA returned positive for trisomy 18 leading to a maternal diagnosis of mosaicism chromosome 18 with normal fetal karyotype. The second case involves a patient with an anomalous fetal ultrasound and cffDNA positive for trisomy 13. Amniocentesis demonstrated a chromosome 8p duplication/deletion. False positive cffDNA may arise in clinical scenarios where diagnostic testing is clearly indicated. Practitioners should recognize the limitations of cffDNA. PMID:25298847

  2. Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Guo Chun; Zhu Faliang [Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Yang Qiaozi [Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States); Gao Guimin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Gong Yaoqin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China)], E-mail: yxg8@sdu.edu.cn; Shao Changshun [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)], E-mail: shao@biology.rutgers.edu

    2009-03-09

    Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis.

  3. Deficiency of DNA fragmentation factor 45 results in reduced oocyte apoptosis in response to doxorubicin

    Institute of Scientific and Technical Information of China (English)

    Mei Dong; Yunxia Fan; Nicholas J Toepfer; Jianhua Zhang

    2007-01-01

    @@ Apoptosis plays a prominent role in ovarian development and function [1-4]. During follicle development, the vast majority of follicles undergo atresia as a consequence of apoptosis of constituent oocyte or follicular cells, or both, failing to complete the maturation process. Atresia occurs at all stages of follicular development during the growth and development of follicles. Atresia of the primordial and primary follicles is initiated by oocyte apoptosis, followed by the death of the granulosa cells [1-4]. In comparison, the reduction of the large number of growing follicles to a single ovulatory follicle is achieved primarily by the cell death of granulosa cells [1-4]. Members of the Bcl-2 and caspase families play important roles in the regulation of ovarian cell death, both in follicle development and in response to chemotherapeutic agents [5].

  4. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3-dependent apoptosis.

    Science.gov (United States)

    Zhao, Yuwan; Jing, Zuolei; Li, Yan; Mao, Weifeng

    2016-07-01

    Berberine (BBR) is an isoquinoline alkaloid extracted from medicinal plants such as Hydrastis canadensis, Berberis aristata and Coptis chinensis. BBR displays a number of beneficial roles in the treatment of various types of cancers, yet the precise mechanisms of its action remain unclear. Cisplatin is an effective cancer chemotherapeutic agent and functions by generating DNA damage, promoting DNA damage-induced cell cycle arrest and apoptosis; however, its efficacy is challenged by the resistance of tumor cells in clinical application. The aim of the present study was to investigate the effects of BBR in combination with cisplatin on human breast cancer cells. MTT assay showed that BBR inhibited breast cancer MCF-7 cell growth with a 50% inhibitory concentration (IC50) value of 52.178±1.593 µM and the IC50 value of cisplatin was 49.541±1.618 µM, while in combination with 26 µM BBR, the IC50 value of cisplatin was 5.759±0.76 µM. BBR sensitized the MCF-7 cells to cisplatin in a time- and dose-dependent manner. After treatment of BBR and cisplatin, the cellular pro-apoptotic capase-3 and cleaved capspase-3 and caspase-9 were upregulated and the anti-apoptotic Bcl-2 was downregulated. Importantly, BBR restrained the expression of cellular PCNA, and immunofluoresence analysis of γH2AX showed that BBR increased the DNA damages induced by cisplatin. Taken together, the results demonstrated that BBR sensitized MCF-7 cells to cisplatin through induction of DNA breaks and caspase-3-dependent apoptosis. PMID:27177238

  5. RRR-α-tocopheryl succinate inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

    Institute of Scientific and Technical Information of China (English)

    Kun Wu; Yan Zhao; Bai-He Liu; Yao Li; Fang Liu; Jian Guo; Wei-Ping Yu

    2002-01-01

    AIM: To investigate the effects of growth inhibition ofhuman gastric cancer SGC-7901 cell with RRR-α-tocopherylsuccinate (VES), a derivative of natural Vitamin E, viainducing apoptosis and DNA synthesis arrest.METHODS: Human gastric cancer SGC-7901 cells wereregularly incubated in the presence of VES at 5, 10 and20mg@ L 1(VES was dissolved in absolute ethanol anddiluted in RPMI 1640 complete condition mediacorrespondingly to a final concentration of VES and 1mL@L-1 ethanol), succinic acid and ethanol equivalents asvehicle (VEH) control andcondition media only asuntreated (UT) control. Trypan blue dye exclusionanalysis and MTT assay were applied to detect the cellproliferation. 37kBq of tritiated thymidine was added tocells and [3H] TdR uptake was measured to observe DNAsynthesis. Apoptotic morphology was observed byelectron microscopy and DAPI staining. Flow cytometryand terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) assay were performed to detectVES-triggered apoptosis.RESULTS: VES inhibited SGC-7901 cell growth in a dose-dependent manner. The growth curve showed suppressionby 24.7%, 49.2% and 68.7% following 24h of VEStreatment at 5, 10 and 20 mg@L 1, respectively, similar tothe findings from MTT assay. DNA synthesis wasevidently reduced by 35%, 45% and 98% after 24h VEStreatment at 20 mg@ L-1 and 48h at 10 and 20 mg@ L 1,respectively. VES induced SGC-7901 cells to undergoapoptosis with typically apoptotic characteristics,including morphological changes of chromatincondensation, chromatin crescent formation/margination,nucleus fragmentation and apoptotic body formation,typical apoptotic sub-G1 peak by flow cytometry andincrease of apoptotic cells by TUNEL assay in which 90%of cells underwent apoptosis after 48h of VES treatment at20 mcg@L-1.CONCLUSION: VES can inhibit human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesisarrest. Inhibition of SGC-7901 cell growth by VES is dose-and time

  6. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    Science.gov (United States)

    Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-01-01

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

  7. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

    Science.gov (United States)

    Arora, Shagun; Tandon, Simran

    2015-01-01

    In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

  8. Antiproliferative effects of goniothalamin on Ca9-22 oral cancer cells through apoptosis, DNA damage and ROS induction.

    Science.gov (United States)

    Yen, Ching-Yu; Chiu, Chien-Chih; Haung, Rou-Wen; Yeh, Chi-Chen; Huang, Kuang-Jing; Chang, Kuo-Feng; Hseu, You-Cheng; Chang, Fang-Rong; Chang, Hsueh-Wei; Wu, Yang-Chang

    2012-09-18

    Goniothalamin (GTN), a plant bioactive styryl-lactone, is a natural product with potent anti-tumorigenesis effects for several types of cancer. Nonetheless, the anticancer effect of GTN has not been examined in oral cancer. The present study was designed to evaluate its potential anticancer effects in an oral squamous cell carcinoma (OSCC) model and to determine the possible mechanisms with respect to apoptosis, DNA damage, reactive oxygen species (ROS) induction, and mitochondrial membrane potential. Our data demonstrated that cell proliferation was significantly inhibited by GTN in Ca9-22 OSCC cancer cells in concentration- and time-dependent manners (pGTN-treated Ca9-22 cancer cells, the sub-G1 population and annexin V-intensity significantly increased in a concentration-dependent manner (pGTN-treated Ca9-22 cancer cells in concentration-response relationship (pGTN significantly induced intracellular ROS levels in Ca9-22 cancer cells in a concentration- and time-dependent manner (pGTN-treated Ca9-22 cancer cells was significantly decreased in concentration- and time-dependent relationships (pGTN against oral cancer cells is valid and GTN-induced growth inhibition and apoptosis influence the downstream cascade including ROS induction, DNA damage, and mitochondria membrane depolarization. Therefore, GTN has potential as a chemotherapeutic agent against oral cancer.

  9. Methacryloxylethyl Cetyl Ammonium Chloride Induces DNA Damage and Apoptosis in Human Dental Pulp Cells via Generation of Oxidative Stress.

    Science.gov (United States)

    Jiao, Yang; Ma, Sai; Wang, Yirong; Li, Jing; Shan, Lequn; Sun, Jinlong; Chen, Jihua

    2016-01-01

    The polymerizable antibacterial monomer methacryloxylethyl cetyl ammonium chloride (DMAE-CB) has provided an effective strategy to combat dental caries. However, the application of such material raises the question about the biological safety and the question remains open. The mechanism of this toxic action, however, is not yet clearly understood. The present study aims at providing novel insight into the possible causal link between cellular oxidative stress and DNA damage, as well as apoptosis in human dental pulp cells exposed to DMAE-CB. The enhanced formation of reactive oxygen species and depletion of glutathione, as well as differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase in DMAE-CB-treated cells indicated oxidative stress. By using substances that can alter GSH synthesis, we found that GSH was the key component in the regulation of cell response towards oxidative stress induced by DMAE-CB. The increase in oxidative stress-sensitive 8-Oxo-2'-deoxyguanosine (8-OHdG) content, formation of γ-H2AX and cell cycle G1 phase arrest indicated that DNA damage occurred as a result of the interaction between DNA base and ROS beyond the capacities of antioxidant mechanisms in cells exposed to DMAE-CB. Such oxidative DNA damage thus triggers the activation of ataxia telangiectasia-mutated (ATM) signaling, the intrinsic apoptotic pathway, and destruction of mitochondrial morphology and function. PMID:27143955

  10. Studies of ruthenium(II) polypyridyl complexes on cytotoxicity in vitro, apoptosis, DNA-binding and antioxidant activity

    Science.gov (United States)

    Huang, Hong-Liang; Liu, Yun-Jun; Zeng, Cheng-Hui; Yao, Jun-Hua; Liang, Zhen-Hua; Li, Zheng-Zheng; Wu, Fu-Hai

    2010-03-01

    Two new ruthenium(II) polypyridyl complexes [Ru(dmb) 2(maip)](ClO 4) 21 (maip = 2-(3-aminophenyl)imizado[4,5-f][1,10]phenanthroline and [Ru(dmb) 2(maip)](ClO 4) 22 (paip = 2-(4-aminophenyl)imidazo[4,5-f][1,10]phenanthroline, dmb = 4,4'-dimethyl-2,2'-bipyridine) have been synthesized and characterized. The DNA-binding behaviors of complexes 1 and 2 were studied by viscosity measurements, thermal denaturation, photocleavage, absorption titration and luminescence spectra. The results show that the two complexes intercalate between the base pairs of DNA. The DNA-binding constants Kb for complexes 1 and 2 were determined to be 1.12 ± 0.11 × 10 5 M -1 ( s = 2.17) and 3.46 ± 0.59 × 10 5 M -1 ( s = 2.11) M -1. The studies on the mechanism of photocleavage demonstrate that superoxide anion radical (O 2rad - ) and singlet oxygen ( 1O 2) may play an important role. The cytotoxicity of these complexes has been evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The IC 50 values are 19.21, 33.15, 38.57 and 21.15 for complex 1 and 41.77, 123.58, 255.44 and 49.11 for complex 2 against BEL-7402, C-6, HepG-2 and MCF-7 cell lines, respectively. The apoptosis assay was carried out with acridine orange/ethidium bromide (AO/EB) staining methods and the results indicate that complexes can induce the apoptosis of BEL-7402 cells. The experiments on antioxidant activity show these complexes exhibit good antioxidant activity against hydroxyl radical (OH rad ).

  11. Simulated Microgravity Promotes Cell Apoptosis Through Suppressing Uev1A/TICAM/TRAF/NF-κB-Regulated Anti-Apoptosis and p53/PCNA- and ATM/ATR-Chk1/2-Controlled DNA-Damage Response Pathways.

    Science.gov (United States)

    Zhao, Tuo; Tang, Xin; Umeshappa, Channakeshava Sokke; Ma, Hong; Gao, Haijun; Deng, Yulin; Freywald, Andrew; Xiang, Jim

    2016-09-01

    Microgravity has been known to induce cell death. However, its underlying mechanism is less studied. In this study, BL6-10 melanoma cells were cultured in flasks under simulated microgravity (SMG). We examined cell apoptosis, and assessed expression of genes associated with apoptosis and genes regulating apoptosis in cells under SMG. We demonstrate that SMG induces cell morphological changes and microtubule alterations by confocal microscopy, and enhances apoptosis by flow cytometry, which was associated with up- and down-regulation of pro-apoptotic and anti-apoptotic genes, respectively. Moreover, up- and down-regulation of pro-apoptotic (Caspases 3, 7, 8) and anti-apoptotic (Bcl2 and Bnip3) molecules was confirmed by Western blotting analysis. Western blot analysis also indicates that SMG causes inhibition of an apoptosis suppressor, pNF-κB-p65, which is complemented by the predominant localization of NF-κB-p65 in the cytoplasm. SMG also reduces expression of molecules regulating the NF-κB pathway including Uev1A, TICAM, TRAF2, and TRAF6. Interestingly, 10 DNA repair genes are down-regulated in cells exposed to SMG, among which down-regulation of Parp, Ercc8, Rad23, Rad51, and Ku70 was confirmed by Western blotting analysis. In addition, we demonstrate a significant inhibition of molecules involved in the DNA-damage response, such as p53, PCNA, ATM/ATR, and Chk1/2. Taken together, our work reveals that SMG promotes the apoptotic response through a combined modulation of the Uev1A/TICAM/TRAF/NF-κB-regulated apoptosis and the p53/PCNA- and ATM/ATR-Chk1/2-controlled DNA-damage response pathways. Thus, our investigation provides novel information, which may help us to determine the cause of negative alterations in human physiology occurring at spaceflight environment. J. Cell. Biochem. 117: 2138-2148, 2016. © 2016 Wiley Periodicals, Inc. PMID:26887372

  12. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples

    OpenAIRE

    Minsoung Rhee; Yooli K Light; Meagher, Robert J.; Anup K. Singh

    2016-01-01

    Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template D...

  13. Common genomic signaling among initial DNA damage and radiation-induced apoptosis in peripheral blood lymphocytes from locally advanced breast cancer patients

    DEFF Research Database (Denmark)

    Henríquez-Hernández, Luis Alberto; Pinar, Beatriz; Carmona-Vigo, Ruth;

    2013-01-01

    PURPOSE: To investigate the genomic signaling that defines sensitive lymphocytes to radiation and if such molecular profiles are consistent with clinical toxicity; trying to disclose the radiobiology mechanisms behind these cellular processes. PATIENTS AND METHODS: Twelve consecutive patients...... suffering from locally advanced breast cancer and treated with high-dose hyperfractionated radiotherapy were recruited. Initial DNA damage was measured by pulsed-field gel electrophoresis and radiation-induced apoptosis was measured by flow cytometry. Gene expression was assessed by DNA microarray. RESULTS...

  14. Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

    Directory of Open Access Journals (Sweden)

    Yasui Akira

    2005-10-01

    Full Text Available Abstract Background UV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations. Methods NER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m2 UVB exposure photoreactivation light (PR, UVA 60 kJ/m2 was provided for photolyase activation and DNA repair. Apoptosis was monitored 24 hours later by flow cytometric analysis of DNA content, using sub-G1 staining to indicate apoptotic cells. To confirm the effects observed with CPD lesions, the molecular mimic of CPD, pTpT, was also tested in vitro and in vivo for its effect on cell cycle and apoptosis. Results The specific repair of 6-4PP lesions after UVB exposure resulted in a dramatic reduction in apoptosis. These findings suggested that 6-4PP lesions may be the primary inducer of UVB-induced apoptosis. Repair of CPD lesions (despite their relative abundance in the UV-damaged cell had little effect on the induction of apoptosis. Supporting these findings, the molecular mimic of CPD, (dinucleotide pTpT could mimic the effects of UVB on cell cycle arrest, but were ineffective to induce apoptosis. Conclusion The primary response of the cell to UV-induced 6-4PP lesions is to trigger an apoptotic program whereas the response of the cell to CPD

  15. Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines

    OpenAIRE

    Leitner, Wolfgang W.; Hwang, Leroy N.; Elke S Bergmann-Leitner; Finkelstein, Steven E.; Frank, Stephan; Restifo, Nicholas P

    2004-01-01

    Alphaviral replicons can increase the efficacy and immunogenicity of naked nucleic acid vaccines. To study the impact of apoptosis on this increased effectiveness, we co-delivered an anti-apoptotic gene (Bcl-XL) with the melanocyte/melanoma differentiation antigen TRP-1. Although cells co-transfected with Bcl-XL lived longer, produced more antigen and elicited increased antibody production in vivo, co-delivery of pro-survival Bcl-XL with antigen significantly reduced the ability of the replic...

  16. UV Differentially Induces Oxidative Stress, DNA Damage and Apoptosis in BCR-ABL1-Positive Cells Sensitive and Resistant to Imatinib

    Directory of Open Access Journals (Sweden)

    Ewelina Synowiec

    2015-08-01

    Full Text Available Chronic myeloid leukemia (CML cells express the active BCR-ABL1 protein, which has been targeted by imatinib in CML therapy, but resistance to this drug is an emerging problem. BCR-ABL1 induces endogenous oxidative stress promoting genomic instability and imatinib resistance. In the present work, we investigated the extent of oxidative stress, DNA damage, apoptosis and expression of apoptosis-related genes in BCR-ABL1 cells sensitive and resistant to imatinib. The resistance resulted either from the Y253H mutation in the BCR-ABL1 gene or incubation in increasing concentrations of imatinib (AR. UV irradiation at a dose rate of 0.12 J/(m2·s induced more DNA damage detected by the T4 pyrimidine dimers glycosylase and hOGG1, recognizing oxidative modifications to DNA bases in imatinib-resistant than -sensitive cells. The resistant cells displayed also higher susceptibility to UV-induced apoptosis. These cells had lower native mitochondrial membrane potential than imatinib-sensitive cells, but UV-irradiation reversed that relationship. We observed a significant lowering of the expression of the succinate dehydrogenase (SDHB gene, encoding a component of the complex II of the mitochondrial respiratory chain, which is involved in apoptosis sensing. Although detailed mechanism of imatinib resistance in AR cells in unknown, we detected the presence of the Y253H mutation in a fraction of these cells. In conclusion, imatinib-resistant cells may display a different extent of genome instability than their imatinib-sensitive counterparts, which may follow their different reactions to both endogenous and exogenous DNA-damaging factors, including DNA repair and apoptosis.

  17. Opposed arsenite-mediated regulation of p53-survivin is involved in neoplastic transformation, DNA damage, or apoptosis in human keratinocytes

    International Nuclear Information System (INIS)

    Highlights: ► Different concentrations of arsenite cause biphasic effects in HaCaT cells. ► p53-survivin signal pathway plays a role in arsenite-induced biphasic effects. ► ERKs inactivate p53, but improve survivin expression by NF-κB/mot-2. ► JNKs block survivin expression by preventing p53 from mdm2-mediated degradation. ► ERKs and JNKs play roles in arsenite-induced biphasic effects. -- Abstract: Biphasic dose–response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose–response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. Our present study shows that, for human keratinocytes (HaCaT) cells, a low concentration of arsenite activates extracellular signal-regulated kinases (ERKs), which leads to up-regulation of nuclear factor κB (NF-κB) binding to DNA and to elevated, NF-κB-dependent expression of mot-2 (a p53 inhibitor) and survivin (an inhibitor of apoptosis). Activation of p53 is blocked, and neoplastic transformation is enhanced. Inhibition of ERKs reduces cell proliferation and neoplastic transformation. In contrast, a high concentration of arsenite activates c-Jun N-terminal kinases (JNKs), positive regulators of p53, by binding to p53 and preventing its murine double minute 2 (mdm2)-mediated degradation. The elevated levels of p53 lead to repair of DNA damage and apoptosis. Inhibition of JNKs increases DNA damage but decreases apoptosis. By identifying a mechanism whereby ERKs and JNKs-mediated regulation of the p53-survivin signal pathway is involved in the biphasic effects of arsenite on human keratinocytes, our data expand understanding of arsenite-induced cell proliferation, neoplastic transformation, DNA damage, and apoptosis.

  18. NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Aruna S Jaiswal

    Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

  19. DAZL Limits Pluripotency, Differentiation, and Apoptosis in Developing Primordial Germ Cells

    Directory of Open Access Journals (Sweden)

    Hsu-Hsin Chen

    2014-11-01

    Full Text Available The scarcity of primordial germ cells (PGCs in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.

  20. JA, a new type of polyunsaturated fatty acid isolated from Juglans mandshurica Maxim, limits the survival and induces apoptosis of heptocarcinoma cells.

    Science.gov (United States)

    Gao, Xiu-Li; Lin, Hua; Zhao, Wei; Hou, Ya-Qin; Bao, Yong-Li; Song, Zhen-Bo; Sun, Lu-Guo; Tian, Shang-Yi; Liu, Biao; Li, Yu-Xin

    2016-03-01

    Juglans mandshurica Maxim (Juglandaceae) is a famous folk medicine for cancer treatment and some natural compounds isolated from it have been studied extensively. Previously we isolated a type of ω-9 polyunsaturated fatty acid (JA) from the bark of J. mandshurica, however little is known about its activity and the underlying mechanisms. In this study, we studied anti-tumor activity of JA on several human cancer cell lines. Results showed that JA is cytotoxic to HepG2, MDA-MB-231, SGC-7901, A549 and Huh7 cells at a concentration exerting minimal toxic effects on L02 cells. The selective toxicity of JA was better than other classical anti-cancer drugs. Further investigation indicated that JA could induce cell apoptosis, characterized by chromatin condensation, DNA fragmentation and activation of the apoptosis-associated proteins such as Caspase-3 and PARP-1. Moreover, we investigated the cellular apoptosis pathway involved in the apoptosis process in HepG2 cells. We found that proteins involved in mitochondrion (cleaved-Caspase-9, Apaf-1, HtrA2/Omi, Bax, and Mitochondrial Bax) and endocytoplasmic reticulum (XBP-1s, GRP78, cleaved-Caspase-7 and cleaved-Caspase-12) apoptotic pathways were up-regulated when cells were treated by JA. In addition, a morphological change in the mitochondrion was detected. Furthermore, we found that JA could inhibit DNA synthesis and induce G2/M cell cycle arrest. The expression of G2-to-M transition related proteins, such as CyclinB1 and phosphorylated-CDK1, were reduced. In contrast, the G2-to-M inhibitor p21 was increased in JA-treated cells. Overall, our results suggest that JA can induce mitochondrion- and endocytoplasmic reticulum-mediated apoptosis, and G2/M phase arrest in HepG2 cells, making it a promising therapeutic agent against hepatoma.

  1. AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

    Science.gov (United States)

    Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

    2006-05-01

    The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability. PMID:25873751

  2. Evaluation of cell proliferation, apoptosis, and dna-repair genes as potential biomarkers for ethanol-induced cns alterations

    Directory of Open Access Journals (Sweden)

    Hicks Steven D

    2012-10-01

    Full Text Available Abstract Background Alcohol use disorders (AUDs lead to alterations in central nervous system (CNS architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Results Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1 was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5 showed a highly significant correlation with AUD-induced decreases in the volume of the left

  3. Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Holst, Bjørn; Tümer, Zeynep;

    2014-01-01

    The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led...... and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion...

  4. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    Directory of Open Access Journals (Sweden)

    Natalia Bailon-Moscoso

    Full Text Available Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL, a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

  5. DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

    OpenAIRE

    Nagao, Toshikage; Oshikawa, Gaku; Wu, Nan; Kurosu, Tetsuya; Miura, Osamu

    2011-01-01

    The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR). Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin...

  6. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

    Directory of Open Access Journals (Sweden)

    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  7. Chromatin and DNA chain fragmentation studies on apoptosis in immune cells induced by 235U and radioprotection of IL—2 or IL—6

    Institute of Scientific and Technical Information of China (English)

    ZhuShou-Peng; ZhangLan-Sheng; 等

    1998-01-01

    The apoptosis in human acute lymphoblastic leukemia cell line Molt-4 cell and macrophage cell line Ana-1 cell are studied after internal irradiation with enriched uranium 235U.The cumulative absorption dose of 235U in cultural cells through different periods are estimated.The fluorescence microscopic observations indicate that Molt-4 and Ana-1 immune cells internally irradiated by 235U displayed significant chromatin fragmentation and marked pyknosis in immune cells nuclei.as well as DNA chain fragmentation and apoptotic bodies formation.It should be noted that DNA chain fragmentation induced by 235U may be inhibited statistically by IL-2(interleukin-2)or IL-6 treatment.

  8. ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jun; Jo, Yong Hwa; Cho, Chang Hoon; Choe, Wonchae; Kang, Insug; Baik, Hyung Hwan [Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of); Yoon, Kyung-Sik, E-mail: sky9999@khu.ac.kr [Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701 (Korea, Republic of)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer A-T cells were not hypersensitive to low levels of DNA DSBs. Black-Right-Pointing-Pointer A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. Black-Right-Pointing-Pointer A-T cells underwent premature senescence after DNA damage accumulated. Black-Right-Pointing-Pointer Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-{beta}-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.

  9. Mitochondrial DNA damage and dysfunction, and oxidative stress are associated with endoplasmic reticulum stress, protein degradation and apoptosis in high fat diet-induced insulin resistance mice.

    Directory of Open Access Journals (Sweden)

    Larysa V Yuzefovych

    Full Text Available BACKGROUND: Recent studies showed a link between a high fat diet (HFD-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR. Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6J male mice were fed either a HFD (60% fat or normal chow (NC (10% fat for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.

  10. SSB protein limits RecOR binding onto single-stranded DNA.

    Science.gov (United States)

    Hobbs, Michael D; Sakai, Akiko; Cox, Michael M

    2007-04-13

    The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action. PMID:17272275

  11. The DNA-mismatch repair enzyme hMSH2 modulates UV-B-induced cell cycle arrest and apoptosis in melanoma cells.

    Science.gov (United States)

    Seifert, Markus; Scherer, Stefan J; Edelmann, Wilfried; Böhm, Markus; Meineke, Viktor; Löbrich, Markus; Tilgen, Wolfgang; Reichrath, Jörg

    2008-01-01

    The mechanisms by which the post-replicative DNA mismatch repair (MMR) enzyme MSH2 is involved in the complex response mechanisms to UV damage are yet to be clarified. Here, we show increased levels of MSH2 mRNA in malignant melanoma, metastases of melanoma, and melanoma cell (MeWo) lines as compared with melanocytic nevi or primary cultured benign melanocytes. UV-B treatment modulated MSH2 expression and silencing of MSH2 gene expression using small interfering RNA technology regulated UV-B-induced cell cycle arrest and apoptosis in human MeWo. We show that MSH2-deficient non-malignant mouse fibroblasts (MEF-/-) are partially resistant against UV-B-induced apoptosis and show reduced S-Phase accumulation. In addition, we show that an Msh2 point mutation (MEFGA) that affects MMR does not affect UV-B-induced apoptosis. In conclusion, we demonstrate that MSH2 modulates in human melanocytes both UV-B-induced cell cycle regulation and apoptosis, most likely via independent, uncoupled mechanisms.

  12. Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis.

    Directory of Open Access Journals (Sweden)

    Qi Wang

    Full Text Available BACKGROUND: To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines. RESULTS: Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352% in BLM-resistant sub-clones (p<0.05 for both. Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05. Significantly reduced DNA damage (COMET and γ-H2AX assays, G2/M arrest, and apoptosis (p<0.05 for each set of comparison following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05. CONCLUSION: Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

  13. SF Treg cells transcribing high levels of Bcl-2 and microRNA-21 demonstrate limited apoptosis in RA

    NARCIS (Netherlands)

    van der Geest, Kornelis S. M.; Smigielska, Katarzyna; Park, Ji-Ah; Abdulahad, Wayel H.; Kim, Hye-Won; Kroesen, Bart-Jan; van den Berg, Anke; Boots, Annemieke M. H.; Lee, Eun-Bong; Brouwer, Elisabeth

    2015-01-01

    Objective. The aim of this study was to investigate the turnover of Treg cells in the SF of RA patients. Methods. Treg cells were enumerated in peripheral blood and SF of RA patients and analysed by flow cytometry for expression of the proliferation marker Ki-67 and binding of the apoptosis marker a

  14. Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia

    Directory of Open Access Journals (Sweden)

    Bitar Fadi F

    2006-07-01

    Full Text Available Abstract Background The aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2. Methods Newborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression. Results TUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days. Conclusion Exposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

  15. α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells.

    Science.gov (United States)

    Lin, Jen-Jyh; Yu, Chien-Chih; Lu, Kung-Wen; Chang, Shu-Jen; Yu, Fu-Shun; Liao, Ching-Lung; Lin, Jaung-Geng; Chung, Jing-Gung

    2014-08-01

    α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro. PMID:25075043

  16. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xinwei Zhao

    2016-06-01

    Full Text Available Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s were investigated. A transmission electron microscopy (TEM assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca2+ levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway.

  17. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway.

    Science.gov (United States)

    Zhao, Xinwei; Tao, Xufeng; Xu, Lina; Yin, Lianhong; Qi, Yan; Xu, Youwei; Han, Xu; Peng, Jinyong

    2016-01-01

    Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s) were investigated. A transmission electron microscopy (TEM) assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca(2+) levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway. PMID:27271587

  18. DNA binding, cytotoxicity and apoptosis induction activity of a mixed-ligand copper(II) complex with taurine Schiff base and imidazole

    Science.gov (United States)

    Li, Mei; kong, Lin Lin; Gou, Yi; Yang, Feng; Liang, Hong

    2014-07-01

    A novel binuclear copper(II) complex (complex 1) with taurine Schiff base and imidazole has been synthesized and structurally characterized by single crystal X-ray diffraction, elemental analysis, ESI-MS spectrometry, UV-vis and IR spectroscopy. Single-crystal analysis revealed that 1 displays the sulfonate-bridged dinuclear copper(II) centers. Both copper atoms are five-coordinated and exhibit slightly distorted square pyramidal geometries. Each of copper atom is surrounded by three oxygen atoms and one nitrogen atom from different taurine Schiff base ligands, and one nitrogen atom from one imidazole ligand. The interaction between 1 and calf thymus DNA (CT-DNA) was investigated by UV-vis, fluorescence, circular dichroism (CD) spectra and agarose gel electrophoresis. The experimental results indicated that 1 could bind to CT-DNA via an intercalative mode and show efficient cleavage activity. In addition, 1 showed an antitumor effect on cell cycle and apoptosis. Flow cytometric analysis revealed that MGC-803 cells were arrested in the S phase after treatment with 1. Fluorescence microscopic observation indicated that 1 could induce apoptosis of MGC-803 cells.

  19. Threshold Dose of Three Types of Quantum Dots (QDs Induces Oxidative Stress Triggers DNA Damage and Apoptosis in Mouse Fibroblast L929 Cells

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    Ting Zhang

    2015-10-01

    Full Text Available Although it has been reported that fluorescent quantum dots (QDs have obvious acute toxic effects in vitro, their toxic effects at low doses or threshold doses are still unknown. Therefore, we evaluated the biological histocompatibility and in vitro toxicity of three types of QDs at threshold doses. Also, we compared the toxic effects of QDs with different raw chemical compositions and sizes. The results showed that low concentrations of QDs (≤7 μg/mL had no obvious effect on cell viability and cell membrane damage, oxidative damage, cell apoptosis or DNA damage. However, QD exposure led to a significant cytotoxicity at higher doses (≥14 μg/mL and induced abnormal cellular morphology. In addition, when comparing the three types of QDs, 2.2 nm CdTe QDs exposure showed a significantly increased proportion of apoptotic cells and significant DNA damage, suggesting that size and composition contribute to the toxic effects of QDs. Based on these discussions, it was concluded that the concentration (7 μg/mL may serve as a threshold level for these three types of QDs only in L929 fibroblasts, whereas high concentrations (above 14 μg/mL may be toxic, resulting in inhibition of proliferation, induction of apoptosis and DNA damage in L929 fibroblasts.

  20. Demethoxycurcumin alters gene expression associated with DNA damage, cell cycle and apoptosis in human lung cancer NCI-H460 cells in vitro.

    Science.gov (United States)

    Ko, Yang-Ching; Hsu, Shu-Chun; Liu, Hsin-Chung; Hsiao, Yung-Ting; Hsia, Te-Chun; Yang, Su-Tso; Hsu, Wu-Huei; Chung, Jing-Gung

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths and new lung cancer cases are continuously emerging around the globe; however, treatment of lung cancer remains unsatisfactory. Demethoxycurcumin (DMC) has been shown to exert cytotoxic effects in human cancer cells via induction of apoptosis. However, the effects of DMC on genetic mechanisms associated with these actions have not been yet elucidated. Human lung cancer NCI-H460 cells were incubated with or without 35 μM of DMC for 24 h and total RNA was extracted for cDNA synthesis labeling and microarray hybridization, followed by fluor-labeled cDNA hybridization on chip. Expression Console software with default Robust Multichip Analysis (RMA) parameters were used for detecting and quantitating the localized concentrations of fluorescent molecules. The GeneGo software was used for investigating key genes involved and their possible interaction pathways. Genes associated with DNA damage and repair, cell-cycle check point and apoptosis could be altered by DMC; in particular, 144 genes were found up-regulated and 179 genes down-regulated in NCI-H460 cells after exposure to DMC. In general, DMC-altered genes may offer information to understand the cytotoxic mechanism of this agent at the genetic level since gene alterations can be useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future. PMID:25600535

  1. Demethoxycurcumin alters gene expression associated with DNA damage, cell cycle and apoptosis in human lung cancer NCI-H460 cells in vitro.

    Science.gov (United States)

    Ko, Yang-Ching; Hsu, Shu-Chun; Liu, Hsin-Chung; Hsiao, Yung-Ting; Hsia, Te-Chun; Yang, Su-Tso; Hsu, Wu-Huei; Chung, Jing-Gung

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths and new lung cancer cases are continuously emerging around the globe; however, treatment of lung cancer remains unsatisfactory. Demethoxycurcumin (DMC) has been shown to exert cytotoxic effects in human cancer cells via induction of apoptosis. However, the effects of DMC on genetic mechanisms associated with these actions have not been yet elucidated. Human lung cancer NCI-H460 cells were incubated with or without 35 μM of DMC for 24 h and total RNA was extracted for cDNA synthesis labeling and microarray hybridization, followed by fluor-labeled cDNA hybridization on chip. Expression Console software with default Robust Multichip Analysis (RMA) parameters were used for detecting and quantitating the localized concentrations of fluorescent molecules. The GeneGo software was used for investigating key genes involved and their possible interaction pathways. Genes associated with DNA damage and repair, cell-cycle check point and apoptosis could be altered by DMC; in particular, 144 genes were found up-regulated and 179 genes down-regulated in NCI-H460 cells after exposure to DMC. In general, DMC-altered genes may offer information to understand the cytotoxic mechanism of this agent at the genetic level since gene alterations can be useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future.

  2. DNA Barcoding for the Identification of Botanicals in Herbal Medicine and Dietary Supplements: Strengths and Limitations.

    Science.gov (United States)

    Parveen, Iffat; Gafner, Stefan; Techen, Natascha; Murch, Susan J; Khan, Ikhlas A

    2016-09-01

    In the past decades, the use of traditional medicine has increased globally, leading to a booming herbal medicine and dietary supplement industry. The increased popularity of herbal products has led to a rise in demand for botanical raw materials. Accurate identification of medicinal herbs is a legal requirement in most countries and prerequisite for delivering a quality product that meets consumer expectations. Traditional identification methods include botanical taxonomy, macroscopic and microscopic examination, and chemical methods. Advances in the identification of biological species using DNA-based techniques have led to the development of a DNA marker-based platform for authentication of plant materials. DNA barcoding, in particular, has been proposed as a means to identify herbal ingredients and to detect adulteration. However, general barcoding techniques using universal primers have been shown to provide mixed results with regard to data accuracy. Further technological advances such as mini-barcodes, digital polymerase chain reaction, and next generation sequencing provide additional tools for the authentication of herbs, and may be successful in identifying processed ingredients used in finished herbal products. This review gives an overview on the strengths and limitations of DNA barcoding techniques for botanical ingredient identification. Based on the available information, we do not recommend the use of universal primers for DNA barcoding of processed plant material as a sole means of species identification, but suggest an approach combining DNA-based methods using genus- or species-specific primers, chemical analysis, and microscopic and macroscopic methods for the successful authentication of botanical ingredients used in the herbal dietary supplement industry.

  3. Catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents by accelerating the degradation of p53.

    Science.gov (United States)

    Bai, Jingxiang; Cederbaum, Arthur I

    2003-02-14

    Oxidants such as H(2)O(2) play a role in the toxicity of certain DNA-damaging agents, a process that often involves the tumor suppressor p53. H(2)O(2) is rapidly degraded by catalase, which protects cells against oxidant injury. To study the effect of catalase on apoptosis induced by DNA-damaging agents, HepG2 cells were infected with adenovirus containing the cDNA of catalase (Ad-Cat). Forty-eight hours after infection, catalase protein and activity was increased 7-10-fold compared with control cells infected with Ad-LacZ. After treatment with Vp16 or mitomycin C, control cells underwent apoptosis in a p53-dependent manner; however, overexpression of catalase inhibited this apoptosis. Basal levels as well as Vp16- or mitomycin C-stimulated levels of p53 and p21 protein were decreased in the catalase-overexpressing cells as compared with control cells; however, p53 mRNA levels were not decreased by catalase. There was no difference in p53 protein synthesis between catalase-overexpressing cells and control cells. However, pulse-chase experiments indicated that p53 protein degradation was enhanced in the catalase-overexpressing cells. Proteasome inhibitors but not calpeptin prevented the catalase-mediated decrease of p53 content. Whereas Vp16 increased, catalase overexpression decreased the phosphorylation of p53. The protein phosphatase inhibitor okadaic acid did not prevent the catalase-mediated down-regulation of p53 or phosphorylated p53. These results demonstrate that catalase protects HepG2 cells from apoptosis induced by DNA-damaging agents in association with decreasing p53 phosphorylation; the latter may lead to an acceleration in the degradation of p53 protein by the proteasome complex. This suggests that the level of catalase may play a critical role in cell-induced resistance to the effects of anti-cancer drugs which up-regulate p53. PMID:12468545

  4. Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis: Mentha extract as a neuroprotective against gamma irradiation

    OpenAIRE

    Hassan, Hanaa A.; Hafez, Hani S.; Goda, Mona S.

    2012-01-01

    Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phas...

  5. DNA damage and apoptosis in blood neutrophils of inflammatory bowel disease patients and in Caco-2 cells in vitro exposed to betanin.

    Science.gov (United States)

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Dobrowolska-Zachwieja, Agnieszka; Łuczak, Michał; Baer-Dubowska, Wanda

    2016-01-01

    Inflammatory bowel diseases (IBD) are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, and continuing colonic inflammation is considered an important risk factor in the development of colorectal cancer. Our previous studies showed that beetroot (Beta vulgaris var. rubra) products and their major component betanin modulate the reactive oxygen species (ROS) production and DNA damage in 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated human polymorphonuclear neutrophils of healthy volunteers. The aim of the present study was to evaluate the effects of betanin on the oxidative DNA damage and apoptosis in neutrophils isolated from blood of patients with inflammatory bowel disease--ulcerative colitis (UC) and Crohn's disease (CD). The results were compared with those obtained in colon carcinoma-derived Caco-2 cells. Betanin treatment at the concentration of 100 μM for 24 h increased DNA damage assessed by comet assay in IBD patients' neutrophils. A similar effect although less pronounced was observed in Caco-2 cells. Treatment of Caco-2 cells with H2O2 caused a 4-fold increase of DNA strand breaks in comparison to untreated cells, but pre-treatment with betanin reduced DNA damage in these cells. Betanin also induced procaspase-3 cleavage and caspase-3 activity accompanied by the loss of mitochondrial transmembrane potential, indicating its pro-apoptotic activity. These results suggest that betanin may support mechanisms that lead to the release of ROS and apoptotic cell death. In this way betanin may exert anti-inflammatory and potentially cancer preventive activity. PMID:27117102

  6. Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields.

    Science.gov (United States)

    Saha, Shreya; Woodbine, Lisa; Haines, Jackie; Coster, Margaret; Ricket, Nicole; Barazzuol, Lara; Ainsbury, Elizabeth; Sienkiewicz, Zenon; Jeggo, Penny

    2014-11-01

    The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10-200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1-5 mGy X-rays.

  7. DNA Damage, Apoptosis and C-myc, C-fos, and C-jun Overexpression Induced by Selenium in Rat Hepatocytes

    Institute of Scientific and Technical Information of China (English)

    RI-AN YU; CHENG-FENG YANG; XUE-MIN CHEN

    2006-01-01

    Objective To study the effects of selenium on DNA damage, apoptosis and c-myc, c-fos, and c-jun expression in rat hepatocytes. Methods Sodium selenite at the doses of 5, 10, and 20 μmol/kg was given to rats by i.p. and there were 5 male SD rats in each group. Hepatocellular DNA damage was detected by single cell gel electrophoresis (or comet assay).Hepatocellular apoptosis was determined by TUNEL (TdT-mediated dUTP nick end labelling) and flow cytometry. C-myc,c-fos, and c-jun expression in rat hepatocytes were assayed by Northern dot hybridization. C-myc, c-fos, and c-jun protein were detected by immunohistochemical method. Results At the doses of 5, 10, and 20 μmol/kg, DNA damage was induced by sodium selenite in rat hepatocytes and the rates of comet cells were 34.40%, 74.80%, and 91.40% respectively. Results also showed an obvious dose-response relationship between the rates of comet cells and the doses of sodium selenite (r=0.9501,P<0.01). Sodium selenite at the doses of 5, 10, and 20 μmol/kg caused c-myc, c-fos, and c-jun overexpression obviously. The positive brown-yellow signal for proteins of c-myc, c-fos, and c-jun was mainly located in the cytoplasm of hepatocytes with immunohistochemical method. TUNEL-positive cells were detected in selenium-treated rat livers. Apoptotic rates (%) of selenium-treated liver cells at the doses of 5, 10, and 20 μmol/kg were (3.72±1.76), (5.82±1.42), and (11.76±1.87) respectively, being much higher than those in the control. Besides an obvious dose-response relationship between apoptotic rates and the doses of sodium selenite (r=0.9897, P<0.01), these results displayed a close relationship between DNA damage rates and apoptotic rates, and the relative coefficient was 0.9021, P<0.01. Conclusion Selenium at 5-20 μmol/kg can induce DNA damage, apoptosis, and overexpression of c-myc, c-fos, and c-jun in rat hepatocytes.

  8. The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

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    Amoroso Antonio

    2009-08-01

    Full Text Available Abstract Background Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach. Methods Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence. Results Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii in the absence of p53, it may rely on p73, and (iii it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB. Conclusion Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies.

  9. The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

    International Nuclear Information System (INIS)

    Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach. Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence. Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB. Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies

  10. The influence of TRP53 in the dose response of radiation-induced apoptosis, DNA repair and genomic stability in murine haematopoietic cells

    International Nuclear Information System (INIS)

    Apoptotic and DNA damage endpoints are frequently used as surrogate markers of cancer risk, and have been well-studied in the Trp53+/- mouse model. We report the effect of differing Trp53 gene status on the dose response of ionizing radiation exposures (0.01-2 Gy), with the unique perspective of determining if effects of gene status remain at extended time points. Here we report no difference in the dose response for radiation-induced DNA double-strand breaks in bone marrow and genomic instability (MN-RET levels) in peripheral blood, between wild-type (Trp53+/+) and heterozygous (Trp53+/-) mice. The dose response for Trp53+/+ mice showed higher initial levels of radiation-induced lymphocyte apoptosis relative to Trp53+/- between 0 and 1 Gy. Although this trend was observed up to 12 hours post-irradiation, both genotypes ultimately reached the same level of apoptosis at 14 hours, suggesting the importance of late-onset p53-independent apoptotic responses in this mouse model. Expected radiation-induced G1 cell cycle delay was observed in Trp53+/+ but not Trp53+/-. Although p53 has an important role in cancer risk, we have shown its influence on radiation dose response can be temporally variable. This research highlights the importance of caution when using haematopoietic endpoints as surrogates to extrapolate radiation-induced cancer risk estimation

  11. Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Rachinger Andrea

    2008-10-01

    Full Text Available Abstract Background In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. Methods We sequenced a 1320-base-pair DNA fragment of the mitochondrial genome (position 16,000-750 in 54 cancer samples and in 44 corresponding germline control samples. In addition, six transcripts (MT-ATP6, MT-CO1, MT-CYB, MT-ND1, MT-ND6, and MT-RNR1 were quantified in 62 cancer tissues by real-time RT-PCR. Results Somatic mutations in the D-loop sequence were found in 57% of ovarian cancers. Univariate analysis showed no association between mitochondrial DNA mutation status or mitochondrial gene expression and any of the examined clinicopathologic parameters. A multivariate logistic regression model revealed that the expression of the mitochondrial gene RNR1 might be used as a predictor of tumour sensitivity to chemotherapy. Conclusion In contrast to many previously published papers, our study indicates rather limited clinical relevance of mitochondrial molecular analyses in ovarian carcinomas. These discrepancies in the clinical utility of mitochondrial molecular tests in ovarian cancer require additional large, well-designed validation studies.

  12. Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

    Science.gov (United States)

    Onodera, Takefumi; Takenaka, Yukiko; Kozaki, Sachiko; Tanahashi, Takao; Mizushina, Yoshiyuki

    2016-03-01

    We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent.

  13. Screening of mammalian DNA polymerase and topoisomerase inhibitors from Garcinia mangostana L. and analysis of human cancer cell proliferation and apoptosis.

    Science.gov (United States)

    Onodera, Takefumi; Takenaka, Yukiko; Kozaki, Sachiko; Tanahashi, Takao; Mizushina, Yoshiyuki

    2016-03-01

    We purified and identified eight xanthones from mangosteen (Garcinia mangostana L.) and investigated whether these compounds inhibited the activities of mammalian DNA polymerases (Pols) and human DNA topoisomerases (Topos). β-Mangostin was the strongest inhibitor of both mammalian Pols and human Topos among the isolated xanthones, with 50% inhibitory concentration (IC50) values of 6.4-39.6 and 8.5-10 µM, respectively. Thermal transition analysis indicated that β-mangostin did not directly bind to double-stranded DNA, suggesting that this compound directly bound the enzyme protein rather than the DNA substrate. β-Mangostin showed the strongest suppression of human cervical cancer HeLa cell proliferation among the eight compounds tested, with a 50% lethal dose (LD50) of 27.2 µM. This compound halted cell cycle in S phase at 12-h treatment and induced apoptosis. These results suggest that decreased proliferation by β-mangostin may be a result of the inhibition of cellular Pols rather than Topos, and β-mangostin might be an anticancer chemotherapeutic agent. PMID:26781450

  14. DNA double-strand break and apoptosis induction in human lymphocytes in different cycle cell phases by 60Co gamma rays and Bragg peak protons of a medical beam

    International Nuclear Information System (INIS)

    A comparative analysis is made of the regularities in the formation of DNA double-strand break and apoptosis induction in peripheral human blood lymphocytes in different cell cycle phases after 60Co gamma and extended Bragg peak proton irradiation. It is shown that the formation of apoptotic cells in a lymphocyte population increases linearly in all the cell cycle stages after proton irradiation. The maximal DNA double-strand break and apoptosis yield in lymphocytes is observed in the S phase of the cell cycle

  15. Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression

    International Nuclear Information System (INIS)

    As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases. The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo®3/7 Assay, respectively. Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008). Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression

  16. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    Directory of Open Access Journals (Sweden)

    Guo Zong-Lun

    2010-02-01

    Full Text Available Abstract Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry, demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299 using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p p 50 of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

  17. Microwave-Assisted Synthesis of Arene Ru(II Complexes Induce Tumor Cell Apoptosis Through Selectively Binding and Stabilizing bcl-2 G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Yanhua Chen

    2016-05-01

    Full Text Available A series of arene Ru(II complexes coordinated with phenanthroimidazole derivatives, [(η6-C6H6Ru(lCl]Cl(1b L = p-ClPIP = 2-(4-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 2b L = m-ClPIP = 2-(3-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 3b L = p-NPIP = 2-(4-Nitrophenylimidazole[4,5f] 1,10-phenanthroline; 4b L = m-NPIP = 2-(3-Nitrophenyl imidazole [4,5f] 1,10-phenanthroline were synthesized in yields of 89.9%–92.7% under conditions of microwave irradiation heating for 30 min to liberate four arene Ru(II complexes (1b, 2b, 3b, 4b. The anti-tumor activity of 1b against various tumor cells was evaluated by MTT assay. The results indicated that this complex blocked the growth of human lung adenocarcinoma A549 cells with an IC50 of 16.59 μM. Flow cytometric analysis showed that apoptosis of A549 cells was observed following treatment with 1b. Furthermore, the in vitro DNA-binding behaviors that were confirmed by spectroscopy indicated that 1b could selectively bind and stabilize bcl-2 G-quadruplex DNA to induce apoptosis of A549 cells. Therefore, the synthesized 1b has impressive bcl-2 G-quadruplex DNA-binding and stabilizing activities with potential applications in cancer chemotherapy.

  18. The SMAC mimetic BV6 sensitizes colorectal cancer cells to ionizing radiation by interfering with DNA repair processes and enhancing apoptosis

    International Nuclear Information System (INIS)

    In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells. Colorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0–4 μM) with or without irradiation (2–8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)). BV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells. The SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells. The online version of this

  19. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

    Directory of Open Access Journals (Sweden)

    Shay Covo

    2010-07-01

    Full Text Available Double-strand break (DSB repair through homologous recombination (HR is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage-induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G(2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage-induced recombinants in G(2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.

  20. DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids

    Directory of Open Access Journals (Sweden)

    Emad A. Ahmed

    2015-12-01

    Full Text Available Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX foci marking DNA double strand breaks (DSBs in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP-ribose polymerase 1 (PARP1 inhibitor (DPQ-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

  1. PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage

    Science.gov (United States)

    Bobrovnikova-Marjon, Ekaterina; Grigoriadou, Christina; Pytel, Dariusz; Zhang, Fang; Ye, Jiangbin; Koumenis, Constantinos; Cavener, Douglas; Diehl, J. Alan

    2010-01-01

    In order to proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential anti-neoplastic targets. However, recent investigations into the role of the ER resident protein kinase PERK have paradoxically suggested both pro- and anti-tumorigenic properties. We have utilized animal models of mammary carcinoma to interrogate PERK contribution in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle due to the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is utilized during both tumor initiation and expansion to maintain redox homeostasis and thereby facilitates tumor growth. PMID:20453876

  2. PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

    Science.gov (United States)

    Bobrovnikova-Marjon, E; Grigoriadou, C; Pytel, D; Zhang, F; Ye, J; Koumenis, C; Cavener, D; Diehl, J A

    2010-07-01

    To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

  3. MicroRNA-related polymorphisms in apoptosis pathway genes are predictive of clinical outcome in patients with limited disease small cell lung cancer

    Science.gov (United States)

    Jiang, Wei; Bi, Nan; Zhang, Wen-Jue; Wu, Li-Hong; Liu, Li-Pin; Men, Yu; Wang, Jing-Bo; Liang, Jun; Hui, Zhou-Guang; Zhou, Zong-Mei; Wang, Lu-Hua

    2016-01-01

    We examined the impact of single nucleotide polymorphisms (SNPs) at miRNA binding sites in the 3′-UTRs of genes in the apoptosis pathway on the prognosis of patients with limited disease-small cell lung cancer (LD-SCLC). Twelve tagSNPs in seven genes were genotyped using blood samples from 146 LD-SCLC patients treated with chemoradiotherapy. Cox proportional hazard regression models and recursive partitioning analysis were performed to identify SNPs significantly associated with overall survival. Three SNPs, CASP8: rs1045494 (C > T), PIK3R1: rs3756668 (A > G) and CASP7: rs4353229 (T > C), were associated with longer overall survival in LD-SCLC patients after chemoradiotherapy. The adjusted hazard ratios (95% confidence intervals) were 0.480 (0.258–0.894), 0.405 (0.173–0.947) and 0.446 (0.247–0.802), respectively, and remained significant after multiple comparison correction. Moreover, subset analysis showed these SNPs were still predictive of overall survival in stage III patients. Recursive partitioning analysis enabled patients to be classified into three risk subgroups based on unfavorable genotype combinations of the rs1045494 and rs4353229 SNPs. These findings suggest miRNA-related polymorphisms in the apoptosis pathway may be useful biomarkers for selection of LD-SCLC patients likely to benefit from chemoradiotherapy. PMID:26988918

  4. Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: a potential new drug for treatment of glioblastoma multiforme.

    Science.gov (United States)

    Lv, Linlin; Zheng, Lingli; Dong, Deshi; Xu, Lina; Yin, Lianhong; Xu, Youwei; Qi, Yan; Han, Xu; Peng, Jinyong

    2013-09-01

    Dioscin, a natural product obtained from medicinal plants shows lipid-lowering, anti-cancer and hepatoprotective effects. However, the effect of it on glioblastoma is unclear. In this study, dioscin significantly inhibited proliferation of C6 glioma cells and caused reactive oxygen species (ROS) generation and Ca²⁺ release. ROS accumulation affected levels of malondialdehyde, nitric oxide, glutathione disulfide and glutathione, and caused cell apoptosis. In addition, ROS generation caused mitochondrial damage including structural changes, increased mitochondrial permeability transition and decreased mitochondria membrane potential, which led to the release of cytochrome C, nuclear translation of programmed cell death-5 and increased activities of caspase-3,9. Simultaneously, dioscin down-regulated protein expression of Bcl-2, Bcl-xl, up-regulated expression of Bak, Bax, Bid and cleaved poly (ADP-ribose) polymerase. Also, oxygen stress induced S-phase arrest of cancer cells by way of regulating expression of DNA Topo I, p53, CDK2 and Cyclin A and caused DNA damage. In a rat allograft model, dioscin significantly inhibited tumor size and extended the life cycle of the rats. In conclusion, dioscin shows noteworthy anti-cancer activity on glioblastoma cells by promoting ROS accumulation, inducing DNA damage and activating mitochondrial signal pathways. Ultimately, we believe dioscin has promise as a new therapy for the treatment of glioblastoma. PMID:23871826

  5. Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Yi Wang; Nam Chun Cho; Xiao-ting Fu; Da-wei Li; Kun Wang; Xin-zhi Wang; Yuan Li; Bao-liang Sun; Xiao-yi Yang; Zun-cheng Zheng

    2016-01-01

    Amyloid beta (Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer’s disease. Cyan-idin, a natural lfavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreat-ment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment signiifcantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial mem-brane potentialvia upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity.

  6. Generation of reactive oxygen species by polyenylpyrroles derivatives causes DNA damage leading to G2/M arrest and apoptosis in human oral squamous cell carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Kuo-Feng Hua

    Full Text Available Oral squamous cell carcinoma (OSCC accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC.

  7. APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain

    OpenAIRE

    Chen, Yuzhi; Liu,Wenyun; McPhie, Donna L.; Hassinger, Linda; Neve, Rachael L

    2003-01-01

    APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons (Chen, Y., D.L. McPhie, J. Hirschberg, and R.L. Neve. 2000. J. Biol. Chem. 275:8929–8935). We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a...

  8. Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells.

  9. The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Heidor, Renato [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Furtado, Kelly Silva; Ortega, Juliana Festa [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Oliveira, Tiago Franco de [Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Purgatto, Eduardo [Laboratory of Food Chemistry and Biochemistry, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Moreno, Fernando Salvador, E-mail: rmoreno@usp.br [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil)

    2014-04-15

    The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200 mg/100 g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100 g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p < 0.05) as well as the ACF with ≥ 4 crypts (p < 0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p < 0.05) and reduced DNA damage (p < 0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p < 0.05). TB administration resulted in increased colonic tissue concentrations of BA (p < 0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB. - Highlights: • Tributyrin is a chemopreventive agent for rat colon aberrant crypt foci. • Tributyrin increased apoptosis in an experimental rat colon carcinogenesis model. • Tributyrin treatment in a rat colon carcinogenesis model decreased DNA damage. • Tributyrin treatment induced H3K9 acetylation in a rat colon carcinogenesis model.

  10. The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

    International Nuclear Information System (INIS)

    The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200 mg/100 g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100 g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p < 0.05) as well as the ACF with ≥ 4 crypts (p < 0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p < 0.05) and reduced DNA damage (p < 0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p < 0.05). TB administration resulted in increased colonic tissue concentrations of BA (p < 0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB. - Highlights: • Tributyrin is a chemopreventive agent for rat colon aberrant crypt foci. • Tributyrin increased apoptosis in an experimental rat colon carcinogenesis model. • Tributyrin treatment in a rat colon carcinogenesis model decreased DNA damage. • Tributyrin treatment induced H3K9 acetylation in a rat colon carcinogenesis model

  11. Determination of apoptosis, proliferation status and O6-methylguanine DNA methyltransferase methylation profiles in different immunophenotypic profiles of diffuse large B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Nilay Şen Türk

    2011-03-01

    Full Text Available Objective: Our aim was to investigate the expression of apoptosis-associated proteins (bcl-2, bcl-xl, bax, bak, bid, apoptotic index (AI and proliferation index (PI in germinal center B-cell-like immunophenotypic profile (GCB and non-GCB of diffuse large B-cell lymphoma (DLBCL. Materials and Methods: The methylation status of the promoter region of O6-methylguanine-DNA yerine O6-methylguanine-DNA methyltransferase (MGMT gene and its relation with immunophenotypic differentiation of DLBCLs were also investigated. 101 cases were classified as GCB (29 cases or non-GCB (72 cases. Apoptosis-associated proteins and PI were determined by IHC, and TUNEL method was used to determine AI. MGMT methylation analysis was performed by real-time PCR. Results: The PI was significantly higher in GCB compared with non-GCB (p=0.011. Percentage of cells stained with bcl-6 was positively correlated with the percentage of cells expressing bcl-2 (p=0.023, AI (p=0.006 and PI (p<0.001, while a significant negative correlation was observed with the percentage of cells expressing bax (p=0.027. The percentage of cells stained with MUM1 showed a significantly positive correlation with the percentage of cells expressing bcl-xl (p=0.003, bid (p=0.002, AI (p<0.001, and PI (p=0.001. MGMT methylation analysis was performed in 95 samples, and methylated profile was found in 31 cases (32.6%. GCB was found in 6 cases (22.2% and non-GCB was determined in 25 cases (36.8% out of 31 with MGMT methylated samples. There was no significant association between MGMT methylation status and immunophenotypic profiles (p=0.173. Conclusion: These results suggest that bcl-6 protein expression may be responsible for the high PI in GCB. Additionally, we found that apoptosis-associated proteins were not significantly associated with immunophenotypic profiles.

  12. Effects of Cinnamon (C. zeylanicum) Bark Oil Against Taxanes-Induced Damages in Sperm Quality, Testicular and Epididymal Oxidant/Antioxidant Balance, Testicular Apoptosis, and Sperm DNA Integrity.

    Science.gov (United States)

    Sariözkan, Serpil; Türk, Gaffari; Güvenç, Mehmet; Yüce, Abdurrauf; Özdamar, Saim; Cantürk, Fazile; Yay, Arzu Hanım

    2016-04-01

    The aim of this study was to investigate whether cinnamon bark oil (CBO) has protective effect on taxanes-induced adverse changes in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular apoptosis, and sperm DNA integrity. For this purpose, 88 adult male rats were equally divided into 8 groups: control, CBO, docetaxel (DTX), paclitaxel (PTX), DTX+PTX, DTX+CBO, PTX+CBO, and DTX+PTX+CBO. CBO was given by gavage daily for 10 weeks at the dose of 100 mg/kg. DTX and PTX were administered by intraperitoneal injection at the doses of 5 and 4 mg/kg/week, respectively, for 10 weeks. DTX+PTX and DTX+PTX+CBO groups were treated with DTX during first 5 weeks and PTX during next 5 weeks. DTX, PTX, and their mixed administrations caused significant decreases in absolute and relative weights of all reproductive organs, testosterone level, sperm motility, concentration, glutathione level, and catalase activity in testicular and epididymal tissues. They also significantly increased abnormal sperm rate, testicular and epididymal malondialdehyde level, apoptotic germ cell number, and sperm DNA fragmentation and significantly damaged the histological structure of testes. CBO consumption by DTX-, PTX-, and DTX+PTX-treated rats provided significant ameliorations in decreased relative weights of reproductive organs, decreased testosterone, decreased sperm quality, imbalanced oxidant/antioxidant system, increased apoptotic germ cell number, rate of sperm with fragmented DNA, and severity of testicular histopathological lesions induced by taxanes. In conclusion, taxanes cause impairments in sperm quality, testicular and epididymal oxidant/antioxidant balance, testicular histopathological structure, and sperm DNA integrity, and long-term CBO consumption protects male reproductive system of rats. PMID:27008095

  13. Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance.

    Science.gov (United States)

    Pytel, Dariusz; Wysocki, Tomasz; Majsterek, Ireneusz

    2006-09-01

    The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.

  14. Anti-proliferative effect of methanolic extract of Gracilaria tenuistipitata on oral cancer cells involves apoptosis, DNA damage, and oxidative stress

    Directory of Open Access Journals (Sweden)

    Yeh Chi-Chen

    2012-08-01

    Full Text Available Abstract Background Methanolic extracts of Gracilaria tenuistipitata (MEGT were obtained from the edible red algae. Previously, we found that water extract of G. tenuistipitata was able to modulate oxidative stress-induced DNA damage and its related cellular responses. Methods In this study, the methanol extraction product MEGT was used to evaluate the cell growth inhibition in oral cancer cells and its possible mechanism was investigated. Results The cell viability of MEGT treated Ca9-22 oral cancer cell line was significantly decreased in a dose–response manner (p p p p p 2(3 intensity for mitochondrial membrane potential (MMP of MEGT-treated Ca9-22 cancer cells was significantly decreased in a dose–response manner (p  Conclusions These results indicated that MEGT had apoptosis-based cytotoxicity against oral cancer cells through the DNA damage, ROS induction, and mitochondrial depolarization. Therefore, MEGT derived from the edible algae may have potential therapeutic effects against oral squamous cell carcinoma (OSCC.

  15. Differential induction of apoptosis and senescence by the DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine in solid tumor cells.

    Science.gov (United States)

    Venturelli, Sascha; Berger, Alexander; Weiland, Timo; Essmann, Frank; Waibel, Michaela; Nuebling, Tina; Häcker, Sabine; Schenk, Martin; Schulze-Osthoff, Klaus; Salih, Helmut R; Fulda, Simone; Sipos, Bence; Johnstone, Ricky W; Lauer, Ulrich M; Bitzer, Michael

    2013-10-01

    Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2'-deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology. PMID:23924947

  16. Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family

    NARCIS (Netherlands)

    S. Greiss (Sebastian); B. Schumacher (Björn); K. Grandien (Kaj); J. Rothblatt (Jonathan); A. Gartner (Anton)

    2008-01-01

    textabstractBackground: In contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activ

  17. Nuclear Apoptosis Contributes to Sarcopenia

    OpenAIRE

    Alway, Stephen E.; Parco M. Siu

    2008-01-01

    Apoptosis results in DNA fragmentation and, subsequently, destruction of cells containing a single nucleus. Our hypothesis is that multinucleated cells such as muscle fibers can experience apoptotic-induced loss of single nuclei (nuclear apoptosis) without destruction of the entire fiber. The loss of nuclei likely contributes to atrophy and sarcopenia. Furthermore, increased chronic activity attenuates apoptotic signaling, which may reduce sarcopenia.

  18. Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

    Directory of Open Access Journals (Sweden)

    Rui Gao

    2015-01-01

    Full Text Available Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease (MJD, is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers

  19. The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells. Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting. Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway. These data demonstrate that the novel curcumin analog FLLL32 has biologic activity

  20. Loss of glucocorticoid receptor expression by DNA methylation prevents glucocorticoid induced apoptosis in human small cell lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Paul Kay

    Full Text Available Human small cell lung cancer (SCLC is highly aggressive, and quickly develops resistance to therapy. SCLC cells are typically insensitive to glucocorticoids due to impaired glucocorticoid receptor (GR expression. This is important as we have previously shown that expression of a GR transgene induces cell death in-vitro, and inhibits tumor growth in-vivo. However, the underlying mechanism for loss of GR expression is unknown. The SCLC cell line, DMS79, has low GR expression, compared to non-SCLC cell lines and normal bronchial epithelial cells. Retroviral GR expression in DMS79 cells caused activation of the apoptotic pathway as evidenced by marked induction of caspase-3 activity. Methylation analysis of the GR promoter revealed some methylation in the 1D, and 1E promoters of the GR gene, however the ubiquitous constitutively active 1C promoter was heavily methylated. In the 1C promoter there was a highly significant increase in DNA methylation in a panel of 14 human SCLC cell lines compared to a mixed panel of GR expressing, and non-expressing cell lines, and to peripheral blood mononuclear cells. Furthermore, within the panel of SCLC cell lines there was a significant negative correlation seen between methylation of the 1C promoter, and GR protein expression. Reversal of GR gene methylation with DNA methyltransferase inhibition caused increased GR mRNA and protein expression in SCLC but not non-SCLC cells. This resulted in increased Gc sensitivity, decreased Bcl-2 expression and increased caspase-3 activity in SCLC cells. These data suggest that DNA methylation decreases GR gene expression in human SCLC cells, in a similar manner to that for conventional tumor suppressor genes.

  1. Triplex DNA-mediated downregulation of Ets2 expression results in growth inhibition and apoptosis in human prostate cancer cells

    OpenAIRE

    Giuseppina M. Carbone; Napoli, Sara; Valentini, Alessandra; Cavalli, Franco; Watson, Dennis K.; Catapano, Carlo V

    2004-01-01

    Ets2 is a member of the Ets family of transcription factors that in humans comprise 25 distinct members. Various Ets-domain transcription factors have been implicated in cancer development. Ets2 is expressed in prostate and breast cancer cells and is thought to have a role in promoting growth and survival in these cell types. However, a definitive role and the mechanisms whereby Ets2 acts in cancer cells are still unclear. Structural and functional similarities as well as overlapping DNA bind...

  2. Endogenous c-Myc is essential for p53-induced apoptosis in response to DNA damage in vivo

    OpenAIRE

    Phesse, T J; Myant, K.B.; Cole, A M; Ridgway, R.A.; Pearson, H; Muncan, V.; van den Brink, G R; Vousden, K H; Sears, R.; Vassilev, L T; Clarke, A R; Sansom, O J

    2014-01-01

    Recent studies have suggested that C-MYC may be an excellent therapeutic cancer target and a number of new agents targeting C-MYC are in preclinical development. Given most therapeutic regimes would combine C-MYC inhibition with genotoxic damage, it is important to assess the importance of C-MYC function for DNA damage signalling in vivo. In this study, we have conditionally deleted the c-Myc gene in the adult murine intestine and investigated the apoptotic response of intestinal enterocytes ...

  3. Molecular DNA variation among Triturus vittatus vittatus (Urodela from different breeding sites at the southern limit of its distribution

    Directory of Open Access Journals (Sweden)

    Oren Pearlson

    2007-11-01

    Full Text Available Molecular DNA variation among Triturus vittatus vittatus (striped newt from different breeding sites at the southern limit of the species distribution (where environmental conditions are most extreme was studied by the random amplification of polymorphic DNA (RAPD method that has been found to be appropriate for other Triturus species. Altitudes of the localities ranged between 15-740 m a.s.l. Of the 20 primers employed, OPA-16 was the only one suitable for T. vittatus, revealing a different band pattern for different populations. Genetic similarity was calculated by band sharing, which demonstrated a high similarity among the Israeli populations.

  4. DNA methyltransferase inhibitor-mediated apoptosis in the Wnt/β-catenin signal pathway in a renal cell carcinoma cell line.

    Science.gov (United States)

    Konac, Ece; Varol, Nuray; Yilmaz, Akin; Menevse, Sevda; Sozen, Sinan

    2013-09-01

    The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3β) is an important regulator of the Wnt/β-catenin signaling pathway. The mechanisms underlying GSK3β regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2'-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3β (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of β-catenin (CTNNB1) expression decreased. The p-GSK3β (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 μM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice. PMID:23975733

  5. Data on the transcriptional regulation of DNA damage induced apoptosis suppressor (DDIAS) by ERK5/MEF2B pathway in lung cancer cells.

    Science.gov (United States)

    Im, Joo-Young; Yoon, Sung-Hoon; Kim, Bo-Kyung; Ban, Hyun Seung; Won, Kyoung-Jae; Chung, Kyung-Sook; Jung, Kyeong Eun; Won, Misun

    2016-12-01

    The data included in this article are associated with the article entitled "DNA-damage-induced apoptosis suppressor (DDIAS) is upregulated via ERK5/MEF2B signaling and promotes β-catenin-mediated invasion" (J.Y. Im, S.H. Yoon, B.K. Kim, H.S. Ban, K.J. Won, K.S. Chung, K.E. Jung, M. Won) [1]. Quantitative RT-PCR data revealed that genetic or pharmacological inhibition of extracellular signal-regulated kinase 5 (ERK5) suppresses DDIAS transcription in response to epidermal growth factor (EGF) in Hela cells. p300 did not interact with myocyte enhancer factor 2B (MEF2B), a downstream target of ERK5 and affect transcription of DDIAS. Moreover, DDIAS transcription is activated by ERK5/MEF2B signaling on EGF exposure in the non-small cell lung cancer cells (NSCLC) NCI-H1703 and NCI-H1299. DDIAS knockdown suppresses lung cancer cell invasion by decreasing β-catenin protein level on EGF exposure. PMID:27660814

  6. Overexpression of TFAM or twinkle increases mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress.

    Directory of Open Access Journals (Sweden)

    Masataka Ikeda

    Full Text Available Mitochondrial DNA (mtDNA copy number decreases in animal and human heart failure (HF, yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM or Twinkle helicase in volume overload (VO-induced HF.Two strains of transgenic (TG mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.

  7. DNA hybridization as a guide to phylogeny: chemical and physical limits.

    Science.gov (United States)

    Schmid, C W; Marks, J

    1990-03-01

    The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance. Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments. As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes. PMID:2109086

  8. Thiosemicarbazone Cu(II) and Zn(II) complexes as potential anticancer agents: syntheses, crystal structure, DNA cleavage, cytotoxicity and apoptosis induction activity.

    Science.gov (United States)

    Shao, Jia; Ma, Zhong-Ying; Li, Ang; Liu, Ya-Hong; Xie, Cheng-Zhi; Qiang, Zhao-Yan; Xu, Jing-Yuan

    2014-07-01

    Four novel thiosemicarbazone metal complexes, [Cu(Am4M)(OAc)]·H2O (1), [Zn(HAm4M)Cl2] (2), [Zn2(Am4M)2Br2] (3) and [Zn2(Am4M)2(OAc)2]·2MeOH (4) [HAm4M=(Z)-2-(amino(pyridin-2-yl)methylene)-N-methylhydrazinecarbothioamide], have been synthesized and characterized by X-ray crystallography, elemental analysis, ESI-MS and IR. X-ray analysis revealed that complexes 1 and 2 are mononuclear, which possess residual coordination sites for Cu(II) ion in 1 and good leaving groups (Cl(-)) for Zn(II) ion in 2. Both 3 and 4 displayed dinuclear units, in which the metal atoms are doubly bridged by S atoms of two Am4M(-) ligands in 3 and by two acetate ions in bi- and mono-dentate forms, respectively, in 4. Their antiproliferative activities on human epithelial cervical cancer cell line (HeLa), human liver hepatocellular carcinoma cell line (HepG-2) and human gastric cancer cell line (SGC-7901) were screened. Inspiringly, IC50 value (11.2±0.9 μM) of complex 1 against HepG-2 cells was nearly 0.5 fold of that against human hepatic cell lines LO2, showing a lower toxicity to human liver cells. Additionally, it displayed a stronger inhibition on the viability of HepG-2 cells than cisplatin (IC50=25±3.1 μM), suggesting complex 1 might be a potential high efficient antitumor agent. Furthermore, fluorescence microscopic observation and flow cytometric analysis revealed that complex 1 could significantly suppress HepG-2 cell viability and induce apoptosis. Several indexes, such as DNA cleavage, reactive oxygen species (ROS) generation, comet assay and cell cycle analysis indicated that the antitumor mechanism of complex 1 on HepG-2 cells might be via ROS-triggered apoptosis pathway. PMID:24690556

  9. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    Science.gov (United States)

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  10. Apoptosis in Pneumovirus Infection

    Directory of Open Access Journals (Sweden)

    Reinout A. Bem

    2013-01-01

    Full Text Available Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  11. Reactive oxygen species and autophagy associated apoptosis and limitation of clonogenic survival induced by zoledronic acid in salivary adenoid cystic carcinoma cell line SACC-83.

    Directory of Open Access Journals (Sweden)

    Xi-Yuan Ge

    Full Text Available Salivary adenoid cystic carcinoma is an epithelial tumor in the head and neck region. Despite its slow growth, patients with salivary adenoid cystic carcinoma exhibit poor long term survival because of a high rate of distant metastasis. Lung and bone are common distant metastasis sites. Zoledronic acid, a third generation bisphosphonate, has been used for tumor-induced osteolysis due to bone metastasis and has direct antitumor activity in several human neoplasms. Here, we observed that zoledronic acid inhibited salivary adenoid cystic carcinoma cell line SACC-83 xenograft tumor growth in nude mice. In vitro, zoledronic acid induced apoptosis and reduced clonogenic survival in SACC-83. Flow cytometry and western blotting indicated that the cell cycle was arrested at G0/G1. Zoledronic acid treatment upregulated reactive oxygen species as well as the autophagy marker protein LC-3B. Reactive oxygen species scavenger N-acetylcysteine and autophagy antagonist 3-methyladenine decreased zoledronic acid-induced apoptosis and increased clonogenic survival. Silencing of the autophagy related gene Beclin-1 also decreased zoledronic acid-induced apoptosis and inhibition of clonogenic formation. In addition, isobolographic analysis revealed synergistic effects on apoptosis when zoledronic acid and paclitaxel/cisplatin were combined. Taken together, our results suggest that zoledronic acid induced apoptosis and reduced clonogenic survival via upregulation of reactive oxygen species and autophagy in the SACC-83 cell line. Thus, zoledronic acid should be considered a promising drug for the treatment of salivary adenoid cystic carcinoma.

  12. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.

    Science.gov (United States)

    Cain, K; Inayat-Hussain, S H; Couet, C; Qin, H M; Oberhammer, F A

    1996-04-01

    This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1. PMID:8900474

  13. Conventional fluorescence microscopy below the diffraction limit with simultaneous capture of two fluorophores in DNA origami

    Science.gov (United States)

    Glasgow, Ben J.

    2016-02-01

    A conventional fluorescence microscope was previously constructed for simultaneous imaging of two colors to gain sub-diffraction localization. The system is predicated on color separation of overlapping Airy discs, construction of matrices of Cartesian coordinates to determine locations as well as centers of the point spread functions of fluorophores. Quantum dots that are separated by as little as 10 nm were resolved in the x-y coordinates. Inter-fluorophore distances that vary by 10 nm could also be distinguished. Quantum dots are bright point light source emitters that excite with a single laser and can serve as a label for many biomolecules. Here, alterations in the method are described to test the ability to resolve Atto 488 and Atto 647 dyes attached to DNA origami at ~40 nm spacing intervals. Dual laser excitation is used in tandem with multi-wavelength bandpass filters. Notwithstanding challenges from reduced intensity in Atto labeled DNA origami helical bundles compared to quantum dots, preliminary data show a mean inter-fluorophore distance of 56 nm with a range (14-148 nm). The range closely matches published results with DNA origami with other methods of subdiffraction microscopy. Sub-diffraction simultaneous two-color imaging fluorescence microscopy acronymically christened (SSTIFM) is a simple, readily accessible, technique for measurement of inter-fluorophore distances in compartments less than 40 nm. Preliminary results with so called nanorulers are encouraging for use with other biomolecules.

  14. Mitochondrial dynamics and apoptosis

    OpenAIRE

    Suen, Der-Fen; Norris, Kristi L.; Youle, Richard J.

    2008-01-01

    In healthy cells, mitochondria continually divide and fuse to form a dynamic interconnecting network. The molecular machinery that mediates this organelle fission and fusion is necessary to maintain mitochondrial integrity, perhaps by facilitating DNA or protein quality control. This network disintegrates during apoptosis at the time of cytochrome c release and prior to caspase activation, yielding more numerous and smaller mitochondria. Recent work shows that proteins involved in mitochondri...

  15. Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

    Directory of Open Access Journals (Sweden)

    Bordón Elisa

    2011-06-01

    Full Text Available Abstract Background Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. Methods Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13 was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp. Radiation-induced apoptosis (RIA at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46. Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively. We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively in multivariate analysis. Conclusions A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low

  16. DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

    Science.gov (United States)

    Mazzanti, Morena; Alessandrini, Federica; Tagliabracci, Adriano; Wells, Jeffrey D; Campobasso, Carlo P

    2010-02-25

    Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

  17. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  18. Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant

    International Nuclear Information System (INIS)

    The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group. A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day-1 and 0.025 day-1) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose. (orig.)

  19. Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line

    International Nuclear Information System (INIS)

    In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1 μM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction. The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occurring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified

  20. Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly increases apoptosis

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque Ahmed; Nexo, Ebba;

    2007-01-01

    : The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR...

  1. Pyrrolidine dithiocarbamate Inhibits NF- κB Activation and Enhance TNFα- Induced Apoptosis in Human Breast Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    TU Gang(涂刚); YAO Zhenxiang(姚榛祥); DONG Pujiang(董浦江)

    2002-01-01

    Objective: Tumor necrosis factor α(TNFα) induced apoptosis is limited by its coactivation of nuclear factor kappa B(NF- κB) -dependent antiapoptosis genes. We examined whether pyrrolidine dithiocarbamate (PDTC) enhance TNFα - induced apoptosis in cultured breast cancer cells and explored the role of NF - κB in TNFα - induced apoptosis. Methods: Human breast cancer cell lines MCF-7 and MDA - MB -435s were treated with TNFα、 PDTC and combination therapy . Induction of apoptosis was detected by TUNEL staining and flow cytometry. NF- κB DNA binding activity was detected using electrophoresis mobility shift assay(EMSA) . Western blots of cytoplasmic lysates were performed to demonstrate IκBα (Inhibitor protein of nuclear factor κB) phosphorylation and degradation. Results:TNFα-induced IκBo phosphorylation and degradation, which was inhibited by PDTC in both cell lines. TNFα-induced apoptosis (TUNEL) increased significantly when both cells were pretreated with PDTC. Flow cytometry also confirmed this. EMSA showed that PDTC continuously inhibited TNFo-induced NF- κB DNA binding activity . Conclusions:PDTC enhances TNFo-induced apoptosis whileinhibiting IκBα phosphorylation and degradation in human breast cancer cells. NF - κB has a protective role on TNFα-induced apoptosis.

  2. Ethanol extract of Hedyotis diffusa willd upregulates G0/G1 phase arrest and induces apoptosis in human leukemia cells by modulating caspase cascade signaling and altering associated genes expression was assayed by cDNA microarray.

    Science.gov (United States)

    Kuo, Yu-Jui; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-09-01

    The authors' previous study has shown that water extract of Hedyotis diffusa Willd (HDW) promoted immune response and exhibited anti-leukemic activity in BALB/c leukemic mice in vivo. In this study, the anti-proliferation effects of ethanol extract of H. diffusa Willd (EEHDW) on lung cancer cell lines (A549, H1355, and LLC), leukemia cell lines (HL-60, WEHI-3), and a mouse melanoma cell line (B16F10) in vitro were investigated. The results demonstrated that EEHDW suppressed the cell proliferation of A549, H1355, HL-60, WEHI-3, and B16F10 cells as well as reduced cell viability in a concentration-dependent manner. We found that EEHDW inhibited the cell proliferation of HL-60 cells in concentration-dependent manner. In addition, EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and sub-G1 population (apoptotic cells). EEHDW provoked DNA condensation and DNA damage in HL-60 cells. The activities of caspase-3, caspase-8, and caspase-9 were elevated in EEHDW-treated HL-60 cells. DNA microarray to investigate and display the gene levels related to cell growth, signal transduction, apoptosis, cell adhesion, cell cycle, DNA damage and repair, transcription and translation was also used. These findings suggest that EEHDW may be a potential herbal medicine and therapeutic agent for the treatment of leukemia. PMID:24677778

  3. Gene transfer by cationic surfactants is essentially limited by the trapping of the surfactant/DNA complexes onto the cell membrane: a fluorescence investigation.

    Science.gov (United States)

    Clamme, J P; Bernacchi, S; Vuilleumier, C; Duportail, G; Mély, Y

    2000-08-25

    The interaction between complexes of plasmid DNA with cetyltrimethylammonium bromide (CTAB) and L929 fibroblasts was first examined using confocal microscopy. The complexes labeled with the DNA intercalator, YOYO-1, were found to be trapped onto the external face of the plasma membrane; a feature that may constitute a major limiting step in transfection. Moreover, since no cytotoxic effect appeared in these conditions, we further inferred that the CTAB molecules remained bound to the DNA. The interaction of the complexes with the membranes was best modeled with neutral vesicles. From anisotropy thermotropic curves of DPHpPC-labeled vesicles and fluorescence resonance energy transfer measurements between these vesicles and YOYO-labeled complexes, we evidenced that the binding of the complexes to the vesicle surface opened the micelle-like domains and unwound DNA. However, DNA was not released but remained stably bound via electrostatic interactions to the CTAB molecules incorporated in the external liposome leaflet. Consequently, the large diameter of the unwound plasmid DNA is likely the major factor that precludes its internalization into the cells by endocytosis. In contrast, anionic vesicles that mimic the cytoplasmic facing monolayer of the plasma membrane rapidly released DNA from the complex. This may explain the previously reported high transfection efficiency of DNA complexed with liposomes composed of neutral lipids and cationic surfactants, since the latter may destabilize the endosomal membrane and induce the release of DNA in the cytoplasm. PMID:11030593

  4. Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision.

    Science.gov (United States)

    Maher, Robyn L; Vallur, Aarthy C; Feller, Joyce A; Bloom, Linda B

    2007-01-01

    The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1. PMID:17018265

  5. Study of apoptosis in human liver cancers

    Institute of Scientific and Technical Information of China (English)

    Chang-Min Shan; Juan Li

    2002-01-01

    AIM: To investigate the action of apoptosis in occurrence ofliver cacinornas in vivo and the biological effect of Solanumlyratum Thumb on BEL-7404 cell line inducing apoptosis invitro.METHODS: The apoptosis in the liver carcinoma wasdetected with terminal deoxynucl neotidyl transferasemediated dUTP nick end labelling (TUNEL); the cancer cellscultured in DMED medium were treated with extract ofSolanum lyratum Thumb and observed under microscope,and their DNA was assayed by gel electrophoresis.RESULTS: In vivo apoptotic cells in the cancer adjacenttissues inceased; in vitro treatment of liver cancers withextract of Solanum lyratum Thumb could induce the cells tomanifest a typical apoptotic morphology. Their DNA wasfractured and a characteristic ladder pattem could be foundusing electrophoresis.CONCLUSION: In vivo the apoptosis of carcinomas waslower; maybe the cells divided quickly and then the cancersoccurred. In the cancer adjacent tissues, the apoptosispricked up, and in vitro Solarium lyratum Thumb couldinduce the apoptosis of BEL-7404 cells.

  6. Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-{kappa}B-STAT3-directed gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu; Ghandhi, Shanaz A.; Zhou, Hongning; Huang, Sarah X.; Chai, Yunfei; Amundson, Sally A.; Hei, Tom K.

    2011-07-01

    Mitochondrial DNA depleted ({rho}{sup 0}) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-{kappa}B and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental {rho}{sup +} HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in {rho}{sup 0} cells compared to {rho}{sup +} HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, {Oota}L17{Beta}, {Oota}L18, {Oota}L19, and {Oota}L28{Beta}) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-{kappa}B and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-{kappa}B/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in {rho}{sup +} HSF, but this response was substantially decreased in {rho}{sup 0} HSF. Suppression of the IKK-NF-{kappa}B pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated {rho}{sup +} HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-{kappa}B activation was partially lost in {rho}{sup 0} HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-{kappa}B targets, further suppressing IL6

  7. Molecular DNA variation among Triturus vittatus vittatus (Urodela) from different breeding sites at the southern limit of its distribution

    OpenAIRE

    Oren Pearlson; Gad Degani

    2007-01-01

    Molecular DNA variation among Triturus vittatus vittatus (striped newt) from different breeding sites at the southern limit of the species distribution (where environmental conditions are most extreme) was studied by the random amplification of polymorphic DNA (RAPD) method that has been found to be appropriate for other Triturus species. Altitudes of the localities ranged between 15-740 m a.s.l. Of the 20 primers employed, OPA-16 was the only one suitable for T. vittatus, revealing a differe...

  8. Apoptosis in cancer: therapeutic implications

    OpenAIRE

    Negoescu, A.

    2000-01-01

    This review outlines the principal limitations of the mechanisms of active cell death (ACD, apoptosis) as the basis of tumorigenesis and the rationale of almost all therapies of malignancy. The concentration of cancer therapy in the directon of ACD induction is presented as both the result of progressive understanding of the mechanisms of apoptosis and that of the favourable tumor environment for ACD signal transmission. The latter property induces the by-stand...

  9. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    OpenAIRE

    Guo Zong-Lun; Tsai Ming-Tz; Lee Alan; Yang Hsin-Ling; Hseu You-Cheng; Hwang Chi-Ching; Chen Jeff; Chang Fang-Rong; Chiu Chien-Chih; Yen Ching-Yu; Cheng Yu-Shan; Liu Yin-Chang; Lan Yu-Hsuan; Chang Yu-Ching; Ko Ying-Chin

    2010-01-01

    Abstract Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkalin...

  10. Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

    Science.gov (United States)

    Routledge, M N; Allan, J M; Garner, R C

    1997-07-01

    To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

  11. Invertebrate Iridovirus Modulation of Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Trevor Williams; Nllesh S. Chitnis; Sh(a)n L. Bilimoria

    2009-01-01

    Programmed cell death (apoptosis) is a key host response to virus infection. Viruses that can modulate host apoptotic responses are likely to gain important opportunities for transmission. Here we review recent studies that demonstrate that particles of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae, genus Iridovirus), or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off host macromolecular synthesis. Induction of apoptosis depends on endocytosis of one or more heat-sensitive virion component(s). Studies with a JNK inh ibitor(SP600125) indicated that the JNK signaling pathway is significantly involved in apoptosis in IIV-6 infections of Choristoneurafumiferana ceils. The genome of IIV-6 codes for an inhibitor of apoptosis iap gene (193R) that encodes a protein of 208 aa with 15% identity and 28% similarity in its amino acid sequence to IAP-3 from Cydia pomonella ganulovirus (CpGV). Transcription of IIV-6 iap did not require prior DNA or protein synthesis, indicating that it is an immediate-early class gene. Transient expression and gene knockdown studies have confirmed the functional nature of the IIV-6 iap gene. We present a tentative model for IIV-6 induction and inhibition of apoptosis in insect cells and discuss the potential applications of these findings in insect pest control.

  12. Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool.

    Science.gov (United States)

    Kalaivani, P; Saranya, S; Poornima, P; Prabhakaran, R; Dallemer, F; Vijaya Padma, V; Natarajan, K

    2014-07-23

    A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis. PMID:24946146

  13. DNA Hypermethylation of CREB3L1 and Bcl-2 Associated with the Mitochondrial-Mediated Apoptosis via PI3K/Akt Pathway in Human BEAS-2B Cells Exposure to Silica Nanoparticles

    Science.gov (United States)

    Zou, Yang; Li, Qiuling; Jiang, Lizhen; Guo, Caixia; Li, Yanbo; Yu, Yang; Li, Yang; Duan, Junchao; Sun, Zhiwei

    2016-01-01

    The toxic effects of silica nanoparticles (SiNPs) are raising concerns due to its widely applications in biomedicine. However, current information about the epigenetic toxicity of SiNPs is insufficient. In this study, the epigenetic regulation of low-dose exposure to SiNPs was evaluated in human bronchial epithelial BEAS-2B cells over 30 passages. Cell viability was decreased in a dose- and passage-dependent manner. The apoptotic rate, the expression of caspase-9 and caspase-3, were significantly increased induced by SiNPs. HumanMethylation450 BeadChip analysis identified that the PI3K/Akt as the primary apoptosis-related pathway among the 25 significant altered processes. The differentially methylated sites of PI3K/Akt pathway involved 32 differential genes promoters, in which the CREB3L1 and Bcl-2 were significant hypermethylated. The methyltransferase inhibitor, 5-aza, further verified that the DNA hypermethylation status of CREB3L1 and Bcl-2 were associated with downregulation of their mRNA levels. In addition, mitochondrial-mediated apoptosis was triggered by SiNPs via the downregulation of PI3K/Akt/CREB/Bcl-2 signaling pathway. Our findings suggest that long-term low-dose exposure to SiNPs could lead to epigenetic alterations. PMID:27362941

  14. Joint effects of cadmium and mercury on DNA damage and apoptosis in human embryo hepatocytes%镉与汞联合作用对人胚肝细胞凋亡和DNA损伤的影响

    Institute of Scientific and Technical Information of China (English)

    蓝晖翔; 江松; 纪存委; 郑丹

    2013-01-01

    目的 研究镉与汞单独及联合染毒对人胚肝细胞(L02细胞)凋亡及DNA损伤的作用.方法 以0.01~100μmol/L的氯化镉、氯化汞以及等浓度的氯化镉、氯化汞混合液对L02细胞染毒24 h,采用MTT法测定细胞的存活率,根据Finney法判断镉与汞对L02细胞的联合作用类型;采用单细胞凝胶电泳技术(SCGE)和流式细胞术(FCM)检测L02细胞的DNA损伤和细胞凋亡情况.结果 0.01 μmol/L的氯化镉、氯化汞单独及联合染毒可以刺激细胞的生长,但≥1μmol/L的氯化镉、氯化汞单独及联合染毒可显著抑制细胞的生长;且联合作用表现为相加作用.镉、汞单独及联合作用可致L02细胞的DNA损伤率和细胞凋亡率均显著高于对照组;且随着染毒浓度的升高,L02细胞的DNA损伤率和细胞凋亡率均呈上升趋势.结论 镉汞联合染毒引起的DNA损伤和细胞凋亡可能存在一定的协同效应,这可能是由于镉、汞诱导细胞发生氧化应激所致.%Objective To study the effects of cadmium and mercury alone and in combination on DNA damage and apoptosis in human embryo hepatocytes (LO2 cells). Methods LO2 cells were exposed to 0.01-100 μmol/L of cadmium chloride (CdCl2), mercury chloride (HgCl2 and their mixtures (CdCl2+HgCl2) for 24 hours. MTT method was used to measure the survival of LO2 cells,and then the joint effects of these mixtures were judged depending on the Finney law. Single cell gel electrophoresis (SCGE) and flow cytometry (FCM) were used to measure the DNA damage and apoptosis of LO2 cells respectively. Results 0.01 μmol/L of cadmium,mercury and their mixtures could stimulate the growth of LO2 cells,but when the concentration of cadmium,mercury and their mixtures were more than 1 μmol/L,they could significantly inhibit the growth of LO2 cells, and the joint effects of cadmium and mercury were additive action. The rates of DNA damage and the percentages of apoptosis were significantly higher than the

  15. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

    Science.gov (United States)

    Lamichhane, Tek N; Raiker, Rahul S; Jay, Steven M

    2015-10-01

    Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

  16. EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity

    Science.gov (United States)

    Li, Yingqian; Takahashi, Yoshimasa; Fujii, Shin-ichiro; Zhou, Yang; Hong, Rongjian; Suzuki, Akari; Tsubata, Takeshi; Hase, Koji; Wang, Ji-Yang

    2016-01-01

    Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance. PMID:26935903

  17. The simultaneous detection of mitochondrial DNA damage from sun-exposed skin of three whale species and its association with UV-induced microscopic lesions and apoptosis.

    Science.gov (United States)

    Bowman, Amy; Martinez-Levasseur, Laura M; Acevedo-Whitehouse, Karina; Gendron, Diane; Birch-Machin, Mark A

    2013-07-01

    Due to life history and physiological constraints, cetaceans (whales) are unable to avoid prolonged exposure to external environmental insults, such as solar ultraviolet radiation (UV). The majority of studies on the effects of UV on skin are restricted to humans and laboratory animals, but it is important to develop tools to understand the effects of UV damage on large mammals such as whales, as these animals are long-lived and widely distributed, and can reflect the effects of UV across a large geographical range. We and others have used mitochondrial DNA (mtDNA) as a reliable marker of UV-induced damage particularly in human skin. UV-induced mtDNA strand breaks or lesions accumulate throughout the lifespan of an individual, thus constituting an excellent biomarker for cumulative exposure. Based on our previous studies in human skin, we have developed for the first time in the literature a quantitative real-time PCR methodology to detect and quantify mtDNA lesions in skin from sun-blistered whales. Furthermore the methodology allows for simultaneous detection of mtDNA damage in different species. Therefore using 44 epidermal mtDNA samples collected from 15 blue whales, 10 fin whales, and 19 sperm whales from the Gulf of California, Mexico, we quantified damage across 4.3 kilobases, a large region of the ~16,400 base pair whale mitochondrial genome. The results show a range of mtDNA damage in the skin of the three different whale species. This previously unreported observation was correlated with apoptotic damage and microscopic lesions, both of which are markers of UV-induced damage. As is the case in human studies, this suggests the potential use of mtDNA as a biomarker for measuring the effect of cumulative UV exposure in whales and may provide a platform to help understand the effects of changing global environmental conditions. PMID:23583579

  18. Design and synthesis of C3-pyrazole/chalcone-linked beta-carboline hybrids: antitopoisomerase I, DNA-interactive, and apoptosis-inducing anticancer agents.

    Science.gov (United States)

    Kamal, Ahmed; Srinivasulu, Vunnam; Nayak, V Lakshma; Sathish, Manda; Shankaraiah, Nagula; Bagul, Chandrakant; Reddy, N V Subba; Rangaraj, Nandini; Nagesh, Narayana

    2014-09-01

    A series of β-carboline hybrids bearing a substituted phenyl and a chalcone/(N-acetyl)-pyrazole moiety at the C1 and C3 positions, respectively, was designed, synthesized, and evaluated for anticancer activity. These new hybrid molecules showed significant cytotoxic activity, with IC50 values ranging from indol-3-yl)prop-2-en-1-one (7 d) and 1-(3-(furan-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (8 d) could effectively cleave pBR322 plasmid DNA upon irradiation with UV light. Active hybrid 8 d inhibited DNA topoisomerase I activity efficiently and preserved DNA in the supercoiled form. To further corroborate the biological activities, as well as to understand the nature of the interaction of these hybrids with DNA, spectroscopic studies were also performed. Unlike simple β-carboline alkaloids, the binding mode of these new hybrid molecules with DNA was not similar, and both biophysical as well as molecular docking studies speculated a combilexin-type of interaction with DNA. Further, an in silico study of these β-carboline hybrids revealed their drug-like properties. PMID:24470122

  19. Constitutive activation of the ATM/BRCA1 pathway prevents DNA damage-induced apoptosis in 5-azacytidine-resistant cell lines.

    Science.gov (United States)

    Imanishi, Satoshi; Umezu, Tomohiro; Ohtsuki, Kazushige; Kobayashi, Chiaki; Ohyashiki, Kazuma; Ohyashiki, Junko H

    2014-06-01

    5-Azacytidine (AZA) exerts its anti-tumor effects by exerting cytotoxicity via its incorporation into RNA and DNA, which causes the reactivation of aberrantly silenced growth-regulatory genes by promoter demethylation, as well as DNA damage. AZA is used for patients with myelodysplastic syndrome and acute myeloid leukemia. However, some patients demonstrate resistance to AZA, the mechanisms of which are not fully elucidated. We therefore sought to better characterize the molecular mechanism of AZA resistance using an in vitro model of AZA resistance. We established AZA-resistant cell lines by exposing the human leukemia cell lines U937 and HL-60 to clinical concentrations of AZA, and characterized these cells. AZA-resistant cells showed a down-regulation of the DNMT3A protein, in correlation with their marked genome-wide DNA hypomethylation. Furthermore, genes involved in pyrimidine metabolism were down-regulated in both AZA-resistant cell lines; AZA sensitivity was restored by inhibition of CTP synthase. Of note is that the DNA damage response pathway is constitutively activated in the AZA-resistant cell lines, but not in the parental cell lines. Inhibition of the DNA damage response pathway canceled the AZA resistance, in association with an increase in apoptotic cells. We found that the molecular mechanism underlying AZA resistance involves pyrimidine metabolism and the DNA damage response through ATM kinase. This study therefore sheds light on the mechanisms underlying AZA resistance, and will enable better understanding of AZA resistance in patients undergoing AZA treatment. PMID:24680865

  20. Leaf extracts from Moricandia arvensis promote antiproliferation of human cancer cells, induce apoptosis, and enhance antioxidant activity.

    Science.gov (United States)

    Skandrani, Ines; Boubaker, Jihed; Bhouri, Wissem; Limem, Ilef; Kilani, Soumaya; Ben Sghaier, Mohamed; Neffati, Aicha; Bouhlel, Ines; Ghedira, Kamel; Chekir-Ghedira, Leila

    2010-01-01

    The in vitro antiproliferative, apoptotic, and antioxidant activities from leaf extracts of Moricandia arvensis, which are used in traditional cooking and medicines, were investigated. The MTT assay revealed that only TOF (total oligomer flavonoids), ethyl acetate (EA), chloroform (Chl), and petroleum ether (PE) extracts inhibited the proliferation of K562 cells. Apoptosis plays a very important role in the treatment of cancer by promoting the apoptosis of cancer cells and limiting the concurrent death of normal cells. Thus, the possible effects of M. arvensis extracts on the induction of apoptosis in human leukemic cells (K562 cells) were investigated. The electrophoretic analysis of DNA fragmentation confirms that TOF, Chl, PE, and EA extracts provoke DNA fragmentation. Using the lipid peroxidation inhibitory assay, the antioxidant capacity of M. arvensis extracts was evaluated by the ability of each extract to inhibit malondialdehyde formation. It was revealed that EA and TOF extracts are the most active in scavenging the hydroxyl radicals. PMID:19995267

  1. Apoptosis in mtDNA-depleted cells and its transmitochondrial cybrids%诱导细胞mtDNA缺失以及再转入线粒体后对肿瘤细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    李歆强; 凌贤龙; 周源; 何玉琦; 李诗伟; 晏斌

    2009-01-01

    目的 对比研究mtDNA缺失以及再转入线粒体后细胞凋亡的变化.方法 在成功构建ρ_0SK-Hepl细胞和转线粒体细胞SK-HeplCyb的基础上,采用Annexin V/PI双染色法检测细胞凋亡;流式细胞仪检测细胞内活性氧(ROS)和线粒体膜电位(△ψm);Western blot检测细胞Bcl-2、Bax表达水平;免疫荧光染色观测Bel-2细胞内分布.结果 SK-Hepl、ρ~0SK-Hepl和sK-HeplCyb细胞凋亡率分别为(2.01±0.11)%、(0.37±0.08)%和(2.10±0.12)%.ρ~0SK-Hepl对细胞凋亡有明显抗性(P0.05).ρ~0SK-Hepl细胞线粒体Bcl-2、Bax表达增多,Bcl-2/Bax比值增加(P<0.01).SK-HeplCyb细胞线粒体Bcl-2/Bax值下降.结论 mtDNA缺失肿瘤细胞对细胞凋亡有明显拮抗.Bcl-2线粒体转位、线粒体Bci-2/Bax值增加、ROS产生增多可能参与细胞凋亡拮抗的形成.%Objective To investigate cell apoptosis in mtDNA-depleted SK-Hepl cells and the transmito-chondrial cybrids on the basis of establishment of ρ~0 SK-Hepl (a mtDNA-depleted cell line derived from human hepatoma cell line SK-Hepl) and the transmitochondrial cybrids, SK-HeplCyb. Methods SK-Hepl cells,ρ~0 SK-Hepl cells, and the transmitochondrial cybrids were cultured in high-glucose DMEM. Apoptosis was de-termined by Annexin V/PI assay. Flow cytometry (FCM) and laser scan confoeal microscopy were used to de-tect reactive oxygen species (ROS) and mitochondrial membrane potential (△ψm) respectively. Western blot analysis was applied to detect Bcl-2 and Bax expressions in the above 3 groups of cells. Distribution of Bcl-2 in the 3 groups was assessed by immunofluorescence analysis. Results Apoptotic ratio of SK-Hepl, ρ~0 SK-Hepl and SK-HeplCyb cells were (2.01±0.11)%, (0.37±0.08)% and (2.10±0.12)%, respectively.ρ~0 SK-Hepl cells were more counteractive against apoptosis than the other 2 cells (P <0.05). Fluorescence in-tensity of ROS in ρ~0 SK-Hepl cells was significantly higher than that of the parent cells (35.5 vs 15.6, P <0.01), but the

  2. Signal transduction and metabolic changes during tumor cell apoptosis following phthalocyanine-sensitized photodynamic therapy

    Science.gov (United States)

    Oleinick, Nancy L.; Agarwal, Munna L.; Berger, Nathan A.; Cheng, Ming-Feng; Chatterjee, Satadel; He, Jin; Kenney, Malcolm E.; Larkin, Hedy E.; Mukhter, Hasan; Rihter, Boris D.; Zaidi, Syed I. A.

    1993-06-01

    Mechanisms of cell death have been explored in cells and tumors treated with photodynamic therapy (PDT). Photosensitizers used for these studies were Photofrin, tetrasulfonated and nonsulfonated aluminum phthalocyanine, and a new silicon phthalocyanine [SiPc(OH)OSi(CH3)2(CH2)3N(CH3)2], referred to as PcIV. In mouse lymphoma L5178Y cells, a dose of PDT sensitized by PcIV which causes a 90% loss of cell survival induces apoptosis (programmed cell death) over a several-hour time course, beginning within 10 minutes of irradiation. Apoptosis is a metabolic process initiated by PDT-induced damage to membranes and triggered by the activation of phospholipases A2 and C and the release of Ca++ from intracellular stores. An endogenous endonuclease is activated and cleaves nuclear DNA in the internucleosomal region of chromatin. Subsequent metabolic events now appear to cause the loss of cellular NAD and ATP, the former a result of the activation of a second nuclear enzyme, poly(ADP-ribose) polymerase, by the endonucleolytically generated DNA strand breaks. Loss of ATP follows upon the loss of NAD needed for energy metabolism. Although the induction of apoptosis is efficiently produced by direct PDT damage to L5178Y cells, we now find that apoptosis is also produced by treatment of certain other lymphoid-derived cells and cells of epithelial origin. Under the limited set of conditions tested, there was no evidence for PDT-induced apoptosis in a fibroblast cell line, in mouse fibrosarcoma RIF-1 and L929 cells, in human adenocarcinoma A549 cells, or in human squamous cell carcinoma cells in culture. The evidence suggests that apoptosis, a form of metabolic cell death, is an important mechanism of tumor ablation in PDT-treated tumors, and that the induction of apoptosis may involve the interaction of direct PDT damage to malignant cells with factors produced by PDT action on vascular and other host cells.

  3. The RNA Splicing Response to DNA Damage.

    Science.gov (United States)

    Shkreta, Lulzim; Chabot, Benoit

    2015-10-29

    The number of factors known to participate in the DNA damage response (DDR) has expanded considerably in recent years to include splicing and alternative splicing factors. While the binding of splicing proteins and ribonucleoprotein complexes to nascent transcripts prevents genomic instability by deterring the formation of RNA/DNA duplexes, splicing factors are also recruited to, or removed from, sites of DNA damage. The first steps of the DDR promote the post-translational modification of splicing factors to affect their localization and activity, while more downstream DDR events alter their expression. Although descriptions of molecular mechanisms remain limited, an emerging trend is that DNA damage disrupts the coupling of constitutive and alternative splicing with the transcription of genes involved in DNA repair, cell-cycle control and apoptosis. A better understanding of how changes in splice site selection are integrated into the DDR may provide new avenues to combat cancer and delay aging.

  4. Resveratrol-3-O-glucuronide and resveratrol-4’-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin

    Science.gov (United States)

    Resveratrol has been reported to inhibit or induce DNA damage depending upon the type of cell and experimental conditions. Dietary resveratrol is present in the body mostly as metabolites and little is known about the activities of these metabolic products. We evaluated physiologically obtainable ...

  5. Few constraints limit the design of quinone methide-oligonucleotide self-adducts for directing DNA alkylation†

    OpenAIRE

    Rossiter, Clifford S.; Modica, Emilia; Kumar, Dalip; Rokita, Steven E

    2010-01-01

    Nucleotide sequences minimally containing adenosine, cytosine or guanosine are sufficient to form intrastrand oligonucleotide quinone methide self-adducts reversibly for subsequent alkylation of complementary DNA. The general lack of sequence restrictions should now allow for alkylation of most any target of interest although reaction is most efficient when the self-adducts contain guanine residues and do not form hairpin structures.

  6. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells.

    Science.gov (United States)

    Kumar, Devbrat; Basu, Soumya; Parija, Lucy; Rout, Deeptimayee; Manna, Sanjeet; Dandapat, Jagneshwar; Debata, Priya Ranjan

    2016-07-01

    Cervical cancer and precancerous lesions of the cervix continue to be a global health issue, and the medication for the treatment for chronic HPV infection so far has not been effective. Potential anticancer and anti HPV activities of two known phytochemicals, Curcumin and Ellagic acid were evaluated in HeLa cervical cancer cells. Curcumin is a natural compound found in the root of Curcuma longa plant and Ellagic acid a polyphenol found in fruits of strawberries, raspberries and walnuts. The combination of Curcumin and Ellagic acid at various concentrations showed better anticancer properties than either of the drug when used alone as evidenced by MTT assay. Besides this, Curcumin and Ellagic acid also restore p53, induce ROS formation and DNA damage. Mechanistic study further indicated that Curcumin and Ellagic acid show anti-HPV activity as evidenced by decrease in the HPV E6 oncoprotein on HeLa cells. PMID:27261574

  7. Monitoring apoptosis in real time.

    Science.gov (United States)

    Green, Allan M; Steinmetz, Neil D

    2002-01-01

    Many therapeutically active anticancer treatments exert their effect by the induction of apoptosis and necrosis. Serial biopsies in breast cancer patients have suggested that response to therapy correlates with early posttreatment increases in tumor apoptotic index. Radiolabeled technetium Tc 99m-recombinant human (rh) annexin V provides a noninvasive technique for imaging treatment-induced cell death. Annexin V is a naturally occurring human protein that binds avidly to membrane-associated phosphatidylserine (PS). PS is normally found only on the inner leaflet of the cell membrane double layer, but it is actively transported to the outer layer as an early event in apoptosis and becomes available for annexin binding. Annexin also gains access to PS as a result of the membrane fragmentation associated with necrosis. In vitro studies of apoptosis using fluorescein annexin have shown good correlation with assessments of apoptosis documented by nuclear DNA degradation and caspase activation. In vivo localization of intravenously administered Tc 99m-annexin V has been demonstrated in numerous preclinical models of apoptosis, including anti-Fas-mediated hepatic apoptosis, rejection of allogeneic heterotopic cardiac allografts, cyclophosphamide treatment of murine lymphoma, cyclophosphamide-induced apoptosis in bone marrow, and leukocyte apoptosis associated with abscess formation. Scintigraphic studies in humans using Tc 99m-rh annexin V have demonstrated the feasibility of imaging cell death in acute myocardial infarction, in tumors with a high apoptotic index, and in response to anti-tumor chemotherapy of non-small cell lung cancer, small-cell lung cancer, breast cancer, lymphoma, and sarcoma. Increased localization of Tc 99m-rh annexin V within 1 to 3 days of chemotherapy has been noted in some, but not all, subjects with these tumors. To date, most subjects showing increased Tc 99m-rh annexin V uptake after the first course of chemotherapy have shown objective

  8. Ciprofloxacin modulates cytokine/chemokine profile in serum, improves bone marrow repopulation, and limits apoptosis and autophagy in ileum after whole body ionizing irradiation combined with skin-wound trauma.

    Directory of Open Access Journals (Sweden)

    Risaku Fukumoto

    Full Text Available Radiation combined injury (CI is a radiation injury (RI combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10. At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6 and KC (i.e., IL-8 in human, but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD(0/10 compared to LD(20/10 for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.

  9. Neem (Azadirachta indica L.) leaf extract deteriorates oocyte quality by inducing ROS-mediated apoptosis in mammals.

    Science.gov (United States)

    Chaube, Shail K; Shrivastav, Tulsidas G; Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ajai K

    2014-01-01

    Neem (Azadirachta indica L.) leaf has been widely used in ayurvedic system of medicine for fertility regulation for a long time. The molecular mechanism by which neem leaf regulates female fertility remains poorly understood. Animal studies suggest that aqueous neem leaf extract (NLE) induces reactive oxygen species (ROS) - mediated granulosa cell apoptosis. Granulosa cell apoptosis deprives oocytes from nutrients, survival factors and cell cycle proteins required for the achievement of meiotic competency of follicular oocytes prior to ovulation. Under this situation, follicular oocyte becomes more susceptible towards apoptosis after ovulation. The increased level of hydrogen peroxide (H2O2) inside the follicular fluid results in the transfer of H2O2 from follicular fluid to the oocyte. The increased level of H2O2 induces p53 activation and over expression of Bax protein that modulates mitochondrial membrane potential and trigger cytochrome c release. The increased cytosolic cytochrome c level induces caspase-9 and caspase-3 activities that trigger destruction of structural and specific proteins leading to DNA fragmentation and thereby oocyte apoptosis. Based on these animal studies, we propose that NLE induces generation of ROS and mitochondria-mediated apoptosis both in granulosa cells as well as in follicular oocyte. The induction of apoptosis deteriorates oocyte quality and thereby limits reproductive outcome in mammals.

  10. Morphologic criteria and detection of apoptosis.

    Science.gov (United States)

    Saraste, A

    1999-05-01

    Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis

  11. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism.

    LENUS (Irish Health Repository)

    Sookhai, S

    2012-02-03

    BACKGROUND: For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. METHODS: PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. RESULTS: There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 mumol\\/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. CONCLUSION: Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism.

  12. Dimerization of two novel apoptosis-inducing proteins and its function in regulating cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    刘青珍; 甘淼; 齐义鹏; 李凌云; 齐兵

    2003-01-01

    Asy (apoptosis/saibousi Yutsudo) is a novel apoptosis-inducing gene found in 1999 by Yutsudo group in Japan. In 2000, Qi Bing et al. cloned another novel gene, named hap (homologue of ASY protein), which encoded the ASY interact ing protein, from human lung cell line (WI-38) cDNA library by using yeast two-h ybrid system. It has been proved that ASY formed homodimer in yeast and human ce ll line, ASY and HAP formed heterodimer in yeast cells, and both induced cell ap optosis in human tumor cell lines Sao2 and CGL4. This paper showed that HAP coul d form homodimer in yeast cells by yeast two-hybrid system; HAP and ASY could pr oduce heterodimer in human cell line by cross-immunoprecipitation test; by using apoptosis-testing technologies such as AnnexinV, TUNEL, DNA ladder and Flow Cyt ometry, the cell apoptosis in human normal or tumor cell lines transfected with hap or asy individually or cotransfected by the both was qualified or quantified . It was firstly demonstrated that ASY or HAP induced cell apoptosis not only in human tumor cell lines, but also in human normal cell lines. Moreover, we prove d that the heterodimer between ASY and HAP decreased apoptosis-inducing activity from the homodimer of ASY or HAP. It revealed that by choosing to form heterodi mer or homodimer between ASY and / or HAP is an important mechanism of regulatin g apoptosis in human cell lines.

  13. Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

    Science.gov (United States)

    Ramesh, Govindarajan; Wu, Honglu

    Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

  14. DETECTION OF B LYMPHOMA CELLS UNDERGOING APOPTOSIS BY ANNEXIN V ASSAY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective.To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. Methods.The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0 μ mol/L dexamethasone (DEX) for 2,4 and 8 h respectively,then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis,and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosis,then apoptotic cells were quantified by flow cytometry (FCM).Furthermore,Annexin+ /PI and Annexin+ /PI+ cells were sorted by fluoresence activated cell sorter (FACS),and identified by electron microscopy (EM) and DNA gel electrophoresis. Results.The percentage of apoptotic cells was found to increase with the incubation time (r=0.97).This method was sensitive with low detection limit (0.02% ),and was reproducible with low coefficient variance (CV)(4.2% ).Meanwhile,the Annexin+ /PI and Annexin+ /PI+ cells were identified as apoptotic and necrotic cells under EM,and DNA extracted from the Annexin+ /PI cells was characterized by " ladder pattern" . Conclusions.Annexin V assay is a specific,sensitive,accurate,reproductive and quantitative method for analyzing apoptotic cells.

  15. Community analysis of ammonia oxidizer in the oxygen-limited nitritation stage of OLAND system by DGGE of PCR amplified 16S rDNA Fragments and FISH

    Institute of Scientific and Technical Information of China (English)

    ZHANG Dan; ZHANG De-min; LIU Yao-ping; CAO Wen-wei; CHEN Guan-xiong

    2004-01-01

    OLAND(oxygen limited autotrophic nitrification and denitrification) nitrogen removal system was constructed by coupling with oxygen limited nitritation stage and anaerobic ammonium oxidation stage. Ammonia oxidizer, as a kind of key bacteria in N cycle, plays an important role at the oxygen limited nitritation stage of OLAND nitrogen removal system. In this study, specific amplification of 16S rDNA fragment of ammonia oxidizer by nested PCR, separation of mixed PCR samples by denaturing gradient gel electrophoresis(DGGE), and the quantification of ammonia oxidizer by Fluorescence in situ hybridization(FISH) were combined to investigate the shifts of community composition and quantity of ammonia oxidizer of the oxygen limited nitritation stage in OLAND system. It showed that the community composition of ammonia oxidizer changed drastically when dissolved oxygen was decreased gradually, and the dominant ammonia oxidizer of the steady nitrite accumulation stage were completely different from that of the early stage of oxygen limited nitritation identified by DGGE . It was concluded that the Nitrosomonas may be the dominant genus of ammonia oxidizer at the oxygen limited nitritation stage of OLAND system characterized by nested PCR-DGGE and FISH, and the percentage of Nitrosomonas was 72.5% ( 0.8% of ammonia oxidizer at the steady nitrite accumulation stage detected by FISH.

  16. Limitations of plasmid vaccines to complex viruses: selected myxoma virus antigens as DNA vaccines were not protective.

    Science.gov (United States)

    Adams, Mathew M; van Leeuwen, Barbara H; Kerr, Peter J

    2004-11-25

    Myxoma virus, a poxvirus of the genus Leporipoxvirus, is the causative agent of the disease myxomatosis which is highly lethal in European rabbits (Oryctolagus cuniculus). Current vaccines to protect against myxomatosis are either attenuated live strains of the virus or the antigenically related rabbit fibroma virus. We examined the immune response of outbred domestic rabbits to the individual myxoma virus antigens M055R, M073R, M115L and M121R, delivered as DNA vaccines co-expressing rabbit interleukin-2 or interleukin-4. M115L and M121R were also delivered simultaneously. None of the vaccine constructs were able to protect the rabbits from disease or reduce mortality after challenge with virulent myxoma virus, despite induction of antigen-specific cell-mediated and humoral immune responses. PMID:15531037

  17. Maternal n-3 polyunsaturated fatty acid deprivation during pregnancy and lactation affects neurogenesis and apoptosis in adult offspring: associated with DNA methylation of brain-derived neurotrophic factor transcripts.

    Science.gov (United States)

    Fan, Chaonan; Fu, Huicong; Dong, Hua; Lu, Yuanyuan; Lu, Yanfei; Qi, Kemin

    2016-09-01

    In this study, we hypothesized that n-3 polyunsaturated fatty acid (PUFA) deficiency during pregnancy and lactation will make a lasting impact on brain neurogenesis and apoptosis of the adult offspring and that these harmful effects cannot be reversed by n-3 PUFA supplementation after weaning. Moreover, the underlying mechanisms may be attributable to the epigenetic changes of brain-derived neurotrophic factor (BDNF). C57BL/6J female mice were fed with n-3 PUFA-deficient diet (n-3 def) or n-3 PUFA-adequate diet (n-3 adq) throughout pregnancy and lactation. At postnatal 21 days, equal numbers of male pups from both groups were fed the opposite diet, and the remaining male pups were fed with the same diets as their mothers until 3 months of age. Feeding the n-3 adq diet to pups from the maternal n-3 def group significantly increased the n-3 PUFA concentration but did not change expressions of calretinin, Bcl2, and Bax in the hippocampus. Feeding the n-3 def diet to pups from the maternal n-3 adq group significantly reduced the n-3 PUFA concentration but did not reduce expressions of calretinin and Bcl2. Similarly, BDNF levels, especially mRNA expressions of BDNF transcripts IV and IX, were also reduced by maternal n-3 def and not reversed by n-3 PUFA supplementation after weaning. The decrease in BDNF expression by maternal n-3 def diet was associated with greater DNA methylation at special CpG sites. These results suggested that the maternal n-3 PUFA deficiency during pregnancy and lactation imprints long-term changes of brain development in adult offspring.

  18. Scope and Limitations of Typical Copper-Free Bioorthogonal Reactions with DNA: Reactive 2'-Deoxyuridine Triphosphates for Postsynthetic Labeling.

    Science.gov (United States)

    Merkel, Marcus; Arndt, Stefanie; Ploschik, Damian; Cserép, Gergely B; Wenge, Ulrike; Kele, Péter; Wagenknecht, Hans-Achim

    2016-09-01

    Four triphosphates of 2'-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic-chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels-Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the "photoclick" cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine-oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45-55 °C. The cyclopropene-oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The "photoclick" cycloaddition between tetrazole-oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2'-deoxyuridine triphosphate. PMID:27513089

  19. APOPTOSIS, OXIDATIVE STRESS AND NEUROLOGICAL DISEASE

    Directory of Open Access Journals (Sweden)

    P. Formichi

    2012-01-01

    Full Text Available Apoptosis is a selective cell deletion process which requires the triggering of a specific cell death programme. Two main pathways determining cell death have been identified: the extrinsic or receptor-mediated pathway, activated in response to extracellular pro-apoptotic signals, and the intrinsic pathway, activated by extracellular receptor-independent stimuli or by intracellular insults, such as DNA damage and oxidative stress. All these stress signals are integrated by mitochondria which participate by releasing the main effectors of this process: a family of aspartic-specific proteases known as caspase. Today there is much evidence to suggest that deregulation of apoptosis is a key feature of many neurodegenerative disease. Our group sought cell models for the study of apoptotic pathways and for the evaluation of the role of apoptosis in specific neurodegenerative diseases. We focused on oxidative stress-induced apoptosis and activation of the intrinsic mitochondrial pathway. In our in-vitro model, lymphocytes from patients and control subjects were cultured both in basal conditions and with 2-deoxy-D-ribose (dRib, a reducing sugar which induces apoptosis through oxidative stress. In the last ten years, we evaluated the role of apoptosis in the pathogenesis of several neurodegenerative diseases: Ataxiatelangiectasia,Rett syndrome, Mitochondrial disease, Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL. Here we report some of our ongoing and recently published articles.

  20. DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions.

    Science.gov (United States)

    Insinga, Alessandra; Cicalese, Angelo; Faretta, Mario; Gallo, Barbara; Albano, Luisa; Ronzoni, Simona; Furia, Laura; Viale, Andrea; Pelicci, Pier Giuseppe

    2013-03-01

    DNA damage leads to a halt in proliferation owing to apoptosis or senescence, which prevents transmission of DNA alterations. This cellular response depends on the tumor suppressor p53 and functions as a powerful barrier to tumor development. Adult stem cells are resistant to DNA damage-induced apoptosis or senescence, however, and how they execute this response and suppress tumorigenesis is unknown. We show that irradiation of hematopoietic and mammary stem cells up-regulates the cell cycle inhibitor p21, a known target of p53, which prevents p53 activation and inhibits p53 basal activity, impeding apoptosis and leading to cell cycle entry and symmetric self-renewing divisions. p21 also activates DNA repair, limiting DNA damage accumulation and self-renewal exhaustion. Stem cells with moderate DNA damage and diminished self-renewal persist after irradiation, however. These findings suggest that stem cells have evolved a unique, p21-dependent response to DNA damage that leads to their immediate expansion and limits their long-term survival.

  1. Induction of apoptosis in frog virus 3-infected cells.

    Science.gov (United States)

    Chinchar, V G; Bryan, Locke; Wang, J; Long, Scott; Chinchar, G D

    2003-02-15

    The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins. PMID:12642103

  2. Apoptosis in the Retina

    OpenAIRE

    Crisanti, Patricia; Lecain, Eric; Omri, Boubaker

    2007-01-01

    Retinal degenerations are a common cause of blindness in Western countries. Despite various origins of retinal degeneration it is well recognised that. Apoptosis is the final pathway of photoreceptor neuron cell death in these diseases. So that Ivana Scovassi presents the historical development of our knowledge in: apoptosis, its difference with other forms of cell death as necrosis and analyses when and how apoptosis arises, discussing also the molecular markers in this form of cell death. T...

  3. Repression of the DNA-binding inhibitor Id3 by Blimp-1 limits CD8+ T cell memory formation

    Science.gov (United States)

    Ji, Yun; Pos, Zoltan; Rao, Mahadev; Klebanoff, Christopher A.; Yu, Zhiya; Sukumar, Madhusudhanan; Reger, Robert N.; Palmer, Douglas C.; Borman, Zachary A.; Muranski, Pawel; Wang, Ena; Schrump, David S.; Marincola, Francesco M.; Restifo, Nicholas P.; Gattinoni, Luca

    2011-01-01

    Blimp-1 is a transcriptional repressor that promotes the differentiation of CD8+ T cells into short-lived KLRG-1+ effector cells (SLEC), but how it operates remains poorly defined. Here we show that Blimp-1 binds and represses the Id3 promoter in SLEC. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited their capacity to persist as memory cells. Enforced expression of Id3 was sufficient to rescue SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of E2a transcriptional activity and induction of genes regulating genome stability. These findings identify a Blimp-1-Id3-E2a axis as a key molecular switch that determines whether effector CD8+ T cells are programmed to die or enter the memory pool. PMID:22057288

  4. Homeostatic imbalance between apoptosis and cell renewal in the liver of premature aging Xpd mice.

    Directory of Open Access Journals (Sweden)

    Jung Yoon Park

    Full Text Available Unrepaired or misrepaired DNA damage has been implicated as a causal factor in cancer and aging. Xpd(TTD mice, harboring defects in nucleotide excision repair and transcription due to a mutation in the Xpd gene (R722W, display severe symptoms of premature aging but have a reduced incidence of cancer. To gain further insight into the molecular basis of the mutant-specific manifestation of age-related phenotypes, we used comparative microarray analysis of young and old female livers to discover gene expression signatures distinguishing Xpd(TTD mice from their age-matched wild type controls. We found a transcription signature of increased apoptosis in the Xpd(TTD mice, which was confirmed by in situ immunohistochemical analysis and found to be accompanied by increased proliferation. However, apoptosis rate exceeded the rate of proliferation, resulting in homeostatic imbalance. Interestingly, a metabolic response signature was observed involving decreased energy metabolism and reduced IGF-1 signaling, a major modulator of life span. We conclude that while the increased apoptotic response to endogenous DNA damage contributes to the accelerated aging phenotypes and the reduced cancer incidence observed in the Xpd(TTD mice, the signature of reduced energy metabolism is likely to reflect a compensatory adjustment to limit the increased genotoxic stress in these mutants. These results support a general model for premature aging in DNA repair deficient mice based on cellular responses to DNA damage that impair normal tissue homeostasis.

  5. Extremely Low Frequency Magnetic Fields Induce Spermatogenic Germ Cell Apoptosis: Possible Mechanism

    Directory of Open Access Journals (Sweden)

    Sang-Kon Lee

    2014-01-01

    Full Text Available The energy generated by an extremely low frequency electromagnetic field (ELF-EMF is too weak to directly induce genotoxicity. However, it is reported that an extremely low frequency magnetic field (ELF-MF is related to DNA strand breakage and apoptosis. The testes that conduct spermatogenesis through a dynamic cellular process involving meiosis and mitosis seem vulnerable to external stress such as heat, MF exposure, and chemical or physical agents. Nevertheless the results regarding adverse effects of ELF-EMF on human or animal reproductive functions are inconclusive. According to the guideline of the International Commission on Non-Ionizing Radiation Protection (ICNIRP; 2010 for limiting exposure to time-varying MF (1 Hz to 100 kHz, overall conclusion of epidemiologic studies has not consistently shown an association between human adverse reproductive outcomes and maternal or paternal exposure to low frequency fields. In animal studies there is no compelling evidence of causal relationship between prenatal development and ELF-MF exposure. However there is increasing evidence that EL-EMF exposure is involved with germ cell apoptosis in testes. Biophysical mechanism by which ELF-MF induces germ cell apoptosis has not been established. This review proposes the possible mechanism of germ cell apoptosis in testes induced by ELF-MF.

  6. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies.

    Science.gov (United States)

    Pistritto, Giuseppa; Trisciuoglio, Daniela; Ceci, Claudia; Garufi, Alessia; D'Orazi, Gabriella

    2016-04-01

    Apoptosis is a form of programmed cell death that results in the orderly and efficient removal of damaged cells, such as those resulting from DNA damage or during development. Apoptosis can be triggered by signals from within the cell, such as genotoxic stress, or by extrinsic signals, such as the binding of ligands to cell surface death receptors. Deregulation in apoptotic cell death machinery is an hallmark of cancer. Apoptosis alteration is responsible not only for tumor development and progression but also for tumor resistance to therapies. Most anticancer drugs currently used in clinical oncology exploit the intact apoptotic signaling pathways to trigger cancer cell death. Thus, defects in the death pathways may result in drug resistance so limiting the efficacy of therapies. Therefore, a better understanding of the apoptotic cell death signaling pathways may improve the efficacy of cancer therapy and bypass resistance. This review will highlight the role of the fundamental regulators of apoptosis and how their deregulation, including activation of anti-apoptotic factors (i.e., Bcl-2, Bcl-xL, etc) or inactivation of pro-apoptotic factors (i.e., p53 pathway) ends up in cancer cell resistance to therapies. In addition, therapeutic strategies aimed at modulating apoptotic activity are briefly discussed.

  7. The phage growth limitation system in Streptomyces coelicolor A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity.

    Science.gov (United States)

    Hoskisson, Paul A; Sumby, Paul; Smith, Margaret C M

    2015-03-01

    The phage growth limitation system of Streptomyces coelicolor A3(2) is an unusual bacteriophage defence mechanism. Progeny ϕC31 phage from an initial infection are thought to be modified such that subsequent infections are attenuated in a Pgl(+) host but normal in a Pgl(-) strain. Earlier work identified four genes required for phage resistance by Pgl. Here we demonstrate that Pgl is an elaborate and novel phage restriction system that, in part, comprises a toxin/antitoxin system where PglX, a DNA methyltransferase is toxic in the absence of a functional PglZ. In addition, the ATPase activity of PglY and a protein kinase activity in PglW are shown to be essential for phage resistance by Pgl. We conclude that on infection of a Pgl(+) cell by bacteriophage ϕC31, PglW transduces a signal, probably via phosphorylation, to other Pgl proteins resulting in the activation of the DNA methyltransferase, PglX and this leads to phage restriction.

  8. Pre-Columbian origins of Native American dog breeds, with only limited replacement by European dogs, confirmed by mtDNA analysis.

    Science.gov (United States)

    van Asch, Barbara; Zhang, Ai-bing; Oskarsson, Mattias C R; Klütsch, Cornelya F C; Amorim, António; Savolainen, Peter

    2013-09-01

    Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.

  9. Pre-Columbian origins of Native American dog breeds, with only limited replacement by European dogs, confirmed by mtDNA analysis.

    Science.gov (United States)

    van Asch, Barbara; Zhang, Ai-bing; Oskarsson, Mattias C R; Klütsch, Cornelya F C; Amorim, António; Savolainen, Peter

    2013-09-01

    Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin. PMID:23843389

  10. ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

    Science.gov (United States)

    Pusapati, Raju V.; Rounbehler, Robert J.; Hong, Sungki; Powers, John T.; Yan, Mingshan; Kiguchi, Kaoru; McArthur, Mark J.; Wong, Paul K.; Johnson, David G.

    2006-01-01

    Overexpression of the c-myc oncogene contributes to the development of a significant number of human cancers. In response to deregulated Myc activity, the p53 tumor suppressor is activated to promote apoptosis and inhibit tumor formation. Here we demonstrate that p53 induction in response to Myc overexpression requires the ataxia-telangiectasia mutated (ATM) kinase, a major regulator of the cellular response to DNA double-strand breaks. In a transgenic mouse model overexpressing Myc in squamous epithelial tissues, inactivation of Atm suppresses apoptosis and accelerates tumorigenesis. Deregulated Myc expression induces DNA damage in primary transgenic keratinocytes and the formation of H2AX and phospho-SMC1 foci in transgenic tissue. These findings suggest that Myc overexpression causes DNA damage in vivo and that the ATM-dependent response to this damage is critical for p53 activation, apoptosis, and the suppression of tumor development. p53 | DNA damage

  11. Measuring Apoptosis by Microscopy and Flow Cytometry.

    Science.gov (United States)

    Hollville, Emilie; Martin, Seamus J

    2016-02-02

    Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, DNA hydrolysis, and nuclear fragmentation. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the efficient recognition and removal of apoptotic cells by phagocytes. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by rapid plasma membrane rupture followed by organelle and cell swelling. Necrosis is often provoked by infectious agents or a severe departure from physiological conditions. This unit describes protocols for the measurement of apoptosis and for distinguishing apoptosis from necrosis.

  12. Inhibitor of apoptosis proteins and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yunbo Wei; Tingjun Fan; Miaomiao Yu

    2008-01-01

    Apoptosis is a physiological cell death process that plays a critical role in development, homeostasis, and immune defense of multicellular animals. Inhibitor of apoptosis proteins (IAPs) constitute a family of proteins that possess between one and three baculovirus IAP repeats. Some of them also have a really interesting new gene finger domain, and can prevent cell death by binding and inhibiting active caspases, but are regulated by IAP antagonists. Some evidence also indicates that IAP can modulate the cell cycle and signal transduction. The three main factors, IAPs, IAP antagonists, and caspases, are involved in regulating the progress of apoptosis in many species. Many studies and assumptions have been focused on the anfractuous interactions between these three main factors to explore their real functional model in order to develop potential anticancer drugs.In this review, we describe the classification, molecular structures, and properties of IAPs and discuss the mechanisms of apoptosis. We also discuss the promising significance of clinical applications of IAPs in the diagnosis and treatment of malignancy.

  13. [GPI-Pr Deficiency and Apoptosis of PNH Granulocytes

    Science.gov (United States)

    Liu, Hai-Li; Xu, Cai-Min; Liu, Fu-Qiang; Lu, Zhao-Jiang; Pan, Hua-Zhen; Zhang, Zhi-Nan

    2001-09-01

    To study the relationship of Glycosyl phosphatidylinositol anchored proteins (GIP-Pr) and apoptosis of paroxysmal nocturnal hemoglobinuria (PNH) cells, we isolated peripheral granulocytes from 10 patients with PNH and 10 normal controls and measured apoptosis induced by serum starvation. The FCM analysis of phosphotidylserine (ps) externalization in granulocytes was determined using Annexin-V-FLUOS labeling. After the cells were induced for apoptosis in serum-free medium for 20 hours, the percentage of externalization was 78.6% in normal control cells but 39.5% in PNH cells. The results of FCM analysis of PI stained granulocytes showed that the PI positive rate was 51.5% in control cells and 30.2% in PNH cells. The gel electrophoresis analysis of DNA fragmentation all indicate that PNH granulocytes were relatively resistant to apoptosis as compared with normal controls. This resistance to apoptosis might not be related to the percentage of CD59 deficient granulocytes. PMID:12578597

  14. Caspases: An apoptosis mediator

    Directory of Open Access Journals (Sweden)

    Tapan Kumar Palai

    2015-03-01

    Full Text Available The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10 or executioner caspases (caspase 3, 6, 7. Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

  15. Neither philopatric nor panmictic: microsatellite and mtDNA evidence suggests lack of natal homing but limits to dispersal in Pacific lamprey.

    Science.gov (United States)

    Spice, Erin K; Goodman, Damon H; Reid, Stewart B; Docker, Margaret F

    2012-06-01

    Most species with lengthy migrations display some degree of natal homing; some (e.g. migratory birds and anadromous salmonids) show spectacular feats of homing. However, studies of the sea lamprey (Petromyzon marinus) indicate that this anadromous species locates spawning habitat based on pheromonal cues from larvae rather than through philopatry. Previous genetic studies in the anadromous Pacific lamprey (Entosphenus tridentatus) have both supported and rejected the hypothesis of natal homing. To resolve this, we used nine microsatellite loci to examine the population structure in 965 Pacific lamprey from 20 locations from central British Columbia to southern California and supplemented this analysis with mitochondrial DNA restriction fragment length polymorphism analysis on a subset of 530 lamprey. Microsatellite analysis revealed (i) relatively low but often statistically significant genetic differentiation among locations (97% pairwise F(ST) values were <0.04 but 73.7% were significant); and (ii) weak but significant isolation by distance (r(2) = 0.0565, P = 0.0450) but no geographic clustering of samples. The few moderate F(ST) values involved comparisons with sites that were geographically distant or far upstream. The mtDNA analysis--although providing less resolution among sites (only 4.7%F(ST) values were significant)--was broadly consistent with the microsatellite results: (i) the southernmost site and some sites tributary to the Salish Sea were genetically distinct; and (ii) southern sites showed higher haplotype and private haplotype richness. These results are inconsistent with philopatry, suggesting that anadromous lampreys are unusual among species with long migrations, but suggest that limited dispersal at sea precludes panmixia in this species. PMID:22564149

  16. DNA Damage and Repair in Vascular Disease.

    Science.gov (United States)

    Uryga, Anna; Gray, Kelly; Bennett, Martin

    2016-01-01

    DNA damage affecting both genomic and mitochondrial DNA is present in a variety of both inherited and acquired vascular diseases. Multiple cell types show persistent DNA damage and a range of lesions. In turn, DNA damage activates a variety of DNA repair mechanisms, many of which are activated in vascular disease. Such DNA repair mechanisms either stall the cell cycle to allow repair to occur or trigger apoptosis or cell senescence to prevent propagation of damaged DNA. Recent evidence has indicated that DNA damage occurs early, is progressive, and is sufficient to impair function of cells composing the vascular wall. The consequences of persistent genomic and mitochondrial DNA damage, including inflammation, cell senescence, and apoptosis, are present in vascular disease. DNA damage can thus directly cause vascular disease, opening up new possibilities for both prevention and treatment. We review the evidence for and the causes, types, and consequences of DNA damage in vascular disease. PMID:26442438

  17. Trace elements and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Koudrine, A.V. [Orenburg State Medical Academy, Orenburg (Russian Federation)

    1998-07-01

    It is known that apoptosis is considered to be responsible for selective deletion of cells during embryogenesis, the homeostasis of cell populations in continuously renewing tissues (i.e., serving as a counterbalance to mitosis), and tissue involution in response to chemical or physical stimuli. There are many publications on these questions. On the other hand, the intracellular processes that contribute to apoptosis are incompletely understood. Therefore, the role of apoptosis in the intracellular accumulation and outflow of minerals is of considerable importance in light of both their essential functions and toxic effects. (orig.)

  18. Amplification of Whole Tumor Genomes and Gene-by-Gene Mapping of Genomic Aberrations from Limited Sources of Fresh-Frozen and Paraffin-Embedded DNA

    OpenAIRE

    Bredel, Markus; Bredel, Claudia; Juric, Dejan; Kim, Young; Vogel, Hannes; Harsh, Griffith R.; Recht, Lawrence D.; Pollack, Jonathan R.; Sikic, Branimir I.

    2005-01-01

    Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of ...

  19. Limits of RNA 2'-OH Mimicry by Fluorine: Crystal Structure of Bacillus halodurans RNase H Bound to a 2'-FRNA:DNA Hybrid.

    Science.gov (United States)

    Pallan, Pradeep S; Prakash, Thazha P; de Leon, Arnie R; Egli, Martin

    2016-09-27

    RNase H1 cleaves the RNA strand of RNA:DNA hybrids. Replacement of RNA 2'-hydroxyls by fluorine (FRNA) is commonly used to stabilize aptamers and siRNAs. However, FRNA:DNA hybrids fail to elicit RNase H activity. The underlying reasons are unclear, as 2'-OH groups are not directly involved in cleavage. We determined the crystal structure of Bacillus halodurans RNase H bound to a FRNA:DNA hybrid. The structure points to dynamic (slippage of the FRNA:DNA hybrid relative to the enzyme), geometric (different curvatures of FRNA:DNA and RNA:DNA hybrids), and electronic reasons (Mg(2+) absent from the active site of the FRNA:DNA complex) for the loss of RNaseH activity.

  20. Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis.

    Science.gov (United States)

    Chandrashekhar, K M; Isloor, Shrikrishna; Veeresh, B H; Hegde, Raveendra; Rathnamma, D; Murag, Shivaraj; Veeregowda, B M; Upendra, H A; Hegde, Nagendra R

    2015-11-01

    Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and β-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis. PMID:25773783

  1. Study on Taxol in Inhibiting Human Leukemia Cell Proliferation and Inducing Apoptosis

    Institute of Scientific and Technical Information of China (English)

    赵小英; 张晓红; 徐磊; 张行

    2004-01-01

    Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations for 12-72 hrs. Cell proliferation was evaluated by MTT assay and morphological changes of apoptosis were examined by microscopy. Cell apoptosis was determined by flow cytometry (FCM) and DNA gel electrophoresis. Results: Growth of K562 cells was inhibited by Taxol with an IC50 value of 0.84 μg/mi.Typical nuclear condensation and apoptosis bodies were observed as early as 24 hrs after a 0.5 μg/ml Taxol treatment; Apoptotic rate of the Taxol-treated K562 cells increased from 3.7% to 24.0% in 24 hrs. No DNA ladder was observed by DNA gel electrophoresis. Conclusion: Taxol could inhibit K562 cell growth and induce apoptosis in vitro.

  2. Apoptosis of motor neurons in the spinal cord after ischemia reperfusion injury delayed paraplegia in rabbits

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Apoptosis is known to occur in the centralnervous system during development and in patho-logical settings such ischemia reperfusion(IR)inju-ry[1].Apoptosis requires an active commit ment ofthe cell to degrade its own DNA,according to aninternal programof self-destruction[2].Newproteinsynthesis is required for apoptosis,and protein syn-thesis inhibitors have been shown to reduce celldeath postischemically[3].Incontrast,necrosis is nota gene-facilitated process but results frominjuriouschanges in the environm...

  3. A novel method for detection of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zagariya, Alexander M., E-mail: zagariya@uic.edu

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  4. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  5. Effects of 5-azacytidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines.

    Science.gov (United States)

    Wang, X M; Wang, X; Li, J; Evers, B M

    1998-01-01

    OBJECTIVE: To determine the cellular effects of 5-azacytidine (5-azaC) and sodium butyrate on two human liver cancers, HepG2 and Hep3B. SUMMARY BACKGROUND DATA: Primary liver cancer is a significant health problem; treatment options are limited and prognosis is poor. Recent studies have focused on the role that programmed cell death (i.e., apoptosis) plays in both normal and neoplastic growth: certain genes can either suppress (e.g., Bcl-2, Bcl-xL) or promote (e.g., Bik, Bax, Bak) apoptosis. The identification of novel agents targeted to specific molecular pathways may be beneficial in the treatment of this disease. METHODS: Human liver cancer cell lines HepG2 and Hep3B were treated with 5-azaC alone, butyrate alone, or 5-azaC and butyrate. Morphologic and proliferative changes were assessed by light microscopy and 5-bromo-2'-deoxyuridine staining; flow cytometry was used to determine cell cycle characteristics. Apoptosis was assessed by DNA laddering and the in situ apoptosis detection assay using the TdT-mediated dUTP nick end labeling method. In addition, total RNA and protein were analyzed by ribonuclease protection and Western blot, respectively, to assess changes in the expression of apoptosis-related genes. RESULTS: Treatment with either 5-azaC or butyrate inhibited cell growth and induced apoptosis in both HepG2 and Hep3B cells; the combination of 5-azaC and butyrate was not more effective than either agent alone. 5-azaC alone resulted in a more differentiated-appearing morphology and G2 cell cycle arrest in both cell lines. Treatment with 5-azaC or butyrate affected the expression levels of proteins of the Bcl-2 family. CONCLUSIONS: Both 5-azaC and butyrate induced apoptosis in the HepG2 and Hep3B liver cancer cells; 5-azaC treatment alone produced G2 arrest in both cell lines. Proteins of the Bcl-2 family may play a role in the cellular changes that occur with treatment, but further studies are required to define this potential role. Products of the

  6. Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

    Directory of Open Access Journals (Sweden)

    Teresa Anglada

    2016-01-01

    Full Text Available In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated defective cell line, as Ataxia-Telangiectasia (AT cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70–85% of the AT viable cells (TUNEL-negative carried ≥10 γH2AX foci/cell, while only 12–27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

  7. LYMPHOCYTE APOPTOSIS IN PSORIASIS

    Directory of Open Access Journals (Sweden)

    О. M. Kapuler

    2006-01-01

    Full Text Available Abstract. Forty-two patients with progressive vulgar psoriasis (PASI = 19.7 ± 1.5 and 40 healthy volunteers were under investigation. Psoriatic patients were characterized by increased number of CD4+ CD95+ peripheral blood T lymphocytes, which correlates with clinical psoriatic score, and by increased levels of soluble Fas (sFas in serum, as compared to controls (resp., 1868.1 ± 186.8 pg/ml vs. 1281.4 ± 142.5 pg/ml, PLSD = 0.019. The levels of spontaneous lymphocyte apoptosis and anti-Fas (Mab-induced apoptosis in psoriatic patients did not differ from the controls. However, apoptosis induced by “oxidative stress” (50 M Н202, 4 hrs was depressed in the patients. Moreover, a simultaneous assessment of cell cycle structure (metachromatic staining with Acridine Orange, apoptosis and Fas receptor expression (AnnV-FITC/antiFas mAbs-PE staining following a short-term mitogenic stimulation (PHA-P, 5 µg/ml, 24 hrs were performed. We found no marked differences in mitogenic reactivity, activation-induced apoptosis, and activation-induced Fas receptor expression when studying lymphocytes from healthy donors and psoriatic patients. However, PHA-activated lymphocytes from psoriatic patients displayed a significantly decreased ratio of AnnV+CD95+ to the total AnnV+ subpopulation, thus suggesting a decreased role of Fas-dependent mechanisms of apoptosis during the cell activation. The data obtained confirm a view, that an abnormal lymphocyte “apoptotic reactivity”, which plays a crucial role in the mechanisms of autoimmunity, may also of importance in the pathogenesis of psoriasis.

  8. Apoptosis: una muerte silenciosa

    OpenAIRE

    Isis Casadelvalle Pérez

    2006-01-01

    La apoptosis o muerte celular programada es un tipo de muerte presente en todas las células eucarióticas. Es un proceso ordenado y esencial del desarrollo normal y de mantenimiento de la homeostasis de un organismo. En el presente trabajo se resumen las principales características fisiológicas, bioquímicas y moleculares de la muerte por apoptosis, evento que ocurre de forma apagada o silenciosa, o sea, sin daño celular aparente diferenciándose claramente del proceso de necrosis celular. En es...

  9. Apoptosis - Methods and Protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-03-01

    Full Text Available Apoptosis - Methods and ProtocolsSecond edition, 2009; Peter Erhardt and Ambrus Toth (Eds; Springer Protocols - Methods in molecular biology, vol. 559; Humana press, Totowa, New Jersey (USA; Pages: 400; €88.35; ISBN: 978-1-60327-016-8The editors rightly begin the preface telling us that: “The ability to detect and quantify apoptosis, to understand its biochemistry and to identify its regulatory genes and proteins is crucial to biomedical research”. Nowadays this is a grounding concept of biology and medicine. What is particularly remarkable...

  10. Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine (FOA): In Vitro and in Vivo Anticancer Activity by Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity, and Induction of Tumor Cell Apoptosis.

    Science.gov (United States)

    Chen, Zhen-Feng; Qin, Qi-Pin; Qin, Jiao-Lan; Zhou, Jie; Li, Yu-Lan; Li, Nan; Liu, Yan-Cheng; Liang, Hong

    2015-06-11

    Three water-soluble ruthenium(II) complexes with chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA) were synthesized and characterized. It was found that these ruthenium(II) complexes exhibited considerable in vitro anticancer activities and that they were the effective stabilizers of telomeric and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. Interestingly, the in vitro anticancer activity of 6 (LC-003) was higher than those of 4 (LC-001) and 5 (LC-002), more selective for BEL-7404 cells than for normal HL-7702 cells, and preferred to activate caspases-3/9. The different biological behaviors of the ruthenium complexes could be correlated with the chiral nature of 4-(2,3-dihydroxypropyl)-formamide oxoaporphine. More significantly, 6 exhibited effective inhibitory on tumor growth in BEL-7402 xenograft mouse model and higher in vivo safety than cisplatin. These mechanistic insights indicate that 6 displays low toxicity and can be a novel anticancer drug candidate.

  11. Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine (FOA): In Vitro and in Vivo Anticancer Activity by Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity, and Induction of Tumor Cell Apoptosis.

    Science.gov (United States)

    Chen, Zhen-Feng; Qin, Qi-Pin; Qin, Jiao-Lan; Zhou, Jie; Li, Yu-Lan; Li, Nan; Liu, Yan-Cheng; Liang, Hong

    2015-06-11

    Three water-soluble ruthenium(II) complexes with chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA) were synthesized and characterized. It was found that these ruthenium(II) complexes exhibited considerable in vitro anticancer activities and that they were the effective stabilizers of telomeric and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. Interestingly, the in vitro anticancer activity of 6 (LC-003) was higher than those of 4 (LC-001) and 5 (LC-002), more selective for BEL-7404 cells than for normal HL-7702 cells, and preferred to activate caspases-3/9. The different biological behaviors of the ruthenium complexes could be correlated with the chiral nature of 4-(2,3-dihydroxypropyl)-formamide oxoaporphine. More significantly, 6 exhibited effective inhibitory on tumor growth in BEL-7402 xenograft mouse model and higher in vivo safety than cisplatin. These mechanistic insights indicate that 6 displays low toxicity and can be a novel anticancer drug candidate. PMID:25988535

  12. Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and"Death factor"family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of"Death factor" family, the transfection experiments with expression vectors pcDNA3-fland pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl2. The data showed that the expression of FasL in pcDNA3fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fltransient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hcpatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

  13. 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...... to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein...

  14. Effect of Celecoxib on Apoptosis of Endometrial Carcinoma Cell

    Institute of Scientific and Technical Information of China (English)

    SHENG Xiu-jie; FANG Zhao

    2007-01-01

    Objective: To investigate the effect of Celecoxib on proliferation and apoptosis of the endometrial carcinoma cell HEC-1B and the effect on the expression of Fas and Survivin mRNA. Methods: The inhibition on the growth of human endometrial carcinoma cell HEC-1B was investigated by cell culture and MTT experiment when treated with different concentrations of Celecoxib. The cell apoptosis was detected by flow cytometry and DNA Ladder Electrophoresis. The change of the expression of Fas and Survivin mRNA after the treatment of Celecoxib was detected With RT-PCR. Results: Celecoxib could effectively inhibit the growth of HEC-1B cells and induce apoptosis. Survivin mRNA expression was decreased and Fas mRNA expression was increased after treating with Celecoxib. Conclusion: Celecoxib could inhibit HEC-1B cell proliferation and induce its apoptosis.

  15. Effects on DNA damage and apoptosis and P53 protein expression induced by phoxim in rat bone marrow stem cells%辛硫磷对大鼠骨髓间充质干细胞DNA损伤、细胞凋亡及P53蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    闫建国; 周亚莉; 邢雪琨; 方方; 朱振东; 王松涛

    2013-01-01

    目的 研究辛硫磷对大鼠骨髓间充质干细胞(BMSCs) DNA的损伤作用及其对氧化损伤、细胞凋亡和P53蛋白表达的影响.方法 Percoll离心法分离培养大鼠BMSCs,正常传代.取第3代BMSCs,调整细胞密度为1.0×106/瓶,当细胞至亚融合状态,分别以0(对照)、0.2、2和20μ g/L的辛硫磷浓度染毒24h.采用MTT法检测BMSCs的存活率,分光光度比色法检测BMSCs超氧化物歧化酶(SOD)、过氧化氢酶(C AT)活性和丙二醛(MDA)含量,单细胞凝胶电泳检测BMSCs的DNA损伤,流式细胞术检测大鼠骨髓间充质干细胞凋亡率,Western blotting检测BMSCs的P53蛋白表达水平.结果 与对照组相比,0.2~20μg/L辛硫磷染毒24h,可诱发大鼠BMSCs的DNA损伤,且具有剂量-效应关系;各染毒组大鼠BMSCs的存活率、SOD和CAT活性均显著下降(P<0.05),细胞凋亡率和MDA含量均显著升高(P<0.05);辛硫磷染毒可以诱导大鼠BMSCs P53蛋白表达水平的增加(P<0.05).结论 辛硫磷可诱导大鼠BMSCs氧化损伤、DNA损伤、细胞凋亡和P53蛋白表达,且具有剂量-效应关系.%Objective To study the DNA damage and oxidative damage and apoptosis and P53 protein expression in rat bone marrow stem cells cultured in vitro induced by phoxim.Methods Rat bone marrow stem cells of P3 cultured in vitro were treated with phoxim of different concentrations(0,0.2,2 and 20μg/L) for 24 h after 48h cultured.The total activity of SOD、CAT and MDA content,survival rate in cell were detected by spectrophotometry,and the DNA damage were detected by single cell gel electrophoresis.The cell apoptosis were detected by flow cytometry,and the P53 protein expression were detected by Western blot.Results With the 0.2 and 20μg/L of phoxim concentrations treated for 24h in culture media,the total activity of SOD and CAT were significantly decreased compared with the controls (P <0.05).With the increase of phoxim concentration in culture media,the MDA contents,DNA

  16. The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei.

    Science.gov (United States)

    Cusick, E L; Milton, J I; Ewen, S W

    1990-04-01

    Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer. Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display. Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted. The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number. The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content. By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei. The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.

  17. Apoptosis and inflammation

    Directory of Open Access Journals (Sweden)

    C. Haanen

    1995-01-01

    Full Text Available During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972 introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.

  18. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  19. [DNA computing].

    Science.gov (United States)

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  20. Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

    Institute of Scientific and Technical Information of China (English)

    JIANGZHENGFAN; SHANZHU; 等

    1999-01-01

    We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments.The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.

  1. Catalase inhibits ionizing radiation-induced apoptosis in hematopoietic stem and progenitor cells.

    Science.gov (United States)

    Xiao, Xia; Luo, Hongmei; Vanek, Kenneth N; LaRue, Amanda C; Schulte, Bradley A; Wang, Gavin Y

    2015-06-01

    Hematologic toxicity is a major cause of mortality in radiation emergency scenarios and a primary side effect concern in patients undergoing chemo-radiotherapy. Therefore, there is a critical need for the development of novel and more effective approaches to manage this side effect. Catalase is a potent antioxidant enzyme that coverts hydrogen peroxide into hydrogen and water. In this study, we evaluated the efficacy of catalase as a protectant against ionizing radiation (IR)-induced toxicity in hematopoietic stem and progenitor cells (HSPCs). The results revealed that catalase treatment markedly inhibits IR-induced apoptosis in murine hematopoietic stem cells and hematopoietic progenitor cells. Subsequent colony-forming cell and cobble-stone area-forming cell assays showed that catalase-treated HSPCs can not only survive irradiation-induced apoptosis but also have higher clonogenic capacity, compared with vehicle-treated cells. Moreover, transplantation of catalase-treated irradiated HSPCs results in high levels of multi-lineage and long-term engraftments, whereas vehicle-treated irradiated HSPCs exhibit very limited hematopoiesis reconstituting capacity. Mechanistically, catalase treatment attenuates IR-induced DNA double-strand breaks and inhibits reactive oxygen species. Unexpectedly, we found that the radioprotective effect of catalase is associated with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway and pharmacological inhibition of STAT3 abolishes the protective activity of catalase, suggesting that catalase may protect HSPCs against IR-induced toxicity via promoting STAT3 activation. Collectively, these results demonstrate a previously unrecognized mechanism by which catalase inhibits IR-induced DNA damage and apoptosis in HSPCs.

  2. p63 is required beside p53 for PERP-mediated apoptosis in uveal melanoma

    Science.gov (United States)

    Awais, Raheela; Spiller, David G; White, Michael R H; Paraoan, Luminita

    2016-01-01

    Background: PERP (p53 apoptosis effector related to PMP-22), a transcriptional target of p53, is downregulated and contributes to the impairment of apoptosis in uveal melanoma (UM). Intriguingly, PERP is not induced in UM despite functional p53. p63, located on chromosome 3, which is characteristically altered in high-risk UM, can transactivate PERP. Here, we determine the functional role of p63 expression in the initiation of p53/PERP-mediated apoptosis in UM. Methods: PERP expression was monitored by quantitative PCR (qPCR) and immunoblotting in UM cell lines treated with DNA-damaging agents. The functional role of p63 was assessed by transient expression of p63-turbo GFP (p63-tGFP) in the apoptosis- resistant, 3q-deficient OCM-1 cells. Expression and localisation of p63, PERP and p53, and induction of apoptosis were characterised by qPCR, immunoblotting and live cell confocal microscopy. Results: PERP expression was significantly downregulated in all UM cell lines. DNA-damaging treatments failed to induce apoptosis and activate PERP in OCM-1 cells, which displayed non-functional levels of p63. Expression of p63-tGFP induced apoptosis with marked increase in PERP expression and associated p53 accumulation. Conclusions: Lack of p63 contributes to reduced PERP levels and impaired p53-mediated apoptosis in UM. p63 expression is required for PERP-mediated apoptosis in UM. PMID:27584665

  3. Apoptosis and survival

    Directory of Open Access Journals (Sweden)

    Manjul Tiwari

    2011-01-01

    Full Text Available The term apoptosis first appeared in the biomedical literature in 1972, to delineate a structurally distinctive mode of cell death responsible for cell loss within living tissues. The cardinal morphological features are cell shrinkage, accompanied by transient but violent bubbling and blebbing from the surface, and culminating in separation of the cell into a cluster of membrane-bounded bodies. Changes in several cell surface molecules also ensure that, in tissues, apoptotic cells are immediately recognised and phagocytosed by their neighbours. However, it is important to note that apoptosis is only one form of cell death and the particular death pathway that is the most important determinant for cancer therapy is not necessarily that which has the fastest kinetics, as is the bias in many laboratories, but rather that which displays the most sensitive dose-response relationship.

  4. Fullerene and apoptosis

    Directory of Open Access Journals (Sweden)

    M. A. Orlova

    2013-01-01

    Full Text Available Fullerene derivatives superfamily attracts a serious attention as antiviral and anticancer agents and drug delivery carriers as well. A large number of such fullerene С60 derivatives obtained to date. However, there is an obvious deficit of information about causes and mechanisms of immediately and long-term consequences of their effects in vivo which is a true obstacle on the way leading to practical medical use of them. First, this concerns their impact on the proliferation, apoptosis and necrosis regulation. Fullerene nanoparticle functionalization type, their sizes and surface nanopathology are of great importance to further promoting of either cytoprotective or cytotoxic effects. This lecture provides modern concept analysis regarding fullerenes effects on apoptosis pathway in normal and tumor cells.

  5. Fullerene and apoptosis

    Directory of Open Access Journals (Sweden)

    M. A. Orlova

    2014-07-01

    Full Text Available Fullerene derivatives superfamily attracts a serious attention as antiviral and anticancer agents and drug delivery carriers as well. A large number of such fullerene С60 derivatives obtained to date. However, there is an obvious deficit of information about causes and mechanisms of immediately and long-term consequences of their effects in vivo which is a true obstacle on the way leading to practical medical use of them. First, this concerns their impact on the proliferation, apoptosis and necrosis regulation. Fullerene nanoparticle functionalization type, their sizes and surface nanopathology are of great importance to further promoting of either cytoprotective or cytotoxic effects. This lecture provides modern concept analysis regarding fullerenes effects on apoptosis pathway in normal and tumor cells.

  6. Apoptosis: una muerte silenciosa

    Directory of Open Access Journals (Sweden)

    Isis Casadelvalle Pérez

    2006-01-01

    Full Text Available La apoptosis o muerte celular programada es un tipo de muerte presente en todas las células eucarióticas. Es un proceso ordenado y esencial del desarrollo normal y de mantenimiento de la homeostasis de un organismo. En el presente trabajo se resumen las principales características fisiológicas, bioquímicas y moleculares de la muerte por apoptosis, evento que ocurre de forma apagada o silenciosa, o sea, sin daño celular aparente diferenciándose claramente del proceso de necrosis celular. En ese proceso se destaca la mitocondria, como organelo celular donde mediado por la activación de las caspasas se inicia el paso hacia la muerte celular programada. En el momento actual, la apoptosis ha cobrado un verdadero valor para la mejor comprensión de los procesos biológicos normales en los que este evento está involucrado y que con anterioridad no era tomado en cuenta. En este sentido, se comentan las principales técnicas de detección de muerte celular programada y se aclara que la elección de algunas de ellas depende del modelo de estudio. Tambi én se dan a conocer algunas de las patologías generales en las que este proceso representa un papel determinante y se discute acerca de cómo algunas alteraciones en los mecanismos de regulación de la apoptosis inducen la aparici ón de varias enfermedades, incluyendo aquellos desórdenes en los que ocurre acumulación celular (cáncer, alteración cardiaca, neurodegeneración y SIDA. El estudio y caracterización de este complejo mecanismo ha cambiado profundamente la comprensión de numerosas patologías en los organismos eucariotas.

  7. Mechanisms of radioinduced apoptosis; Mecanismes de l'apoptose radio-induite

    Energy Technology Data Exchange (ETDEWEB)

    Baatout, S.; Derradji, H.; Petitfour, Ol.; Von Schodoletz, H.; Mergeay, M. [Lab. de Radiobiologie, Centre d' Etude de l' Energie Nucleaire, SCK-CEN, Boeretang, Mol (Belgium)

    2002-07-01

    A general overview of the activation mechanisms of programmed cell death or apoptosis following an irradiation is given in this review. First, are summarized the main induction pathways of radiation-induced apoptosis by which extracellular (tumor necrosis factor (TNF), Fas ligand, TNF-related apoptosis-inducing ligand (TRAIL)) and intracellular (mitochondria and caspases) signals are integrated. A second part is then devoted to the importance of p53 and of its regulators (ATR, ATM, DNA-PKcs) in the process of radiation-induced apoptosis. Thereafter, signal transduction pathways and more specially the role of some protein kinases (MEKK, SAPK/JNK, p38-MAPK) is treated. At last, a chapter concerns the clinical interest of radiation-induced apoptosis and the implication of apoptosis in the treatment of certain diseases. (author)

  8. Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy.

    Science.gov (United States)

    Fernandes, Alinda R; Chari, Divya M

    2016-09-28

    Genetically engineered neural stem cell (NSC) transplant populations offer key benefits in regenerative neurology, for release of therapeutic biomolecules in ex vivo gene therapy. NSCs are 'hard-to-transfect' but amenable to 'magnetofection'. Despite the high clinical potential of this approach, the low and transient transfection associated with the large size of therapeutic DNA constructs is a critical barrier to translation. We demonstrate for the first time that DNA minicircles (small DNA vectors encoding essential gene expression components but devoid of a bacterial backbone, thereby reducing construct size versus conventional plasmids) deployed with magnetofection achieve the highest, safe non-viral DNA transfection levels (up to 54%) reported so far for primary NSCs. Minicircle-functionalized magnetic nanoparticle (MNP)-mediated gene delivery also resulted in sustained gene expression for up to four weeks. All daughter cell types of engineered NSCs (neurons, astrocytes and oligodendrocytes) were transfected (in contrast to conventional plasmids which usually yield transfected astrocytes only), offering advantages for targeted cell engineering. In addition to enhancing MNP functionality as gene delivery vectors, minicircle technology provides key benefits from safety/scale up perspectives. Therefore, we consider the proof-of-concept of fusion of technologies used here offers high potential as a clinically translatable genetic modification strategy for cell therapy.

  9. Safrole oxide inhibits angiogenesis by inducing apoptosis.

    Science.gov (United States)

    Zhao, Jing; Miao, Junying; Zhao, Baoxiang; Zhang, Shangli; Yin, Deling

    2005-06-01

    Our previous studies indicate that 3, 4-(methylenedioxy)-1-(2', 3'-epoxypropyl)-benzene (safrole oxide), a newly synthesized compound, induces apoptosis in vascular endothelial cells (VECs) and A549 lung cancer cells. To our knowledge, the inhibition of angiogenesis by safrole oxide has not been reported yet. We report here that cultured rat aorta treated with safrole oxide exhibited a significant microvessel reduction as determined by counting the number of microvessels in a phase contrast microscope. There were more microvessels formed in the presence of A549 lung cancer cells in rat aorta model, while a dramatic inhibition of angiogenesis was obtained by adding 220-450 micromol l(-1) of safrole oxide to the growth medium (Psafrole oxide produced only some abortive endothelial cells but not microvessels. Furthermore, safrole oxide induced antiangiogenic effect in the chorioallantoic membranes (CAM) as a dose dependent manner. Eggs treated with 2-11 micromol 100 microl(-1) per egg of the safrole oxide for 48 h exhibited a significant reduction in blood vessel area of the CAM, a process likely mediated by apoptosis as demonstrated by DNA fragmentation. Our results suggest that safrole oxide has antiangiogenic activity and this effect might occur by induction of cellular apoptosis.

  10. Progress in studies on the DEK protein and its involvement in cellular apoptosis

    Institute of Scientific and Technical Information of China (English)

    HUA Ying; HU HongGang; PENG XiangLei

    2009-01-01

    DEK protein is an ubiquitous phosphorylated nuclear protein. Specific binding of DEK to DNA could change the topology of DNA and then affect the gene activity of the underlying DNA sequences. It is speculated that there might be some potential relationship between the stress reaction of cells and DEK proteins. The phosphorylation status of DEK protein is altered during death-receptor-mediated cell apoptosis. Both phosphorylation and poly(ADP-ribosyl)aUon could promote the release of DEK from apoptotic nuclei to extracellular environment, and in this case DEK becomes a potential autoantigen of some autoimmune diseases. The available evidence powerfully suggests that DEK protein is closely relevant to apoptosis. The overexpression of DEK protein has dual function in cell apoptosis, in terms of inhibiting or triggering cell apoptosis.

  11. Limited phylogeographic signal in sex-linked and autosomal loci despite geographically, ecologically, and phenotypically concordant structure of mtDNA variation in the Holarctic avian genus Eremophila.

    Directory of Open Access Journals (Sweden)

    Sergei V Drovetski

    Full Text Available Phylogeographic studies of Holarctic birds are challenging because they involve vast geographic scale, complex glacial history, extensive phenotypic variation, and heterogeneous taxonomic treatment across countries, all of which require large sample sizes. Knowledge about the quality of phylogeographic information provided by different loci is crucial for study design. We use sequences of one mtDNA gene, one sex-linked intron, and one autosomal intron to elucidate large scale phylogeographic patterns in the Holarctic lark genus Eremophila. The mtDNA ND2 gene identified six geographically, ecologically, and phenotypically concordant clades in the Palearctic that diverged in the Early-Middle Pleistocene and suggested paraphyly of the horned lark (E. alpestris with respect to the Temminck's lark (E. bilopha. In the Nearctic, ND2 identified five subclades which diverged in the Late Pleistocene. They overlapped geographically and were not concordant phenotypically or ecologically. Nuclear alleles provided little information on geographic structuring of genetic variation in horned larks beyond supporting the monophyly of Eremophila and paraphyly of the horned lark. Multilocus species trees based on two nuclear or all three loci provided poor support for haplogroups identified by mtDNA. The node ages calculated using mtDNA were consistent with the available paleontological data, whereas individual nuclear loci and multilocus species trees appeared to underestimate node ages. We argue that mtDNA is capable of discovering independent evolutionary units within avian taxa and can provide a reasonable phylogeographic hypothesis when geographic scale, geologic history, and phenotypic variation in the study system are too complex for proposing reasonable a priori hypotheses required for multilocus methods. Finally, we suggest splitting the currently recognized horned lark into five Palearctic and one Nearctic species.

  12. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis

  13. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang, E-mail: wangdang511@126.com; Xiao, Shaobo; Chen, Huanchun

    2014-10-03

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

  14. Protection against apoptosis in chicken bursa and thymus cells by phorbol ester in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Asakawa, J.; Thorbecke, G.J. (Univ. of New York, New York (United States))

    1991-03-15

    Programmed suicide or apoptosis, due to activation of endogenous nucleases, occurs in immature CD4{sup {minus}}85{sup {minus}} mammalian thymus cells. Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study the authors have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hrs with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed with both bursa and thymus cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristic acetate during culture and, more effectively, by PMA plus ionomycin, but not by ionomycin alone or by anti-{mu}. PMA did not affect the patterns of DNA fragmentation seen with spleen cells. Addition of the protein kinase C inhibitor staurosporin inhibited the preventive effect of PMA on apoptosis. PMA also greatly promoted the survival of bursa cells in culture, as assayed by percentage cell death and by {sup 3}H-thymidine incorporation. It is concluded that bursa and thymus cells from the chicken exhibit apoptosis. The data further suggest that protein kinase C activation protects apoptosis in cultured bursa cells.

  15. Detection of radiation-induced apoptosis using the comet assay

    International Nuclear Information System (INIS)

    The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)

  16. Differential Apoptosis Radiosensitivity of Neural Progenitors in Adult Mouse Hippocampus

    Directory of Open Access Journals (Sweden)

    Yu-Qing Li

    2016-06-01

    Full Text Available Mammalian tissue-specific stem cells and progenitors demonstrate differential DNA damage response. Neural progenitors in dentate gyrus of the hippocampus are known to undergo apoptosis after irradiation. Using a mouse model of hippocampal neuronal development, we characterized the apoptosis sensitivity of the different neural progenitor subpopulations in adult mouse dentate gyrus after irradiation. Two different bromodeoxyuridine incorporation paradigms were used for cell fate mapping. We identified two apoptosis sensitive neural progenitor subpopulations after irradiation. The first represented non-proliferative and non-newborn neuroblasts and immature neurons that expressed doublecortin, calretinin or both. The second consisted of proliferative intermediate neural progenitors. The putative radial glia-like neural stem cells or type-1 cells, regardless of proliferation status, were apoptosis resistant after irradiation. There was no evidence of radiation-induced apoptosis in the absence of the Trp53 (p53 gene but absence of Cdkn1a (p21 did not alter the apoptotic response. Upregulation of nuclear p53 was observed in neuroblasts after irradiation. We conclude that adult hippocampal neural progenitors may demonstrate differential p53-dependent apoptosis sensitivity after irradiation.

  17. HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

    Directory of Open Access Journals (Sweden)

    Joshua L Andersen

    2006-12-01

    Full Text Available The HIV-1 accessory protein viral protein R (Vpr causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.

  18. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  19. THE LIMITS OF DNA TESTING USED AS EVIDENCE IN COURT. A REPORT BASED ON THE PRACTICE OF THE INSTITUTE OF LEGAL MEDICINE CLUJ-NAPOCA

    OpenAIRE

    Dan Perju-Dumbrava; Ioana Mic; Ovidiu Sorin Chiroban

    2013-01-01

    The evaluation of the evidentiary value of scientific evidence is the assessment of thestrength of the link between a finding and a person. It is usually a statisticalassessment but its presentation is full of pitfalls. The evaluation of scientific evidencemust be based on an established methodology to both evaluate, expose and interpretthe evidence. The formidable expansion in the use of DNA has not only increased theextent of interaction between forensic scientists and lawyers but more impo...

  20. Cerebral ischemia—induced neuronal apoptosis mediated by nitric oxide

    Institute of Scientific and Technical Information of China (English)

    NomuY

    2002-01-01

    To elucidate the cellular and molecular mechanism of cerebral ischemia-induced neuronal apoptosis mediated by nitric oxide (NO) in the brain,we investigated:(1)cell death in hippocampal CA1 neurons of rats after a rransient four vessel occlusion (4VO)/reperfusion and (2) apoptosis induced by NOC18(NO releaser) using SHSY5Y cells,a human neuroblastoma cell line.We found that 4VO caused expression of inducible type of NO synthase (iNOS) in glial cells and neuronal apoptosis in CA1 region of rats.Next we examined in vitro apoptotic effects of NOC18 on SHSY5Y cells and suggest that NO decrease mitochondrial membrane potential,release cytochrome C from mitochondria,activates caspase-3,degrade inhibitor of caspase-activated DNase(Icad),and activated DNase translocate into nucleus and induce DNA fragmentation.Thus we conclude that the excess amount of NO produced by glial iNOS at cerebral ischemia could be involved in neuronal apoptosis in CA1 region.Regarding NO action on neurons,we further obtained that NO propects neuronal apoptosis in PC12 cells perhaps by nitrosylation of caspase,subsequent reduction of proteolytic activity.Taken together,we suggest that NO seem to exert dual effects(toxic and beneficial) on neuronal apoptosis,the one (toxic);apoptosis-induction throuth the decrease in mitochondrial membrane potentials and cytochrome C release and the othe (beneficial);protection against apoptosis through the inhibition of caspase activity.

  1. Mitochondria in human pluripotent stem cell apoptosis.

    Science.gov (United States)

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

    2016-04-01

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

  2. Enhancing microRNA transfection to inhibit survivin gene expression and induce apoptosis: could it be mediated by a novel combination of sonoporation and polyethylenimine?

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhi-yi; LIANG Kun; QIURi-xiang; LUO Liang-ping

    2011-01-01

    Apoptosis is a physiologically essential mechanism of cell and plays an important role in reducing the development and progression of tumors.The appealing strategy for cancer therapy is to target the lesions that induce apoptosis in cancer colls.Survivin,the smallest member of the mammalian inhibitors of the apoptosis protein family,is upregulated in various malignancies to protect cells from apoptosis.Survivin knockdown could induco cancer cell apoptosis and inhibit tumor-angiogenesis.Survivin expression would be silenced by microRNA (miRNA)-mediated RNA interference.However,noninvasive and tissue-specific gene delivery techniques remain absent recently and the utilizations of miRNA expression vectors have been limited by inefficient delivery technique,especially in vivo.On the other hand,safe and promising technologies of gene transfection would be valuable in clinical gene therapy.Successful treatment of gene transfer method would lead to a new and readily available approach in the anticancer research.Sonoporation is an alternative technique of gene delivery that uses ultrasound targeted microbubble destruction to create pores in the cell membrane.Based on our previous studies,in this article,we postulated that the transfection of miRNA could be mediated by the combination of sonoporation and polyethylenimine (PEI) which was one of the most effective poly-cationic gene vectors and enhance the endocytosis of plasmids DNA and hypothesized that the gene silencing and apoptosis induction with miRNA targeting human Survivin would be improved by this novel technique.In our opinion,this novel combination of sonoporation and PEI could enhance targeted gene delivery effectively and might be a feasible,novel candidate for gene therapy.

  3. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner

    Directory of Open Access Journals (Sweden)

    Gretel G. Pellegrini

    2016-07-01

    Full Text Available Oats contain unique bioactive compounds known as avenanthramides (AVAs with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT and Nrf2 Knockout (KO osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast

  4. Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitro in an Nrf2-Independent Manner.

    Science.gov (United States)

    Pellegrini, Gretel G; Morales, Cynthya C; Wallace, Taylor C; Plotkin, Lilian I; Bellido, Teresita

    2016-01-01

    Oats contain unique bioactive compounds known as avenanthramides (AVAs) with antioxidant properties. AVAs might enhance the endogenous antioxidant cellular response by activation of the transcription factor Nrf2. Accumulation of reactive oxygen species plays a critical role in many chronic and degenerative diseases, including osteoporosis. In this disease, there is an imbalance between bone formation by osteoblasts and bone resorption by osteoclasts, which is accompanied by increased osteoblast/osteocyte apoptosis and decreased osteoclast apoptosis. We investigated the ability of the synthethic AVAs 2c, 2f and 2p, to 1-regulate gene expression in bone cells, 2-affect the viability of osteoblasts, osteocytes and osteoclasts, and the generation of osteoclasts from their precursors, and 3-examine the potential involvement of the transcription factor Nrf2 in these actions. All doses of AVA 2c and 1 and 5 µM dose of 2p up-regulated collagen 1A expression. Lower doses of AVAs up-regulated OPG (osteoprotegerin) in OB-6 osteoblastic cells, whereas 100 μM dose of 2f and all concentrations of 2c down-regulated RANKL gene expression in MLO-Y4 osteocytic cells. AVAs did not affect apoptosis of OB-6 osteoblastic cells or MLO-Y4 osteocytic cells; however, they prevented apoptosis induced by the DNA topoisomerase inhibitor etoposide, the glucocorticoid dexamethasone, and hydrogen peroxide. AVAs prevented apoptosis of both wild type (WT) and Nrf2 Knockout (KO) osteoblasts, demonstrating that AVAs-induced survival does not require Nrf2 expression. Further, KO osteoclast precursors produced more mature osteoclasts than WT; and KO cultures exhibited less apoptotic osteoclasts than WT cultures. Although AVAs did not affect WT osteoclasts, AVA 2p reversed the low apoptosis of KO osteoclasts. These in vitro results demonstrate that AVAs regulate, in part, the function of osteoblasts and osteocytes and prevent osteoblast/osteocyte apoptosis and increase osteoclast apoptosis; further

  5. Radiation-induced apoptosis in human lymphocytes: Potential as a biological dosimeter

    International Nuclear Information System (INIS)

    We have tested the possibility of using apoptosis (programmed cell death) in human peripheral blood lymphocytes as a short-term biological dosimeter. Lymphocytes isolated from whole blood were irradiated in culture with 250 kVp x-rays or 60Co gamma rays. Two assays were used to measure apoptosis in lymphocytes after irradiation: in situ terminal deoxynucleotidyl transferase assay and fluorescence analysis of DNA unwinding assay. Similar qualitative and quantitative results were produced by the assays, supporting the notion that the fluorescence analysis of DNA unwinding assay measured DNA fragmentation associated with apoptosis. Induction of apoptosis in lymphocytes irradiated in vitro was proportional to dose and could be detected following exposures as low as 0.05 Gy. Lymphocytes irradiated in vitro was proportional to dose and could be detected following exposures as low as 0.05 Gy. Lymphocytes from individual donors had reproducible dose responses. There was, however, variation between donors. X-ray and gamma-ray exposures induced similar levels of apoptosis at similar doses. The induction kinetics of apoptosis in vitro indicate a maximum is reached about 72 h after irradiation. In conclusion, the in vitro experimental evidence indicates that radiation-induced apoptosis in human lymphocytes has the kinetics, sensitivity, and reproductibility to be a potential biological dosimeter. 29 refs., 5 figs

  6. Tributyltin stimulates apoptosis in rat thymocytes.

    Science.gov (United States)

    Aw, T Y; Nicotera, P; Manzo, L; Orrenius, S

    1990-11-15

    Treatment of rat thymocytes with micromolar concentrations of tributyltin caused a rapid increase in the cytosolic free Ca2+ concentration that was inhibited by Ni2+, which blocks Ca2+ influx through membrane channels. The elevation of cytosolic Ca2+ was associated with extensive DNA fragmentation, which was prevented by pretreatment of the cells with either of the intracellular Ca2+ chelators quin-2 or 1,2-bis(2-amino-phenoxy)ethane-N',N',N',N',-tetraacetic acid. Loss of thymocyte viability, which followed DNA fragmentation, was also prevented by the two Ca2+ chelators or by removing extracellular Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. The pattern of DNA fragmentation was characteristic of that produced by agents which activate a Ca2(+)- and Mg2(+)-dependent endogenous endonuclease during apoptosis or programmed cell death. Additional studies showed that other organotin compounds, including trimethyltin, triphenyltin, and dibutyltin had minimal effects on cytosolic Ca2+, DNA fragmentation, and cell viability. These results are consistent with a greater susceptibility of thymocytes to tributyltin and provide a basis for understanding its selective immunotoxicity in vivo. PMID:2241174

  7. Neuropeptide Y protects kidney against cisplatin-induced nephrotoxicity by regulating p53-dependent apoptosis pathway

    Science.gov (United States)

    Kim, Namoh; Min, Woo-Kie; Park, Min Hee; Lee, Jong Kil; Jin, Hee Kyung; Bae, Jae-sung

    2016-01-01

    Cisplatin is a platinum-based chemotherapeutic drug for treating various types of cancers. However, the use of cisplatin is limited by its negative effect on normal tissues, particularly nephrotoxicity. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and apoptosis are involved in the adverse effect induced by cisplatin treatment. Several studies have suggested that neuropeptide Y (NPY) is involved in neuroprotection as well as restoration of bone marrow dysfunction from chemotherapy induced nerve injury. However, the role of NPY in chemotherapy-induced nephrotoxicity has not been studied. Here, we show that NPY rescues renal dysfunction by reducing the expression of pro-apoptotic proteins in cisplatin induced nephrotoxicity through Y1 receptor, suggesting that NPY can protect kidney against cisplatin nephrotoxicity as a possible useful agent to prevent and treat cisplatin-induced nephrotoxicity. [BMB Reports 2016; 49(5): 288-292] PMID:26728272

  8. Suppression of peroxiredoxin 4 in glioblastoma cells increases apoptosis and reduces tumor growth.

    Directory of Open Access Journals (Sweden)

    Tae Hyong Kim

    Full Text Available Glioblastoma multiforme (GBM, the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4 is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future.

  9. Requirement for SAPK-JNK signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.

    Science.gov (United States)

    Johnson, C R; Jiffar, T; Fischer, U M; Ruvolo, P P; Jarvis, W D

    2003-11-01

    The present studies examined performance of SAPK cascades and apoptotic commitment following ribosomal trauma in REH lymphoid leukemia cells. Ribostatic insults included disruption of ribosomal activity by mechanistically dissimilar agents such as blasticidin-S (BCS) (which binds 28S-rRNA to block peptidyl bond formation), kasugamycin (KSM) (which binds 18S-rRNA to prevent translational initiation), and cycloheximide (CHX) (which blocks A-site to P-site translocation of peptidyl-tRNA). Exposure of REH cells to BCS elicited DNA degradation and apoptotic cytolysis. BCS stimulated JNK1/JNK2 and p38, and their shared targets c-Jun and ATF2. Inhibition of JNK1/JNK2 (but not of p38) antagonized blasticidin-induced apoptosis, whereas targeting alternative ribosomal sites with KSM or CHX limited translation, but failed to activate the SAPK cascade or initiate apoptosis. Our findings indicate that interference with 28S-rRNA by BCS initiates apoptosis in REH cells through recruitment of SAPK-JNK signaling. Disparities between the lethal actions of BCS, KSM, and CHX appear to reflect established differences in the subribosomal targets of these agents. We propose that the SAPK cascade comprises an essential mechanism for the transduction of specific lethal stress signals emanating from active ribosomes, and that interference with the 28S-rRNA, rather than the peptidyl transfer center of the large subunit, is critical to apoptotic commitment. PMID:12970763

  10. [Level of nitric oxide in the kidneys during apoptosis activation].

    Science.gov (United States)

    Komarievtseva, I O; Orlova, O A; Blahodarenko, Ie A

    2002-01-01

    The content of nitric oxide stable metabolites in a tissue of kidneys of rats in conditions of activation of apoptosis was investigated. Research was carried out in two models: acute renal failure and a hypertrophy of a unique kidney after a unilateral nephrectomy. Detection of apoptosis was carried out by definition of DNA fragmentation. Substantial increase of the nitric oxide stable metabolites contents is revealed at activation of apoptosis in both models. Change of a ratio of the contents of nitrite--anions in relation to the general contents of NO2- + NO3- is revealed, indicating the role of peroxide processes in effect of nitric oxide and its metabolites on the cell. PMID:14964872

  11. Study of progesterone mechanisms in radio-induced apoptosis prevention

    International Nuclear Information System (INIS)

    The purpose of this work was to study the modulation of radiation-induced cell death of human mammary tumoral cells by progesterone. On the one hand, we observed that progesterone protects cells against radiation-induced apoptosis and increases the proportion of surviving and proliferating damaged cells. On the other hand, a transcriptome analysis was undertaken in irradiated cells treated by progesterone, using DNA micro-arrays. This let us highlight several transcriptional dis-regulations that are likely to explain the protective effect of the hormone; in particular, we showed that progesterone regulates the expression of genes implicated in apoptosis signaling by death receptors. Knowing the crucial role of hormonal control and of apoptosis regulation in breast cancer initiation, our results may help to achieve a better understanding of the implication of progesterone in mammary carcinogenesis. (author)

  12. Apoptosis Resistance in Endometriosis

    Directory of Open Access Journals (Sweden)

    Liselotte Mettler

    2011-08-01

    Full Text Available Introduction: In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. Methods: Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5. Results: Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. Conclusion: A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures.

  13. Comparison of HIV-1 resistance profiles in plasma RNA versus PBMC DNA in heavily treated patients in Honduras, a resource-limited country

    OpenAIRE

    Diallo, Karidia; Murillo, Wendy E; de Rivera, Ivette Lorenzana; Albert, Jan; Zhou, Zhiyong; Nkengasong, John; Zhang, Guoqing; Sabatier, Jennifer F; Yang, Chunfu

    2012-01-01

    The World Health Organization currently does not recommend the use of dried blood spot specimens for drug resistance testing in patients undergoing antiretroviral therapy (ART). Therefore, HIV-1 resistance testing using peripheral blood mononuclear cells (PBMCs) may be of value in resource-limited settings. We compared genotypic resistance profiles in plasma and PBMCs from patients failing ART in two cities of Honduras (Tegucigalpa and San Pedro Sula), a resource-limited country. One hundred ...

  14. Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells

    DEFF Research Database (Denmark)

    Larsen, Claus Morten; Døssing, M G; Papa, S;

    2006-01-01

    IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen......-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study...

  15. Apoptosis : Target of cancer therapy

    NARCIS (Netherlands)

    Ferreira, CG; Epping, M; Kruyt, FAE; Giaccone, G

    2002-01-01

    Recent knowledge on apoptosis has made it possible to devise novel approaches, which exploit this process to treat cancer. In this review, we discuss in detail approaches to induce tumor cell apoptosis, their mechanism of action, stage of development, and possible drawbacks. Finally, the obstacles y

  16. Cardiovascular molecular imaging of apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wolters, S.L.; Reutelingsperger, C.P.M. [Maastricht University, Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht (Netherlands); Corsten, M.F.; Hofstra, L. [Maastricht University, Department of Cardiology, Cardiovascular Research Institute Maastricht, P.O. Box 616, Maastricht (Netherlands); Narula, J. [University of California Irvine, Department of Cardiology, Irvine (United States)

    2007-06-15

    Molecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible. Apoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far. This review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies. (orig.)

  17. Dividing roles of prion protein in staurosporine-mediated apoptosis.

    Science.gov (United States)

    Zhang, Ying; Qin, Kefeng; Wang, Jianwei; Hung, Tao; Zhao, Richard Y

    2006-10-20

    Prion protein (PrPC) is a normal cellular glycoprotein that is expressed in almost all tissues including the central nervous system. Much attention has been focused on this protein because conversion of the normal PrPC to the diseased form (PrPSc) plays an essential role in transmissible spongiform encephalopathies such as mad cow disease and Creutzfeldt-Jakob disease. In spite of the extensive effort, the normal physiological function of PrPC remains elusive. Emerging evidence suggests that PrPC plays a protective role against cellular stresses including apoptosis induced by various pro-apoptotic agents such as Bax and staurosporine (STS), however, other reports showed overexpression of PrPC enhances STS-mediated apoptosis. In this study, we took a different approach by depleting endogenous PrPC using specific interfering RNA technique and compared the depleting and overproducing effects of PrPC on STS-induced apoptosis in neuro-2a (N2a) cells. We demonstrate here that down-regulation of PrPC sensitizes N2a cells to STS-induced cytotoxicity and apoptosis. The enhanced apoptosis induced by STS was shown by increased DNA fragmentation, immunoreactivity of Bax, and caspase-3 cleavage. We also showed that overproduction of PrPC had little or no effect on STS-mediated DNA fragmentation in N2a cells but it augments STS-mediated apoptosis in HEK293 cells, suggesting a cell line-specific effect. In addition, the inhibitory effect of PrPC on STS-mediated cellular stress appears to be modulated in part through induction of cell cycle G2 accumulation. Together, our data suggest that physiological level of endogenous PrPC plays a protective role against STS-mediated cellular stress. Loss of this protection could render cells more prone to cellular insults such as STS. PMID:16950206

  18. Apoptosis Evaluation by Electrochemical Techniques.

    Science.gov (United States)

    Yin, Jian; Miao, Peng

    2016-03-01

    Apoptosis has close relevance to pathology, pharmacology, and toxicology. Accurate and convenient detection of apoptosis would be beneficial for biological study, clinical diagnosis, and drug development. Based on distinct features of apoptotic cells, a diversity of analytical techniques have been exploited for sensitive analysis of apoptosis, such as surface plasmon resonance, electrochemical methods, flow cytometry, and some imaging assays. Among them, the features of simplicity, easy operation, low cost, and high sensitivity make electrochemical techniques powerful tools to investigate electron-transfer processes of in vitro biological systems. In this contribution, a general overview of current knowledge on various technical approaches for apoptosis evaluation is provided. Furthermore, recently developed electrochemical biosensors for detecting apoptotic cells and their advantages over traditional methods are summarized. One of the main considerations focuses on designing the recognition elements based on various biochemical events during apoptosis.

  19. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  20. Apoptosis by Direct Current Treatment in Tumor Cells and Tissues

    Science.gov (United States)

    Kim, Hongbae; Sim, Sungbo; Ahn, Saeyoung

    2003-10-01

    Electric field induces cell fusion, electroporation on biological cells, including apoptosis. Apoptosis is expressed in a series of natural enzymatic reactions for the natural elimination of unhealthy, genetically damaged, or otherwise aberrant cells that are not needed or not advantageous to the well-being of the organism. Its markers involve cell shrinkage, activation of intracellular caspase proteases, externalization of phosphatidylserine at the plasma membrane, and fragmentation of DNA. Direct electric fields using direct current have been exploited recently to investigate its effects on tumor cells and tissues, but the mechanism of direct electric fields has not been exhibited clearly other than by electroosmosis or pH changes. Direct electric field induces apoptosis in tumor cells cultured and tumor tissues as indicated by cell shrinkage, DNA fragmentation and tumor suppression. In our experiment that direct electric field was applied to tumor tissues via two needle electrodes inserted into tumor tissue 5mm at distance in parallel, pH changes resulted from electrochemical reaction, exhibiting about pH 9.0, 1.83, 2.0 in the vicinity of cathodic and anodic electrode, and at their mid-point, respectively. DNA fragmentation of tumor tissues destructed by direct electric field was analyzed by Tunel assay by ApopTag technology. As a result of this analysis, it showed that apoptosis in tumor tissue destructed was increased up to 59.1normal(control) tissues, showing 41.1, 31.1cathodic tissues. In vitro cell survival was exhibited that it was decreased with enhancing electric current intensity in the same condition of electrical charge 5C having different time applied. We will show results of apoptosis analyzed by flow cytometry in vitro.

  1. Gene Analysis of Arsenic Trioxide—induced Apoptosis of Lymphoma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGZidong; LIWeiyu; 等

    2002-01-01

    Objective The effect of arsenic trioxide on apoptosis gene expression of Raji cell was explored when Raji cells were incubated with 0.5μmol/L of arsenic trioxide for 6h。Methods Cell culture,extraction and isolation of mRNA,preparation of probes labeled with fluorescence,hybridization technique of DNA chip(each chip containing 200 apoptosis genes,Chinese Shanghai Biostar,In.)were used.Results Arsenic trioxide induced significant changes in 10%(20/200 genes)of the apoptosis genes:18 genes were downregulated,only two upregulated.In particular,inhibitors of apoptosis protein,such as X-linked inhibitor of apoptosis protein,were significantly downregulated.P53 and the other apoptosis genes were also downregulatec.Of the upregulated genes,high expression of heat-shock protein could promote apoptosis of Raji cells.Conclusion The inhibitors of apoptosis protein play an important role in the process of arsenic trioxide-induced apoptosis of Raji cells.

  2. Design of Ionizable Lipids To Overcome the Limiting Step of Endosomal Escape: Application in the Intracellular Delivery of mRNA, DNA, and siRNA.

    Science.gov (United States)

    Habrant, Damien; Peuziat, Pauline; Colombani, Thibault; Dallet, Laurence; Gehin, Johan; Goudeau, Emilie; Evrard, Bérangère; Lambert, Olivier; Haudebourg, Thomas; Pitard, Bruno

    2016-04-14

    The intracellular delivery of nucleic acid molecules is a complex process involving several distinct steps; among these the endosomal escape appeared to be of particular importance for an efficient protein production (or inhibition) into host cells. In the present study, a new series of ionizable vectors, derived from naturally occurring aminoglycoside tobramycin, was prepared using improved synthetic procedures that allow structural variations on the linker and hydrophobic domain levels. Complexes formed between the new ionizable lipids and mRNA, DNA, or siRNA were characterized by cryo-TEM experiments and their transfection potency was evaluated using different cell types. We demonstrated that lead molecule 30, bearing a biodegradable diester linker, formed small complexes with nucleic acids and provided very high transfection efficiency with all nucleic acids and cell types tested. The obtained results suggested that the improved and "universal" delivery properties of 30 resulted from an optimized endosomal escape, through the lipid-mixing mechanism. PMID:26943260

  3. Mitochondrial DNA in Sensitive Forensic Analysis

    OpenAIRE

    Nilsson, Martina

    2007-01-01

    Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA. DNA quan...

  4. Coxsackievirus B3-induced apoptosis and Caspase-3

    Institute of Scientific and Technical Information of China (English)

    JIAN PING YUAN; WEI ZHAO; HONG TAO WANG; KAI YU WU; TAO LI; XIAO KUI GUO; SHAN QING TONG

    2003-01-01

    Cell death can be classified into two categories: apoptosis and necrosis. Apoptotic pathway can beeither caspase-dependent or caspase-independent. Caspase-independent cytopathic effect (CPE) has beendescribed. In order to evaluate the pattern of HeLa cell death induced by Coxsackievirus B3 (CVB3)and whether apoptosis involves caspase activation, we co-cultivated HeLa cells with CVB3 and detectedthe cytopathic changes, the alteration of mRNA and protein expression of caspase-3 gene plus caspase-3activity, as well as analyzing DNA fragmentation before and after caspase-3 activity inhibition. Accordingto the results, we propose that CVB3 may induce apoptosis and necrosis in HeLa cells, the latter appearingmuch earlier. Caspase-3 is activated at the levels of both transcription and translation, and procaspase-3 isproteolytically cleaved, thus leading to the continuous increasing of both caspase-3 precursor protein and itssubunit. However, besides CPE, apoptosis induced by CVB3 is not a direct consequence of the activationof caspase-3, or caspase-3 is not the only effector molecule in apoptotic cell death, for caspase-3 inhibitorcan not decrease DNA fragmentation. Some other biochemical mechanisms may participate in the process,whose role weakens the effect of inhibiting caspase-3 activity.

  5. ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Bianca M Sirbu

    Full Text Available Homologous recombination (HR is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB. However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

  6. DNA repair and redox activities and inhibitors of apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1): a comparative analysis and their scope and limitations toward anticancer drug development.

    Science.gov (United States)

    Kaur, Gagandeep; Cholia, Ravi P; Mantha, Anil K; Kumar, Raj

    2014-12-26

    The apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional enzyme involved in DNA repair and activation of transcription factors through its redox function. The evolutionarily conserved C- and N-termini are involved in these functions independently. It is also reported that the activity of APE1/Ref-1 abruptly increases several-fold in various human cancers. The control over the outcomes of these two functions is emerging as a new strategy to combine enhanced DNA damage and chemotherapy in order to tackle the major hurdle of increased cancer cell growth and proliferation. Studies have targeted these two domains individually for the design and development of inhibitors for APE1/Ref-1. Here, we have made, for the first time, an attempt at a comparative analysis of APE1/Ref-1 inhibitors that target both DNA repair and redox activities simultaneously. We further discuss their scope and limitations with respect to the development of potential anticancer agents.

  7. The MAPK pathway as an apoptosis enhancer in melanoma.

    Science.gov (United States)

    Haydn, Johannes M; Hufnagel, Anita; Grimm, Johannes; Maurus, Katja; Schartl, Manfred; Meierjohann, Svenja

    2014-07-15

    Inhibition of RAF/MEK/ERK signaling is beneficial for many patients with BRAF(V600E)-mutated melanoma. However, primary and secondary resistances restrict long-lasting therapy success. Combination therapies are therefore urgently needed. Here, we evaluate the cellular effect of combining a MEK inhibitor with a genotoxic apoptosis inducer. Strikingly, we observed that an activated MAPK pathway promotes in several melanoma cell lines the pro-apoptotic response to genotoxic stress, and MEK inhibition reduces intrinsic apoptosis. This goes along with MEK inhibitor induced increased RAS and P-AKT levels. The protective effect of the MEK inhibitor depends on PI3K signaling, which prevents the induction of pro-apoptotic PUMA that mediates apoptosis after DNA damage. We could show that the MEK inhibitor dependent feedback loop is enabled by several factors, including EGF receptor and members of the SPRED family. The simultaneous knockdown of SPRED1 and SPRED2 mimicked the effects of MEK inhibitor such as PUMA repression and protection from apoptosis. Our data demonstrate that MEK inhibition of BRAF(V600E)-positive melanoma cells can protect from genotoxic stress, thereby achieving the opposite of the intended anti-tumorigenic effect of the combination of MEK inhibitor with inducers of intrinsic apoptosis.

  8. Relationship between Cell Proliferation and Apoptosis in Cervical Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To study the relationship between cell proliferation and apoptosis in cervical carcinoma and its clinical significance.Methods The cell proliferation and apoptosis of cervical epithelial cells in archival formalin-fixed,paraffin-embedded tissue sections of normal cervix ,cervical intraepithelial neoplasms(CN) and cervical squamous carcinoma were tested by using immunohistochemistry assay and DNA nick end-labeling technigue.The proliferation index(PI) and apoptosis index(AI) were calculated and their correlation with clinical and pathological data was analyzed. Results PI was gradually increased,but the AI and AI/PI ratio decreased from normal cervical epithelium,CIN to cervical carcinoma. There was no significant relationship among cell proliferation,apoptosis,clinical stages and pathological grades.High AI was always asso-ciated with a poor prognosis of the patients. Conclusion Cell proliferation and apoptosis allow to distinguish among normal epithelium,CIN and cervical carcinoma and are useful for the assessment of the malignant potential of tumor tissues.

  9. Puma and p21 represent cooperating checkpoints limiting self-renewal and chromosomal instability of somatic stem cells in response to telomere dysfunction.

    Science.gov (United States)

    Sperka, Tobias; Song, Zhangfa; Morita, Yohei; Nalapareddy, Kodandaramireddy; Guachalla, Luis Miguel; Lechel, André; Begus-Nahrmann, Yvonne; Burkhalter, Martin D; Mach, Monika; Schlaudraff, Falk; Liss, Birgit; Ju, Zhenyu; Speicher, Michael R; Rudolph, K Lenhard

    2011-12-04

    The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-cycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs.

  10. The Hsp90 inhibitor SNX-7081 is synergistic with fludarabine nucleoside via DNA damage and repair mechanisms in human, p53-negative chronic lymphocytic leukemia.

    Science.gov (United States)

    Kaufman, Kimberley L; Jenkins, Yiping; Alomari, Munther; Mirzaei, Mehdi; Best, O Giles; Pascovici, Dana; Mactier, Swetlana; Mulligan, Stephen P; Haynes, Paul A; Christopherson, Richard I

    2015-12-01

    Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered. PMID:26556860

  11. Poliovirus protease 3C(pro) kills cells by apoptosis.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  12. Role of Calcium Ion in Apoptosis of MD Cancer Cells Induced by Arsenic Trioxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jiuli; WANG Jintao; XU Shiwen

    2008-01-01

    In order to observe the role of calcium ion in apoptosis of MD cancer cells induced by arsenic trioxide, inhibition percentage was detected by MTT assay;morphology changes were examined by fluorescence microscope;apoptosis was examined by DNA Ladder;[Ca2+]i was investigated by spectrofluorimeter in vitro on MDCC-MSB1 cells. The results showed that As2O3 inhibited the proliferation of MDCC-MSB1 cells in concentration dependent manner (P<0.05 or P<0.01);typical apoptosis character was observed by fluorescence microscope;DNA Ladder was observed;the [Ca2+]i was elevated significantly after the treatment of As203 (P<0.05 or P<0.01) and showed a dose-dependent manner. It is concluded that the calcium may play an important role in apoptosis of MD cancer cells induced by arsenic trioxide.

  13. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang

    2004-01-01

    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  14. HCA520, A NOVEL TUMOR ASSOCIATED ANTIGEN, INVOLVED IN CELL PROLIFERATION AND APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    杨美香; 曲迅; 刘福利; 郑广娟

    2003-01-01

    Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis. Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.

  15. Changes of Apoptosis in Rats of Acute Ischemic Renal Injury under Treatment of Tetrandrine

    Institute of Scientific and Technical Information of China (English)

    钱玲梅; 王笑云; 冷静

    2002-01-01

    ObjectiveTo elucidate the effect of tetrandrine on acute ischemic renal injury and its relation with apoptosis.MethodsA model for bilateral post-ischemic renal injury in rats was developed by clamping renal pedicles for 45 min.Renal tissular DNA fragmentation analysis and renal tissular HE staining were used.Also quantitative analysis of apoptosis in injured renal tubular epithelium was carried out by using TdT-mediated dUTP nick and labeling (TUNEL).ResultsApoptosis of renal tubular epithelium increased in acute ischemic renal injury.Tetrandrine could remarkably decrease the level of apoptosis in injured renal tubule while protecting renal tissue against the ischemic injuries.ConclusionTetrandrine could adjust the level of apoptosis in renal tubular epithelium and alleviate renal tissular injury.``

  16. Changes of Apoptosis in Rats of Acute Ischemic Renal Injury under Treatment of Tetrandrine

    Institute of Scientific and Technical Information of China (English)

    钱玲梅; 王笑云; 等

    2002-01-01

    Objective To elucidate the effect of tetrandrine on acute ischemic renal injury and its relation with apoptosis.Methods A model for bilateral post-ischemic renal injury in rats was developed by clamping renal pedicles for 45 min.Renal tissular DNA fragmentation analysis and renal tissular HE staining were used.Also quantitative analysis of apoptosis in injured renal tubular epithelium was carried out by using TdT-mediated dUTP nick and labeling(TUNEL).Results Apoptosis of renal tubular epithelium increased in acute ischemic renal injury.Tetrandrine could remarkably decrease the level of apoptosis in injured renal tubule while protecting renal tissue against the ischemic injuries.Conclusion Tetrandrine could adjust the level of apoptosis in renal tubular epithelium and alleviate renal tissular injury.

  17. ARF and ATM/ATR cooperate in p53-mediated apoptosis upon oncogenic stress

    International Nuclear Information System (INIS)

    Induction of apoptosis is pivotal for eliminating cells with damaged DNA or deregulated proliferation. We show that tumor suppressor ARF and ATM/ATR kinase pathways cooperate in the induction of apoptosis in response to elevated expression of c-myc, β-catenin or human papilloma virus E7 oncogenes. Overexpression of oncogenes leads to the formation of phosphorylated H2AX foci, induction of Rad51 protein levels and ATM/ATR-dependent phosphorylation of p53. Inhibition of ATM/ATR kinases abolishes both induction of Rad51 and phosphorylation of p53, and remarkably reduces the level of apoptosis induced by co-expression of oncogenes and ARF. However, the induction of apoptosis is downregulated in p53-/- cells and does not depend on activities of ATM/ATR kinases, indicating that efficient induction of apoptosis by oncogene activation depends on coordinated action of ARF and ATM/ATR pathways in the regulation of p53

  18. Effective chemotherapy induce apoptosis in vivo in patients with leukemia

    Institute of Scientific and Technical Information of China (English)

    岑溪南; 朱平; 虞积仁; 石永进; 马明信

    2003-01-01

    Objective To investigate apoptosis in vivo in patients with leukemia at different stages of the first cycle of chemotherapy.Methods We detected apoptosis of HL-60 cells and peripheral blood leukemia cells in 17 patients at different stages, using in situ terminal deoxynucleotidyl transferase (TdT) fluorescence measurement and DNA electrophoresis. Results When HL-60 cells were incubated with 0.02 mg/L harringtonine for 0 to 48 hours, agarose gel electrophoresis showed that DNA ladder patterns became evident only at 12 hour into the treatment. In situ TdT assay showed that apoptotic cells occurred after one hour of the treatment. Apoptotic cells were few (0-3.3%) before chemotherapy, but increased substantially (11.4%-87.5%) during chemotherapy in patients with complete remission (CR) or partial remission (PR). Apoptotic cells were few (0-6.1%) during chemotherapy in ten patients with no remission (NR). DNA ladder cannot be detected by agarose gel electrophoresis either before, during or after chemotherapy. Wilcoxon signed rank test shows: P=0.0012<0.01, apoptotic cells during chemotherapy were present in greater quantity than prior to chemotherapy. Wilcoxon rank sum test shows: P=0.0011<0.01, with the median of apoptotic cells during chemotherapy in patients with CR or PR more than with NR.Conclusions TdT assay can be used to detect apoptotic cells earlier and more sensitively than DNA agarose gel electrophoresis. In situ TdT assay is useful to detect apoptosis in vivo in the initial phase of chemotherapy for immediate modification of the chemotherapy regimen, whereas electrophoretic analysis is not sensitive enough to detect apoptotic cell in vivo. Where the median of apoptotic cells during chemotherapy in patients with CR or PR were greater than with NR, only effective drug therapy could induce apoptosis.

  19. Fas-associated factor 1 interacts with protein kinase CK2 in vivo upon apoptosis induction

    DEFF Research Database (Denmark)

    Guerra, B; Boldyreff, B; Issinger, O G

    2001-01-01

    We show here that in several different cell lines protein kinase CK2 and Fas-associated factor 1 (FAF1) exist together in a complex which is stable to high monovalent salt concentration. The CK2/FAF1 complex formation is significantly increased after induction of apoptosis with various DNA damaging...... the view that protein kinase CK2 plays an important role in certain steps of apoptosis....

  20. Gene promoter DNA methylation patterns have a limited role in orchestrating transcriptional changes in the fetal liver in response to maternal folate depletion during pregnancy

    Science.gov (United States)

    Adriaens, Michiel; Evelo, Chris T.; Ford, Dianne; Mathers, John C.

    2016-01-01

    Scope Early‐life exposures are critical in fetal programming and may influence function and health in later life. Adequate maternal folate consumption during pregnancy is essential for healthy fetal development and long‐term offspring health. The mechanisms underlying fetal programming are poorly understood, but are likely to involve gene regulation. Epigenetic marks, including DNA methylation, regulate gene expression and are modifiable by folate supply. We observed transcriptional changes in fetal liver in response to maternal folate depletion and hypothesized that these changes are concomitant with altered gene promoter methylation. Methods and results Female C57BL/6J mice were fed diets containing 2 or 0.4 mg folic acid/kg for 4 wk before mating and throughout pregnancy. At 17.5‐day gestation, genome‐wide gene expression and promoter methylation were measured by microarray analysis in male fetal livers. While 989 genes were differentially expressed, 333 promoters had altered methylation (247 hypermethylated, 86 hypomethylated) in response to maternal folate depletion. Only 16 genes had both expression and methylation changes. However, most methylation changes occurred in genomic regions neighboring expression changes. Conclusion In response to maternal folate depletion, altered expression at the mRNA level was not associated with altered promoter methylation of the same gene in fetal liver. PMID:27133805

  1. 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.

  2. Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method

    Science.gov (United States)

    Lakshmanan, Imayavaramban; Batra, Surinder K

    2016-01-01

    This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium Iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

  3. Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields

    OpenAIRE

    Saha, Shreya; Woodbine, Lisa; Haines, Jacqueline; Coster, Margaret; Ricket, Nicole; Barazzuol, Lara; Ainsbury, Elizabeth; Sienkiewicz, Zenon; Jeggo, Penny

    2014-01-01

    The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radios...

  4. TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells.

    Science.gov (United States)

    Guelen, Lars; Paterson, Hugh; Gäken, Joop; Meyers, Michelle; Farzaneh, Farzin; Tavassoli, Mahvash

    2004-02-01

    Apoptin has been described to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it an interesting candidate for the development of novel therapeutic strategies. Apoptin was generated and cloned into several mammalian expression vectors. Transfection or microinjection of apoptin cDNA resulted in its expression, initially in the cytoplasm with a filamentous pattern. Subsequently, apoptin entered the nucleus and efficiently induced apoptosis in several cancer cell lines. Nuclear localization was shown to be required for induction of apoptosis. Apoptin expression level was found to be an important determinant of the efficiency of induction of apoptosis. Surprisingly, expression of apoptin or GFP-apoptin cDNA induced apoptosis in some normal cells. When fused to the HIV-TAT protein transduction domain and delivered as a protein, TAT-apoptin was transduced efficiently (>90%) into normal and tumour cells. However, TAT-apoptin remained in the cytoplasm and did not kill normal 6689 and 1BR3 fibroblasts. In contrast TAT-apoptin migrated from the cytoplasm to the nucleus of Saos-2 and HSC-3 cancer cells resulting in apoptosis after 24 h. This study shows that apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy. PMID:14691460

  5. Role of Mitochondria in Neuron Apoptosis during Ischemia-Reperfusion Injury

    Institute of Scientific and Technical Information of China (English)

    段秋红; 王西明; 王忠强; 卢涛; 韩义香; 何善述

    2004-01-01

    To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media.DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (△ψm)were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVDinhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could comthat there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.

  6. Biological dosimetry: the potential use of radiation-induced apoptosis in human T-lymphocytes

    International Nuclear Information System (INIS)

    An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% ± 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter. (orig.)

  7. DNA display I. Sequence-encoded routing of DNA populations.

    OpenAIRE

    Halpin, David R; Pehr B Harbury

    2004-01-01

    Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools ...

  8. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

    Directory of Open Access Journals (Sweden)

    Adriana Díaz

    2009-01-01

    Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

  9. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus) leukocytes measured with the Comet and DNA diffusion assays.

    Science.gov (United States)

    Díaz, Adriana; Carro, Sandra; Santiago, Livia; Estévez, Juan; Guevara, Celia; Blanco, Miriam; Sánchez, Laima; Sánchez, Liena; López, Nirka; Cruz, Danilo; López, Ronar; Cuetara, Elizabeth B; Fuentes, Jorge Luis

    2009-04-01

    The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3) to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29%) of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins. PMID:21637693

  10. Apoptosis of matured T lymphocytes induced by mouse sertoli cells in cocultures in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yang; LIN Zi-hao; ZHU Xiao-hai; LIU Shan-rong

    2001-01-01

    Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes + culture medium of sertoli cells), group B (T lymphocytes + sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P<0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P<0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.

  11. APOPTOSIS IN WHOLE MOUSE OVARIES

    Science.gov (United States)

    Apoptosis in Whole Mouse Ovaries Robert M. Zucker Susan C. Jeffay and Sally D. Perreault Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  12. Protooncogenes as mediators of apoptosis.

    Science.gov (United States)

    Teng, C S

    2000-01-01

    Apoptosis has been well established as a vital biological phenomenon that is important in the maintenance of cellular homeostasis. Three major protooncogene families and their encoded proteins function as mediators of apoptosis in various cell types and are the subject of this chapter. Protooncogenic proteins such as c-Myc/Max, c-Fos/c-Jun, and Bcl-2/Bax utilize a synergetic effect to enhance their roles in the pro- or antiapoptotic action. These family members activate and repress the expression of their target genes, control cell cycle progression, and execute programmed cell death. Repression or overproduction of these protooncogenic proteins induces apoptosis, which may vary as a result of either cell type specificity or the nature of the apoptotic stimuli. The proapoptotic and antiapoptotic proteins exert their effects in the membrane of cellular organelles. Here they generate cell-type-specific signals that activate the caspase family of proteases and their regulators for the execution of apoptosis.

  13. Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

    Institute of Scientific and Technical Information of China (English)

    LIANG Xiaohong; SUN Wensheng; GAO Lifen; MA Chunhong; HAN Lihui; CHEN Youhai

    2005-01-01

    The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test,caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.

  14. Cell Survival and Apoptosis Signaling as Therapeutic Target for Cancer: Marine Bioactive Compounds

    Directory of Open Access Journals (Sweden)

    Kim Se-Kwon

    2013-01-01

    Full Text Available Inhibition of apoptosis leads to activation of cell survival factors (e.g., AKT causes continuous cell proliferation in cancer. Apoptosis, the major form of cellular suicide, is central to various physiological processes and the maintenance of homeostasis in multicellular organisms. A number of discoveries have clarified the molecular mechanism of apoptosis, thus clarifying the link between apoptosis and cell survival factors, which has a therapeutic outcome. Induction of apoptosis and inhibition of cell survival by anticancer agents has been shown to correlate with tumor response. Cellular damage induces growth arrest and tumor suppression by inducing apoptosis, necrosis and senescence; the mechanism of cell death depends on the magnitude of DNA damage following exposure to various anticancer agents. Apoptosis is mainly regulated by cell survival and proliferating signaling molecules. As a new therapeutic strategy, alternative types of cell death might be exploited to control and eradicate cancer cells. This review discusses the signaling of apoptosis and cell survival, as well as the potential contribution of marine bioactive compounds, suggesting that new therapeutic strategies might follow.

  15. Nicotinamide-Induced Apoptosis Can Be Enhanced by Melatonin in Mouse Myeloma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guiyou; SHENG Hongzhi; LIU Jia

    2006-01-01

    The mechanism of apoptosis induced by nicotinamide was investigated by treating mouse myeloma cells (Sp2/0) with various concentrations of nicotinamide. The typical hallmarks of apoptosis, including chromatin condensation and DNA fragmentation, were detected when cells were treated with nicotinamide at concentrations of 30, 40, 50, and 60 mmol/L. The apoptosis percentage increased with increasing nicotinamide concentration. Interestingly, the strong antioxidant melatonin did not restrain the apoptosis induced by nicotinamide in mouse myeloma cells but greatly increased the induction of nicotinamide on apoptosis. When cells were preincubated with 0.1, 1, and 10 mmol/L melatonin before nicotinamide induction, the percentage of apoptosis induced by 50 mmol/L nicotinamide markedly increased with increasing melatonin concentration. These results suggest that apoptosis induced by nicotinamide has no relationship with oxidative stress and melatonin could enhance nicotinamide-induced apoptosis in mouse myeloma cells by stimulating cell division in a certain manner. Nicotinamide may provide a new method to treat some kinds of tumors with no damage to normal tissues.

  16. Effects of Sterigmatocystin, Deoxynivalenol and Aflatoxin G1 on Apoptosis of Human Peripheral Blood Lymphocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To explore the effects of Sterigmatocystin (ST), Deoxynivalenol (DON) and Aflatoxin G1 (AFG1) on apoptosis of human peripheral blood lymphocytes (HPBLs) in vitro and thus to further elucidate the putative roles of these three mycotoxins on human immunosystem. Methods The effects of ST, DON and AFG1 on apoptosis of HPBLs were studied with cell culture, flow cytometric (FCM) DNA analysis and DNA agarose gel electrophoresis. Results DNA agarose gel electrophoresis results showed the characteristic "ladder" pattern of apoptosis in HPBLs treated with ST, DON and AFG1. Flow cytometric DNA analysis revealed that typical subdiploid peaks of apoptosis in DNA histogram could be seen in all groups treated with the three mycotoxins. Significant time-effect and dose-effect relationships were found between the apoptosis rates and treatment time as well as concentrations of the three mycotoxins. Conclusion ST, DON and AFG1 can induce apoptosis of HPBLs in vitro and may have some negative effects on human immunosystem.

  17. Influence of vitamin D on cell cycle, apoptosis, and some apoptosis related molecules in systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Nafise Tabasi

    2015-11-01

    Full Text Available Objective(s:Genetic and environmental factors are involved in the pathogenesis of systemic lupus erythematosus (SLE. Autoreactive lymphocytes are cleared through apoptosis and any disturbance in the apoptosis or clearance of apoptotic cells may disturb tolerance and lead to autoimmunity. Vitamin D has anti-proliferative effects and controls cell cycle progression. In this study we investigated the effects of vitamin D on cell cycle and apoptosis induction in lupus patients. Materials and Methods:Isolated peripheral blood mononuclear cells (PBMCs from 25 SLE patients were cultured in the presence of 50 nM of 1,25(OH2D3; then one part of the cells were stained with FITC labeled Annexin V and PI and were analyzed for apoptosis determination. For gene expression assessment of FasL, Bcl-2 and Bax, RNA was extracted from one another part of the cells, cDNA was synthesized and gene expression analysis was performed using Real time PCR. An additional part of the cells were treated with PI and the cell cycle was analyzed using flowcytometer. Results: The mean number of early apoptotic cells in vitamin D treated cells decreased significantly (18.48±7.9% compared to untreated cells (22.02±9.4% (P=0.008. Cell cycle analysis showed a significant increase in G1 phase in vitamin D treated cells (67.33±5.2% compared to non treated ones (60.77±5.7% (P =0.02. Vitamin D up-regulated the expression levels of Bcl-2 by (18.87 fold increase, and down-regulated expression of Bax (23% and FasL (25%. Conclusion:Vitamin D has regulatory effects on cell cycle progression, apoptosis and apoptosis related molecules in lupus patients.

  18. 16S rDNA-based analysis reveals cosmopolitan occurrence but limited diversity of two cyanobacterial lineages with contrasted patterns of intracellular carbonate mineralization

    Directory of Open Access Journals (Sweden)

    Marie eRagon

    2014-07-01

    Full Text Available Cyanobacteria are mainly thought to induce carbonate precipitation extracellularly via their photosynthetic activity combined with the nucleation potential of exopolymeric substances. The discovery in microbialites of the alkaline lake Alchichica (Mexico of Candidatus Gloeomargarita lithophora, a cyanobacterium forming large amounts of intracellular Mg-Ca-Sr-Ba carbonate spherules, showed that intracellular biomineralization in cyanobacteria is also possible. A second cyanobacterium isolated from the same environment, Candidatus Synechococcus calcipolaris G9, has been recently shown to also form intracellular calcium carbonates at the cell poles, a capability shared by all cultured species of the Thermosynechococcus clade, to which it belongs. To explore the diversity of these two distant cyanobacterial lineages representing two different patterns of intracellular calcification, we designed specific primers against their 16S rRNA genes and looked for their occurrence in a wide variety of samples. We identified the presence of members of the Gloeomargarita and Thermosynechococcus/S. calcipolaris lineages in microbialites collected from Lake Alchichica and three other neighboring Mexican lakes. The two clades also occurred in karstic areas and in some thermophilic or hypersaline microbial mats collected in South America and/or Southern Europe. Surprisingly, the within-group diversity in the two clades was low, especially within the S. calcipolaris clade, with all 16S rRNA gene sequences retrieved sharing more than 97% identity. This suggests that these clades are composed of a limited number of species with cosmopolitan distribution. Moreover, scanning electron microscopy coupled with energy dispersive x-ray spectrometry showed the presence of intracellularly calcifying Gloeomargarita-like cyanobacteria in fresh samples where this clade was relatively abundant, suggesting that these cyanobacteria do precipitate carbonates intracellularly under

  19. Early Contact Stage of Apoptosis: Its Morphological Features and Function

    Directory of Open Access Journals (Sweden)

    Etheri Mikadze

    2006-01-01

    Full Text Available Apoptosis has been a biological phenomenon of intense interest for 20 years, but the earlier morphological features of apoptosis have not been determined hitherto. Using the methods of semi- and ultrathin sections, the livers of intact embryos and young rats have been studied under the effect of cycloheximide to determine morphological features of an early stage of apoptosis. It is discovered that both in hepatoblasts and hepatocytes, apoptosis, besides the well-known stages, also includes an early contact stage, distinguishing features of which are agglutination of bound ribosomes (breaking of translation, elimination of the nucleolus, reduction of free polysomes (and in hepatocytes, reduction of cisterns of rough endoplasmic reticulum, formation of cytoplasmic excrescences, and cell shape changes. The early stage of apoptosis is characterized by close contact with neighboring cells. At a certain phase of the contact stage of apoptosis, the nucleolus reappears in the nucleus and the number of free polysomes in the cytoplasm increases, which suggests the renewal of synthesis of new RNA and proteins. Close contact of differentiating and mitotic hepatoblasts with apoptotic cells indicates a certain functional relationship between these cells that is realized not only by micropinocytosis, but through gap junctions as well. We assume that the apoptotic cell, besides proteolytic products, can contain newly synthesized, low-molecular substances, the relocation of which from apoptotic to neighboring cells may contribute to both functional activity and proliferation of adjacent hepatoblasts and, therefore, the function of apoptosis may not be limited only to the elimination of harmful, damaged, and unwanted cells.

  20. Synthetic lethal approaches exploiting DNA damage in aggressive myeloma

    OpenAIRE

    Cottini, Francesca; Hideshima, Teru; Suzuki, Rikio; Tai, Yu-Tzu; Bianchini, Giampaolo; Richardson, Paul G.; Anderson, Kenneth C.; Tonon, Giovanni

    2015-01-01

    Ongoing DNA damage is a common feature of epithelial cancers. Here we show that tumor cells derived from multiple myeloma (MM), a disease of clonal plasma cells, demonstrate DNA replicative stress leading to DNA damage. We identified a poor prognosis subset of MM with extensive chromosomal instability and replicative stress which rely on ATR to compensate for DNA replicative stress; conversely, silencing of ATR or treatment with a specific ATR inhibitor triggers MM cell apoptosis. We show tha...

  1. Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus)

    OpenAIRE

    Blanco, M.; Cruz, D.; Carro, S.; López, R.; Díaz, A.; Santiago, L; Guevara, C.; Sánchez, L.; Cuetara, E.B.; López, N.

    2009-01-01

    The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosi...

  2. Cleaving DNA with DNA

    Science.gov (United States)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  3. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  4. Involvement of Prohibitin Upregulation in Abrin-Triggered Apoptosis

    Directory of Open Access Journals (Sweden)

    Yu-Huei Liu

    2012-01-01

    Full Text Available Abrin (ABR, a protein purified from the seeds of Abrus precatorius, induces apoptosis in various types of cancer cells. However, the detailed mechanism remains largely uncharacterized. By using a cDNA microarray platform, we determined that prohibitin (PHB, a tumor suppressor protein, is significantly upregulated in ABR-triggered apoptosis. ABR-induced upregulation of PHB is mediated by the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK pathway, as demonstrated by chemical inhibitors. In addition, ABR significantly induced the expression of Bax as well as the activation of caspase-3 and poly(ADP-ribose polymerase (PARP in Jurkat T cells, whereas the reduction of PHB by specific RNA interference delayed ABR-triggered apoptosis through the proapoptotic genes examined. Moreover, our results also indicated that nuclear translocation of the PHB-p53 complex may play a role in the transcription of Bax. Collectively, our data show that PHB plays a role in ABR-induced apoptosis, which may be helpful for the development of diagnostic or therapeutic agents.

  5. ClC-3 chloride channel in hippocampal neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Lijuan Xu; Shuling Zhang; Hongling Fan; Zhichao Zhong; Xi Li; Xiaoxiao Jin; Quanzhong Chang

    2013-01-01

    Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol-lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was tablished by 0.5 mmol/L 3-morpholinosyndnomine (SIN-1), a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4’-di sothiocyanostilbene-2,2’-disulfonic acid (DIDS;the chloride channel blocker) for 18 hours. Neuronal survival was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunoche-nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted ClC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-1. Our findings indicate that the increased activities of the ClC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.

  6. Phytoconstituents as apoptosis inducing agents: strategy to combat cancer.

    Science.gov (United States)

    Kumar, Manish; Kaur, Varinder; Kumar, Subodh; Kaur, Satwinderjeet

    2016-08-01

    Advancement in the field of cancer molecular biology has aided researchers to develop various new chemopreventive agents which can target cancer cells exclusively. Cancer chemopreventive agents have proficiency to inhibit, reverse and delay process of carcinogenesis during its early and later course. Chemopreventive agents can act as antioxidative, antimutagenic/antigenotoxic, anti-inflammatory agents or via aiming various molecular targets in a cell to induce cell death. Apoptosis is a kind of cell death which shows various cellular morphological alterations such as cell shrinkage, blebbing of membrane, chromatin condensation, DNA fragmentation, formation of apoptotic bodies etc. Nowadays, apoptosis is being one of the new approaches for the identification and development of novel anticancer therapies. For centuries, plants are known to play part in daily routine from providing food to management of human health. In the last two decades, diverse phytochemicals and various botanical formulations have been characterized as agents that possess potential to execute cancer cells via inducing apoptosis. Data obtained from the research carried out globally pointed out that natural products are the potential candidates which have capability to combat cancer. In the present review, we surveyed literature on natural products which throws light on the mechanism through which these phytochemicals induce apoptosis in cancer cells. PMID:26239338

  7. Apoptosis-based therapy to treat pulmonary arterial hypertension

    Science.gov (United States)

    Suzuki, Yuichiro J.; Ibrahim, Yasmine F.; Shults, Nataliia V.

    2016-01-01

    Pulmonary arterial hypertension (PAH) is rare, but patients who are diagnosed with this disease still suffer from a lack of satisfactory treatment strategies to prolong survival. While currently approved drugs for PAH have some benefits, these vasodilators only have limited efficacy for eliminating pulmonary vascular remodeling and reducing mortality. Thus, our laboratory has been exploring the use of aggressive drugs, which are capable of causing apoptotic cell death, to treat PAH. We have so far found that three classes of anti-tumor agents, including anthracyclines, taxanes, and proteasome inhibitors, are capable of reducing pulmonary vascular thickness in rats with PAH. These drugs kill cells in remodeled pulmonary vessels without affecting the normal, healthy pulmonary vasculature, revealing that proliferating vascular cells in PAH patients are more sensitive to drug-induced apoptosis compared to the differentiated phenotype that is physiologically important for smooth muscle contraction. Since many apoptosis-inducing drugs cause cardiotoxicity in cancer patients, and because PAH patients already have a weakened heart, we focus on finding biological mechanisms that may reverse pulmonary vascular remodeling without promoting cardiotoxicity. We found two agents, dexrazoxane and pifithrin-α, that selectively inhibit cardiac muscle apoptosis without affecting the drug-induced apoptosis of the proliferating pulmonary vascular cells. Thus, we propose that the addition of apoptosis-inducing drugs and cardioprotectants to PAH therapies may be effective in treating patients and preventing right heart failure.

  8. RADIATION-INDUCED APOPTOSIS OF TWO NASOPHARANGEAL CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    WANG Feng-wei; LIANG Ke; YIN Wei-bo; SHEN Yu; SHENG Xiu-gui

    1999-01-01

    Objective: To study apoptosis induced by radiation in two nasopharyngeal carcinoma (NPC) cell lines, CNE and CNE-2. Methods: Hoechst 33342 staining, immunohistochemical staining, RT-PCR, DNA dot blotting and Southern blotting were used to identify apoptosis.Results: A single dose of X-irradiation resulted in apoptosis, the apoptotic index (AI) was time- and dosedependent. Different apoptotic responses existed in the two cell lines. Immunohistochemical staining showed that bcl-2 protein was strongly positive in CNE but negative in CNE-2. However, RT-PCR revealed p53mRNA in CNE-2 but not in CNE. P53 and bcl-2 genes were both present in the two cell lines as shown by DNA blotting, but the 2.8 kb fragment of the p53 gene was much lower than the 5.6 kb fragment on CNE which was clearly shown in Southern hybridization, suggestive of partial deletion of p53 gene in CNE. Conclusion:Apoptotic response to radiation is different in two NPC cell lines. CNE is more radioresistant than CNE-2.Overexpression of bcl-2 protein and partial deletion of p53 gene may explain their difference in radiosensitivity.

  9. Development and vulnerability of rat brain and testes reflected by parameters for apoptosis and ornithine decarboxylase activity

    DEFF Research Database (Denmark)

    Lam, Henrik Rye; Dalgaard, Majken; Ladefoged, Ole;

    2002-01-01

    of apoptosis (DNA laddering and Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling-staining) were investigated. Results: Brain ODC activity reaches maximum at G19 and thereafter rapidly decreases until P7. Apoptotic DNA laddering occurs in the brain from G17 to P7. Significant...... apoptotic ladders were not detected between P9 and 60. In the testes, apoptotic laddering was weak from G21 to P15, but increased significantly from P15 to 60. Histologic examination and DNA laddering analyses revealed low-level germ cell apoptosis from G15 to P11. At onset of spermatogenesis at P15...

  10. Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jinshun 赵进顺; Alexandra Schmid-Kotsas; Hans-Juergen Gross; Adolf Gruenert; Max G. Bachem

    2003-01-01

    Objective To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.Methods Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.Results One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis.Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50%-90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or AnnexinV-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.Conclusions Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA

  11. Ellagic acid induces apoptosis through inhibition of nuclear factor in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    Mouad Edderkaoui; Irina Odinokova; Izumi Ohno; Ilya Gukovsky; Vay Liang W Go; Stephen J Pandol; Anna S Gukovskaya

    2008-01-01

    AIM: To determine the effect of ellagic acid on apoptosis and proliferation in pancreatic cancer cells and to determine the mechanism of the pro-survival effects of ellagic acid.METHODS: The effect of ellagic acid on apoptosis was assessed by measuring Phosphatidylserine externalization, caspase activity, mitochondrial membrane potential and DNA fragmentation; and proliferation by measuring DNA thymidine incorporation. Mitochondrial membrane potential was measured in permeabilized cells, and in isolated mitochondria. Nuclear factor kB (NF-kB) activity was measured by electromobility shift assay (EMSA).RESULTS: We show that ellagic acid, a polyphenolic compound in fruits and berries, at concentrations 10 to 50 mmol/L stimulates apoptosis in human pancreatic adenocarcinoma cells. Further, ellagic acid decreases proliferation by up to 20-fold at 50 mmol/L Ellagic acid stimulates the mitochondrial pathway of apoptosis associated with mitochondrial depolarization, cytochrome C release, and the downstream caspase activation. Ellagic acid does not directly affect mitochondria. Ellagic acid dose-dependently decreased NF-kB binding activity. Furthermore, inhibition of NF-kB activity using IkB wild type plasmid prevented the effect of ellagic acid on apoptosis.CONCLUSION: Our data indicate that ellagic acid stimulates apoptosis through inhibition of the prosurvival transcription factor NF-kB.

  12. MECHANISM OF TAXOL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    Chen Lirong; Zheng Shu; MC Willingham; Fan Weimin

    1998-01-01

    Objective: To investigate the mechanism by which taxol induces apoptosis in human breast cancer cells.Methods: Cell morphology, agarose gel electrophoresis,flow cytometry, video time-lapse monitor and Western blot were performed for investigating taxol-induced apoptosis in human breast cancer cells (BCap 37).Results: BCap 37 cells treated with taxol (100 nm) underwent the arrests of cell mitosis at metaphase of mitosis and induction of apoptosis. Apoptotic cells demonstrated cell shrinkage, condensation or fragmentation of chromosomes. Nuclear DNA of apoptotic cells displayed ladder bands characteristic of internucleosomal DNA fragmentation. The expression of bcl-2, inhibitor of apotosis, was decreased with modification, while that of bax, inducer of apoptosis, increased only at early stage of the apoptotic pathway and decreased later. Conclusion:In human breast cancer cells the induction of apoptosis by taxol was closely associated with mitotic arrest of cell cycle, and altered expressions of bcl-2 and bax gene possibly played an important role in regulating taxolinduced apoptosis.

  13. Dietary flavonoid fisetin targets caspase-3-deficient human breast cancer MCF-7 cells by induction of caspase-7-associated apoptosis and inhibition of autophagy.

    Science.gov (United States)

    Yang, Pei-Ming; Tseng, Ho-Hsing; Peng, Chih-Wen; Chen, Wen-Shu; Chiu, Shu-Jun

    2012-02-01

    The outcome of producing apoptotic defects in cancer cells is the primary obstacle that limits the therapeutic efficacy of anticancer agents, and hence the development of novel agents targeting novel non-canonical cell death pathways has become an imperative mission for clinical research. Fisetin (3,3',4',7-tetrahydroxyflavone) is a naturally occurring flavonoid commonly found in fruits and vegetables. In this study, we investigated the potential anticancer effects of fisetin on breast cancer cells. The result showed fisetin induced higher cytotoxicity in human breast cancer MCF-7 than in MDA-MB-231 cells otherwise it did not exert any detectable cytotoxicity in non-tumorigenic MCF-10A cells. We found fisetin can trigger a novel form of atypical apoptosis in caspase-3-deficient MCF-7 cells, which was characterized by several apoptotic features, including plasma membrane rupture, mitochondrial depolarization, activation of caspase-7, -8 and -9, and PARP cleavage; however, neither DNA fragmentation and phosphotidylserine (PS) externalization was observed. Although p53 was also activated by fisetin, the fisetin-induced apoptosis was not rescued by the p53 inhibitor pifithrin-α. In contrast, the fisetin-induced apoptosis was abrogated by pan-caspase inhibitor z-VAD-fmk. Furthermore, inhibition of autophagy by fisetin was shown as additional route to prompt anticancer activity in MCF-7 cells. These data allow us to propose that fisetin appears as a new potential anticancer agent which can be applied to develop a clinical protocol of human breast cancers. PMID:21922137

  14. Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant.

    Directory of Open Access Journals (Sweden)

    Martina Bauer

    Full Text Available Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs. In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs. Furthermore, monocytes accumulated DNA double-strand breaks (DSBs following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective

  15. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2012-01-01

    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  16. PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia.

    Science.gov (United States)

    Guirguis, A A; Slape, C I; Failla, L M; Saw, J; Tremblay, C S; Powell, D R; Rossello, F; Wei, A; Strasser, A; Curtis, D J

    2016-06-01

    Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with resultant cytopenias. Increased apoptosis and aberrantly functioning progenitors are thought to contribute to this phenotype. As is the case for other malignancies, overcoming apoptosis is believed to be important in progression toward acute myeloid leukemia (AML). Using the NUP98-HOXD13 (NHD13) transgenic mouse model of MDS, we previously reported that overexpression of the anti-apoptotic protein BCL2, blocked apoptosis and improved cytopenias, paradoxically, delaying leukemic progression. To further understand this surprising result, we examined the role of p53 and its pro-apoptotic effectors, PUMA and NOXA in NHD13 mice. The absence of p53 or PUMA but not NOXA reduced apoptosis and expanded the numbers of MDS-repopulating cells. Despite a similar effect on apoptosis and cell numbers, the absence of p53 and PUMA had diametrically opposed effects on progression to AML: absence of p53 accelerated leukemic progression, while absence of PUMA significantly delayed progression. This may be explained in part by differences in cellular responses to DNA damage. The absence of p53 led to higher levels of γ-H2AX (indicative of persistent DNA lesions) while PUMA-deficient NHD13 progenitors resolved DNA lesions in a manner comparable to wild-type cells. These results suggest that targeting PUMA may improve the cytopenias of MDS without a detrimental effect on leukemic progression thus warranting further investigation. PMID:26742432

  17. Cardiomyocytic apoptosis and heart failure

    Institute of Scientific and Technical Information of China (English)

    Quanzhou Feng

    2008-01-01

    Heart failure is a major disease seriously threatening human health.Once left ventricular dysfunction develops,cardiac function usually deteriorates and progresses to congestive heart failure in several months or years even if no factors which accelerate the deterioration repeatedly exist.Mechanism through which cardiac function continually deteriorates is still unclear.Cardiomyocytic apoptosis can occur in acute stage of ischemic heart diseases and the compensated stage of cardiac dysfunction.In this review,we summarize recent advances in understanding the role of cardiomyocytic apoptosis in heart failure.

  18. Doxorubicin Differentially Induces Apoptosis, Expression of Mitochondrial Apoptosis-Related Genes, and Mitochondrial Potential in BCR-ABL1-Expressing Cells Sensitive and Resistant to Imatinib

    Directory of Open Access Journals (Sweden)

    Ewelina Synowiec

    2015-01-01

    Full Text Available Imatinib resistance is an emerging problem in the therapy of chronic myeloid leukemia (CML. Because imatinib induces apoptosis, which may be coupled with mitochondria and DNA damage is a prototype apoptosis-inducing factor, we hypothesized that imatinib-sensitive and -resistant CML cells might differentially express apoptosis-related mitochondrially encoded genes in response to genotoxic stress. We investigated the effect of doxorubicin (DOX, a DNA-damaging anticancer drug, on apoptosis and the expression of the mitochondrial NADH dehydrogenase 3 (MT-ND3 and cytochrome b (MT-CYB in model CML cells showing imatinib resistance caused by Y253H mutation in the BCR-ABL1 gene (253 or culturing imatinib-sensitive (S cells in increasing concentrations of imatinib (AR. The imatinib-resistant 253 cells displayed higher sensitivity to apoptosis induced by 1 μM DOX and this was confirmed by an increased activity of executioner caspases 3 and 7 in those cells. Native mitochondrial potential was lower in imatinib-resistant cells than in their sensitive counterparts and DOX lowered it. MT-CYB mRNA expression in 253 cells was lower than that in S cells and 0.1 μM DOX kept this relationship. In conclusion, imatinib resistance may be associated with altered mitochondrial response to genotoxic stress, which may be further exploited in CML therapy in patients with imatinib resistance.

  19. Apoptosis-like death, an extreme SOS response in Escherichia coli.

    Science.gov (United States)

    Erental, Ariel; Kalderon, Ziva; Saada, Ann; Smith, Yoav; Engelberg-Kulka, Hanna

    2014-07-15

    In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. Importance: The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree

  20. DNA display I. Sequence-encoded routing of DNA populations.

    Directory of Open Access Journals (Sweden)

    David R Halpin

    2004-07-01

    Full Text Available Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery.

  1. DNA media storage

    Institute of Scientific and Technical Information of China (English)

    Christy M.Bogard; Eric C.Rouchka; Benjamin Arazi

    2008-01-01

    In 1994. University of Southern California computer scientist,Dr.Leonard Adleman solved the Hamiltonian path problem using DNA as a computational mechanism.He proved the principle that DNA computing could be used to solve computationally complex problems.Because of the limitations in discovery time,resource requirements,and sequence mismatches,DNA computing has not yet become a commonly accepted practice.However,advancements are continually being discovered that are evolving the field of DNA computing.Practical applications of DNA are not restricted to computation alone.This research presents a novel approach in which DNA could be used as a means of storing files.Through the use of multiple sequence alignment combined with intelligent heuristics,the most probabilistic file contents can be determined with minimal errors.

  2. Apoptosis in immune cells induced by fission fragment 147Pm

    Institute of Scientific and Technical Information of China (English)

    ZhuShou-Peng; ZhangLan-Sheng; 等

    1997-01-01

    Apoptosis in human acute lymphoblastic leukemia cell line Molt-4 cell and macrophage cell line Ana-1 cell could be induced by fission fragment 147Pm,The cumulative absorption dose of 147Pm in cultural cells through different periods were estimated.By using fluorescence microscopy and microautoradiographic tracing it can be found that Molt-4 and Anal-1 cells internally irradiated by 147Pm,displayed an obvious nuclear fragmentation and a marked phknosis in immune cell nucei,as well as DNA chain fragmentation and apoptotic bodies formation.The microautoradiographic study showed that 147Pm could infiltrate thourgh cell membrane and displayed membrane-seeking condensation in cells.At the same time.the membrane-bounded apoptotic bodies were observed.Experimental results in recent study provide evidence that Molt-4 and Ano-1 immune cells undergo apoptosis while internally irradiated with 147Pm.

  3. Induction of apoptosis in lung cancer cells by isorhamnetin

    Institute of Scientific and Technical Information of China (English)

    LingZHU; Li-mingZHOU; Chun-leiYANG; Zun-zhenZHANG; JingXIAO; Zheng-rongWANG

    2005-01-01

    AIM The aim of the present study was to explore cytotoxic activity and the mechanism of tumor cell killing by isorhamnetin and to investigate the effect of isorhamnetin on tumor growth, cell prolification and apoptosis in transplantation tumor of lung cancer of Lewis cell line in C57BL/6 mice. METHODS Human A549 cells were treated with 10-320(g/ml isorhamnetin, C57BL/6 mice were subcutaneously inoculated Lewis cells 0.2ml/each (1×107cells/ml) below the right forelimb armpit and were treated with 50 (g/ml isorhamnetin isorhamnetin.The results were observed and analyzed under light-microscope, electronic microscopy, growth inhibition was analyzed by MTT, clonogenic asssays and growth curve;the apoptosis and the expression-associated genes peaks were detected with flow cytometry (FCM), DNA fragmentation, single cell gel electrophoresis (comet) assay,

  4. Induction of Apoptosis by Superoxide Anion and the Protective Effects of Selenium and Vitamin E

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective The purpose of this study is to investigate the effect of superoxide anion on the apoptosis of cultured fibroblasts and the protective role of selenium and Vitamin E. Methods Cultured fibroblasts (NIH3T3), with or without selenium or vitamin E in the medium, were treated by superoxide anion produced by xanthine/xanthine oxidase reaction system and changes in cell structure and DNA were observed microscopically and electrophoretically. Results Apoptosis was observed when superoxide anion at a concentration of 5 nmol/L or 10 nmol/L had acted on the fibroblasts for 5-10 h. Selenium and Vitamin E in the medium inhibited the apoptosis significantly when their concentrations reached 1.15 mol/L and 2.3 mol/L respectively. Conclusion Selenium and vitamin E have protective effect against the apoptosis induced by superoxide anion. The effect of selenium is more remarkable than that of vitamin E.

  5. APOPTOSIS INDUCED BY HYPERTHERMIA IN HUMAN GLIOBLASTOMA CELL LINE AND MURINE GLIOBLASTOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To study the role of apoptosis in tumor cell of malignant glioma death following treatment with hyperthermia and calcium ionophore. Methods: The apoptosis induced by hyperthermia and calcium ionophore, A23187, in human glioblastoma cell line TJ905 and murine glioblastoma G422 was evaluated by characteristic findings in DNA agarose gel electrophresis, ultrastructural examination and flow cytometric analysis. Results: Apoptosis could be induced by moderate hyperthermia, but not by mild hyperthermia, calcium ionophore enhanced significantly the effect of mild hyperthermia on the induction of apoptosis. Conclusion: This result indicates that apoptotic cell death is one of the mechanisms of hyperthermic therapy for malignant glioma and taking measures to increase the cytolic calcium may enhance the effect of hyperthermia.

  6. THE EFFECTS OF LOW CONCENTRATIONS OF ZINC ON ETOPOSIDE INDUCED HL-60 CELLS APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    盛晓阳; 沈立松; 徐翀; 吴湘如; 陈宏新; 洪照毅

    2001-01-01

    Objective Observation of the effects of lower concentrations of zinc (20~400μmol/L), which is nearby the physiological concentration, on etoposide induced HL-60 cells apoptosis. Methods Using the flow cytometry , DNA extraction, electrophoresis , and fiuoresence microscopic observation. Results We demonstrated that the low concentrations of zinc also affect cells apoptosis. If zinc was added at early time, even 200μmol/L could inhibit VP16 induced HL-60 cell apoptosis completely in 4h. But at this concentration, zinc also seems to have cell toxicity, not prolong the time of protection effect. Conclusion Zinc plays multiple roles in cellular functions and in protecting cells from exogenous deleterious factors. Zinc plays a complex, dose- and time-dependent role in apoptosis.

  7. Identification of inhibitor of apoptosis specific DNase in Xenopus egg extract

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    When added with cytochrome c, Xenopus laevis egg extract XS-150 can induce exogenous nuclei undergoing apoptosis. Apoptosis specific DNase XAD was activated during this process, and cut chromatin between nucleosome,leading to DNA Ladder in electrophoresis. Our results showed that an inhibitor of XAD, IXAD, exists abundantly in normal egg extract, its molecular weight is about 40 ku.Normally, IXAD exists either in the form of dimmer or in complex with XAD. It was degraded during apoptosis, releasing active XAD. The results of Western assay and cross-inhibition showed that IXAD was likely homologous to DFF45 in structure and function. At the same time, these results also indicated that the pathway in apoptosis was conserved in evolution.``

  8. Signaling-based apoptosis therapy: potential for improving the outcome in clinical radiotherapy

    International Nuclear Information System (INIS)

    The most prevalent mechanism of cell kill by radiation in mitosis-dependent and results from lethal DNA double strand breaks and failure to maintain normal replication. Apoptosis is believed to represent a minor component of the clinical effects of radiation. Apoptosis is a pre-programmed death pathway that is constitutively expressed in many cells, albeit in an inactive form, regulated by anti-apoptotic mechanisms. Data will be presented to demonstrate that in irradiated cells, the balance between pro- and anti-apoptotic signaling may determine the apoptotic outcome in vitro and in vivo. This balance can be modulated by pharmacological intervention to produce a more pro-apoptotic phenotype, increasing apoptotic cell kill by radiation in vivo. These studies establish the basic principles of signaling-based apoptosis therapy, designed to overcome the relative resistance to radiation-induced apoptosis and to improve the therapeutic ratio in the treatment of human tumors with fractionated radiation

  9. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    Science.gov (United States)

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  10. β-Sitosterol sensitizes MDA-MB-231 cells to TRAIL-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Cheol PARK; Dong-oh MOON; Chung-ho RYU; Byung tae CHOI; Won ho LEE; Gi-young KIM; Yung hyun CHOI

    2008-01-01

    Aim:To investigate whether subtoxic concentration of β-sitosterol (SITO) com-bined with TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in TRAIL-resistant MDA-MB-231 breast cancer cells.Methods:Cell viability and growth were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphnyl-2H-tetrazolim bromide assays,chromatin condensation,release of lactate dehydrogenase (LDH),and Annexin V+ cells.The apoptosis-related proteins were detected by Western blotting.Results:Treatment with TRAIL in combination with subtoxic concen-trations of SITO sensitized MDA-MB-231 breast cancer cells to TRAIL-mediated apoptosis.The synergistic treatment induced chromatin condensation,DNA fragmentation,the release of LDH,and Annexin V cells.The indicators of apoptosis are correlated to the induction of caspase activities,which results in the cleavage ofpoly(ADP-ribose)polymerase.Both the cytotoxic effects and apoptotic characteristics induced by the synergistic treatment were significantly inhibited by a pan-caspase inhibitor z-VAD-fmk,demonstrating the important role of caspases.These results indicate that caspases are crucial regulators of apoptosis induced by the combined treatment of SITO and TRAIL in MDA-MB-231 cells.Conclusion:The synergistic treatment of SITO and TRAIL induces apoptosis,which can serve as a potential preventive and therapeutic agent.

  11. Influence of oxygen limitation, absence of the cytochrome bc(1) complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology.

    Science.gov (United States)

    Hanke, Tanja; Richhardt, Janine; Polen, Tino; Sahm, Hermann; Bringer, Stephanie; Bott, Michael

    2012-02-10

    The genome-wide transcriptional responses of the strictly aerobic α-proteobacterium Gluconobacter oxydans 621H to oxygen limitation, to the absence of the cytochrome bc(1) complex, and to low pH were studied using DNA microarray analyses. Oxygen limitation caused expression changes of 486 genes, representing 20% of the chromosomal genes. Genes with an increased mRNA level included those for terminal oxidases, the cytochrome bc(1) complex, transhydrogenase, two alcohol dehydrogenases, heme biosynthesis, PTS proteins, proteins involved in cyclic diGMP synthesis and degradation, two sigma factors, flagella and chemotaxis proteins, several stress proteins, and a putative exporter protein. The downregulated genes comprised those for respiratory dehydrogenases, enzymes of central metabolism, PQQ biosynthesis, outer membrane receptors, Sec proteins, and proteins involved in transcription and translation. A ΔqrcABC mutant of G. oxydans showed a growth defect during cultivation on mannitol at pH 4 under oxygen saturation. Comparison of the transcriptomes of this mutant versus the wild type under these conditions revealed 51 differentially expressed genes. Interestingly, almost all of the 45 genes with increased expression in the ΔqrcABC mutant at pH 4 were also upregulated in the wild type grown at pH 6 under oxygen limitation. These results support an active role of the cytochrome bc(1) complex in G. oxydans respiration. The transcriptome comparison of G. oxydans wild type at pH 4 versus pH 6 in mannitol medium under oxygen-saturated conditions uncovered only 72 differentially expressed genes. The 35 upregulated genes included those for cytochrome bd oxidase, major polyol dehydrogenase, iron storage and oxidative stress proteins. Among the 37 downregulated genes were some encoding enzymes dealing with carbon dioxide, such as biotin carboxylase, biotin carboxyl carrier protein, and carboanhydrase. These results give first insights into global transcriptional responses of

  12. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  13. Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

    Institute of Scientific and Technical Information of China (English)

    Yanan Cai; Xiaodong Yuan; Ya Ou; Yanhui Lu

    2011-01-01

    β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro. However, because of the short survival time of the differentiated cells, clinical applications for this technique are limited. As such, we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended. Taken together, these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death. However, the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

  14. DEK Is a Poly(ADP-Ribose) Acceptor in Apoptosis and Mediates Resistance to Genotoxic Stress

    OpenAIRE

    Kappes, Ferdinand; Fahrer, Jörg; Khodadoust, Michael A.; Tabbert, Anja; Strasser, Christine; Mor-Vaknin, Nirit; Moreno-Villanueva, María; Bürkle, Alexander; Markovitz, David M; May, Elisa

    2008-01-01

    DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA- and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells com...

  15. Lonidamine induces apoptosis in drug-resistant cells independently of the p53 gene.

    OpenAIRE

    Del Bufalo, D; Biroccio, A; Soddu, S; Laudonio, N; D'Angelo, C.; Sacchi, A; Zupi, G.

    1996-01-01

    Lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid, was shown to play a significant role in reversing or overcoming multidrug resistance. Here, we show that exposure to 50 microg/ml of lonidamine induces apoptosis in adriamycin and nitrosourea-resistant cells (MCF-7 ADR(r) human breast cancer cell line, and LB9 glioblastoma multiform cell line), as demonstrated by sub-G1 peaks in DNA content histograms, condensation of nuclear chromatin, and internucleosomal DNA fragmentatio...

  16. Interdependence of Bad and Puma during ionizing-radiation-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Cristhian Toruno

    Full Text Available Ionizing radiation (IR-induced DNA double-strand breaks trigger an extensive cellular signaling response that involves the coordination of hundreds of proteins to regulate DNA repair, cell cycle arrest and apoptotic pathways. The cellular outcome often depends on the level of DNA damage as well as the particular cell type. Proliferating zebrafish embryonic neurons are highly sensitive to IR-induced apoptosis, and both p53 and its transcriptional target puma are essential mediators of the response. The BH3-only protein Puma has previously been reported to activate mitochondrial apoptosis through direct interaction with the pro-apoptotic Bcl-2 family proteins Bax and Bak, thus constituting the role of an "activator" BH3-only protein. This distinguishes it from BH3-only proteins like Bad that are thought to indirectly promote apoptosis through binding to anti-apoptotic Bcl-2 family members, thereby preventing the sequestration of activator BH3-only proteins and allowing them to directly interact with and activate Bax and Bak. We have shown previously that overexpression of the BH3-only protein Bad in zebrafish embryos supports normal embryonic development but greatly sensitizes developing neurons to IR-induced apoptosis. While Bad has previously been shown to play only a minor role in promoting IR-induced apoptosis of T cells in mice, we demonstrate that Bad is essential for robust IR-induced apoptosis in zebrafish embryonic neural tissue. Moreover, we found that both p53 and Puma are required for Bad-mediated radiosensitization in vivo. Our findings show the existence of a hierarchical interdependence between Bad and Puma whereby Bad functions as an essential sensitizer and Puma as an essential activator of IR-induced mitochondrial apoptosis specifically in embryonic neural tissue.

  17. Apoptosis in barley aleurone during germination and its inhibition by abscisic acid.

    Science.gov (United States)

    Wang, M; Oppedijk, B J; Lu, X; Van Duijn, B; Schilperoort, R A

    1996-12-01

    During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as alpha-amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.

  18. Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa.

    Science.gov (United States)

    Sheng, Y; Pero, R W; Amiri, A; Bryngelsson, C

    1998-01-01

    Growth inhibitory activities of novel water extracts of Uncaria tomentosa (C-Med-100) were examined in vitro using two human leukemic cell lines (K562 and HL60) and one human EBV-transformed B lymphoma cell line (Raji). The proliferative capacities of HL60 and Raji cells were strongly suppressed in the presence of the C-Med-100 while K562 was more resistant to the inhibition. Furthermore, the antiproliferative effect was confirmed using the clonogenic assay, which showed a very close correlation between C-Med-100 concentration and the surviving fraction. The suppressive effect of Uncaria tomentosa extracts on tumor cell growth appears to be mediated through induction of apoptosis which was demonstrated by characteristic morphological changes, internucleosomal DNA fragmentation after agarose gel electrophoresis and DNA fragmentation quantification. C-Med-100 induced a delayed type of apoptosis becoming most dose-dependently prominent after 48 hours of exposure. Both DNA single and double strand breaks were increased 24 hours after C-Med-100 treatment, which suggested a well-established linkage between the DNA damage and apoptosis. The induction of DNA strand breaks coupled to apoptosis may explain the growth inhibition of the tumor cells by Uncaria tomentosa extracts. These results provide the first direct evidence for the antitumor properties of Uncaria tomentosa extracts to be via a mechanism of selective induction of apoptosis.

  19. Modulation of the BRCA1 Protein and Induction of Apoptosis in Triple Negative Breast Cancer Cell Lines by the Polyphenolic Compound Curcumin

    Directory of Open Access Journals (Sweden)

    Danica L. Rowe

    2009-09-01

    Full Text Available In the current study, we sought to examine the effects of curcumin in a specific type of breast cancer called triple negative breast cancer. These cancers lack expression of the estrogen and progesterone receptors and do not over-express HER2. Current treatment for triple negative breast cancers is limited to cytotoxic chemotherapy, and upon relapse, there are not any therapies currently available. We demonstrate here that the bioactive food compound curcumin induces DNA damage in triple negative breast cancer cells in association with phosphorylation, increased expression, and cytoplasmic retention of the BRCA1 protein. In addition, curcumin promotes apoptosis and prevents anchorage-independent growth and migration of triple negative breast cancer cells. Apoptosis and BRCA1 modulation were not observed in non-transformed mammary epithelial cells, suggesting curcumin may have limited non-specific toxicity. This study suggests that curcumin and potentially curcumin analogues should be tested further in the context of triple negative breast cancer. These results are novel, having never been previously reported, and suggest that curcumin could provide a novel, non-toxic therapy, which could lead to improved survival for patients with triple negative breast cancer. Curcumin should be studied further in this subset of breast cancer patients, for whom treatment options are severely limited.

  20. Preparation of monoclonal antibody against apoptosis-associated antigens of hepatoma cells by subtractive immunization

    Institute of Scientific and Technical Information of China (English)

    Lian-Jun Yang; Wen-Liang Wang

    2002-01-01

    AIM: To elucidate the expression of the apoptosis-associatedmolecules in human primary hepatocellular carcinoma (HCC)cells, and prepare the monoclonal antibodies (mAb) againstthe apoptosis-associated antigens of HCC cells.METHODS: Human HCC cell line HCC-9204 cells wereinduced apoptosis with 60 mL.L-1 ethanol for 6 h and theirmorphological changes were observed by transmissionelectron microscope. The cell DNA fragmentations weredetected by Terminal Deoxynucleotidyl transferase-mediateddUTP nick end labeling (TUNEL) assay, and the cell DNAcontents by flow cytometry. Ten mice were immunized withethanol-induced apoptotic HCC-9204 cells with the methodof subtractive immunization, while the other 10 mice usedas the control were immunized by the routine procedures.The tail blood of all the mice were prepared after the lastimmunization, and the produced antibodies were determinedby the immunocytochemical ABC staining. The splenic cellsof the mice whose tail blood sera-HCC-9204 cells serumreactions were most different between the apoptotic andthe non-apoptotic were prepared and fused with the mousemyeloma cell line SP2/0 cells. The positive antibodies wereselected by ELISA assay. The fusion rates of hybridoma cellsand the producing rates of antibodies were calculated. Thefused cells that secreted candidate objective antibody werecloned continually with the of limited dilution method, andthen selected and analyzed further by theimmunocytochemical ABC staining. The chromosomes of thecloned hybridoma cells that secreted objective mAb and themAb immunoglobulin (Ig) subtype of the prepared mAb werealso determined. The molecular mass of the mAb associatedantigen was analyzed by Western blot assay.RESULTS: HCC-9204 cells treated with 60 mL.L-1 ethanolfor 6 h, manifested obvious apoptotic morphological changes,the majority of the cells were TUNEL-positive, and the sub-G1 apoptotic peak was evident. There were 2 mice in theexperimental group whose tail blood serum reacted

  1. CIRRHOSIS INDUCES APOPTOSIS IN RENAL TISSUE THROUGH INTRACELLULAR OXIDATIVE STRESS

    Directory of Open Access Journals (Sweden)

    Keli Cristina Simões da SILVEIRA

    2015-03-01

    Full Text Available Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis.

  2. Phorbol Esters Isolated from Jatropha Meal Induced Apoptosis-Mediated Inhibition in Proliferation of Chang and Vero Cell Lines

    Directory of Open Access Journals (Sweden)

    Syahida Ahmad

    2012-10-01

    Full Text Available The direct feeding of Jatropha meal containing phorbol esters (PEs indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang and African green monkey kidney (Vero cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC50 of 125.9 and 110.3 μg/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA values as positive control i.e., 124.5 and 106.3 μg/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC50 concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-δ and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-δ and caspase-3 as their mode of actions.

  3. Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

    Science.gov (United States)

    Ghorai, Atanu; Sarma, Asitikantha; Bhattacharyya, Nitai P; Ghosh, Utpal

    2015-04-01

    High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

  4. Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b6/f Complex

    Institute of Scientific and Technical Information of China (English)

    张贵友; 李萍; 朱瑞宇; 田瑞华; 戴尧仁

    2004-01-01

    An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco suspension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei,evidenced by DNA electrophoresis and fluorescence microscopy observations.Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still exhibited the apoptosis-inducing activity,but the supernatant had no effect,which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble.The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO,the specific inhibitor of Caspase-3,implying involvement of a Caspase-3-like protease during the process.Furthermore,extensive apoptosis in nuclei was induced by cytochrome b6/f on the thylakoid membrane,indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.

  5. DNA Microarrays

    Science.gov (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  6. Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy

    DEFF Research Database (Denmark)

    So, Jonathan; Pasculescu, Adrian; Dai, Anna Y.;

    2015-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous secreted peptide and, in preclinical studies, preferentially induces apoptosis in tumor cells rather than in normal cells. The acquisition of resistance in cells exposed to TRAIL or its mimics limits their clinical e...

  7. Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes

    OpenAIRE

    Ito, Minoru; Nagasawa, Michiaki; Hara, Tomoko; Ide, Tomohiro; Murakami, Koji

    2010-01-01

    Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDE...

  8. Immunofluorescence analysis of cytokeratin 8/18 staining is a sensitive assay for the detection of cell apoptosis

    OpenAIRE

    DONG, QIAO-MEI; LING, CHUN; Zhao, Li

    2015-01-01

    Apoptosis is one of the major types of programmed cell death. During this process, cells experience a series of morphological and biochemical changes. Flow cytometric analysis of Annexin V staining of cell surface phosphatidylserine, in combination with a DNA-staining dye to probe the permeability of the cell membrane, is an established method for detecting apoptosis. The present study aimed to show that the immunofluorescence analysis of cytokeratin (CK) 8/18 staining may provide a new and s...

  9. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    Science.gov (United States)

    Björklund, H V; Johansson, T R; Rinne, A

    1997-07-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor. PMID:9188644

  10. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    OpenAIRE

    Björklund, H V; Johansson, T R; Rinne, A

    1997-01-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor.

  11. Norcantharidin induces apoptosis in HeLa cells through caspase, MAPK, and mitochondrial pathways

    Institute of Scientific and Technical Information of China (English)

    Wei-weiAN; Xian-fengGONG; Min-weiWANG; Shin-ichiTASHIRO; SatoshiONODERA; TakashiIKEJIMA

    2004-01-01

    AIM: To investigate the mechanism of norcantharidin (NCTD)-induced HeLa cell apoptosis. METHODS: HeLa cell growth inhibition was measured by MTT method. Apoptosis was detected by Hoechst 33258 staining and agarose gel electrophoresis. Caspase activities were assayed using caspase apoptosis detection kit. Western blot analysis was used to evaluate the level of ICAD, ERK/p-ERK, JNK/p-JNK, and Bcl-X.L/Bax expression. RESULTS: Norcantharidin inhibited HeLa cell growth in a time- and dose-dependent manner. HeLa cells treated with norcantharidin showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevent norcantharidin-induced apoptosis, but initiator caspase-1 inhibitor (Ac-YVAD-fmk) did not. The activities of caspase-3, -8, and -9 were up-regulated after norcantharidin treatment. Furthermore, NCTD-induced activation of caspase-3 resulted in the degradation of the inhibitor of caspase-activated DNase (ICAD). Up-regulation of mitochondrial Bax expression and down-regulation of Bcl-xLexpression also participated in the apoptosis induced by NCTD. Although p38 MAPK inhibitor (SB203580) failed to block cell death, ERK MAPK inhibitor (PD98059) and JNK MAPK inhibitor (SP600125) had marked inhibitory effects on norcantharidin-induced apoptosis. Moreover, the phosphorylation of JNK were up-regulated followed by delayed ERK phosphorylation after treatment with NCTD, suggesting that ERK and JNK were both responsible for NCTD-induced apoptosis in HeLa cells and worked at different stages. CONCLUSION: The cytotoxic effect of NCTD on HeLa cells was mainly due to apoptosis. The anti-tumor mechanism of NCTD might involve caspses, mitochondrial, and MAPKs pathways.

  12. Effect of mitochondrial energetic state on radiation-induced DNA internucleosomal fragmentation in thymocytes

    International Nuclear Information System (INIS)

    To determine the potential effect of energetic state of mitochondria on apoptosis in thymocytes induced by ionizing radiation, the effect of various mitochondria energy potential on DNA internucleosome fragmentation is studied. Thymocytes of rats-males were exposed to radiation of GUPOS installation (137Cs) at 8 Gy dose, dose rate - 1.8 Gy/min. It is shown that the irradiation stimulates the DNA fragmentation during thymocytes incubation, but in case of incubation with mitochondria energy potential inhibitors the amount of dead cells does not exceed the reference level. It is proposed that internucleosome fragmentation suppression by mitochondria energy potential inhibitors is connected with the limitation of energy incoming in the ATP form

  13. Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

    OpenAIRE

    Li Li; Hongping Tan; Zhengtao Gu; Zhifeng Liu; Yan Geng; Yunsong Liu; Huasheng Tong; Youqing Tang; Junmin Qiu; Lei Su

    2014-01-01

    Background Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined. Methods The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, pho...

  14. Cigarette smoking induces heat shock protein 70 kDa expression and apoptosis in rat brain: Modulation by bacoside A.

    Science.gov (United States)

    Anbarasi, K; Kathirvel, G; Vani, G; Jayaraman, G; Shyamala Devi, C S

    2006-01-01

    Cigarette smoking is associated with the development of several diseases and antioxidants play a major role in the prevention of smoking-related diseases. Apoptosis is suggested as a possible contributing factor in the pathogenesis of smoking-induced toxicity. Therefore the present study was designed to investigate the influence of chronic cigarette smoke exposure on apoptosis and the modulatory effect of bacoside A (triterpenoid saponin isolated from the plant Bacopa monniera) on smoking-induced apoptosis in rat brain. Adult male albino rats of Wistar strain were exposed to cigarette smoke and simultaneously administered with bacoside A (10 mg/kg b.w./day, orally) for a period of 12 weeks. Expression of brain hsp70 was analyzed by Western blotting. Apoptosis was identified by DNA fragmentation, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick end labeling (TUNEL) staining and transmission electron microscopy. The results showed that exposure to cigarette smoke induced hsp70 expression and apoptosis as characterized by DNA laddering, increased TUNEL-positive cells and ultrastructural apoptotic features in the brain. Administration of bacoside A prevented expression of hsp70 and neuronal apoptosis during cigarette smoking. We speculate that apoptosis may be responsible for the smoking-induced brain damage and bacoside A can protect the brain from the toxic effects of cigarette smoking.

  15. Bisabololoxide A, one of the main constituents in German chamomile extract, induces apoptosis in rat thymocytes.

    Science.gov (United States)

    Ogata, Ikuko; Kawanai, Takuya; Hashimoto, Erika; Nishimura, Yumiko; Oyama, Yasuo; Seo, Hakaru

    2010-01-01

    German chamomile (Matricaria recutita L.), one of the popular ingredients in herbal teas, has been traditionally used for medicinal purposes. Bisabololoxide A (BSBO) is one of the main constituents in this herb. BSBO is supposed to be principle in some bioactivities of German chamomile such as anti-inflammatory, gastrointestinal, and antipruritic actions. Although the use of German chamomile has spread, the information related to toxicity of BSBO is very limited. In present study, the cytotoxic effect of micromolar BSBO was cytometrically examined on rat thymocytes by using appropriate fluorescent dyes. When the cells were incubated with BSBO for 24 h, BSBO at concentrations of 30 microM or more significantly increased populations of dead cells, shrunken cells, and cells with phosphatidylserine exposed on membrane surface. Both cell shrinkage and externalization of membrane phosphatidylserine are general features in an early stage of apoptosis. In addition, BSBO significantly increased population of cells containing hypodiploid DNA, and the increase was completely attenuated by Z-VAD-FMK, a pan-inhibitor for caspases, indicating an involvement of caspase activation. Thus, it is likely that the type of cell death induced by BSBO is apoptosis. The significant changes in cellular parameters of rat thymocytes by BSBO were not observed when the concentration was 10 microM or less. Furthermore, the short incubation (3 h) of cells even with 30-100 microM BSBO did not significantly affect the cells. Therefore, it may be suggested that BSBO is practically safe when German chamomile is conventionally used. PMID:19834689

  16. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Rossi Adriano G

    2009-05-01

    Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

  17. A benzophenanthridine alkaloid, chelerythrine induces apoptosis in vitro in a Dalton′s lymphoma

    Directory of Open Access Journals (Sweden)

    Sanjay Kumar

    2013-01-01

    Full Text Available Purpose: The aim of this study was to investigate the effect of chelerythrine on DL cell apoptosis in an in vitro experimental setup. Materials and Methods: For tumor model, spontaneous occurring T-cell lymphoma designated as Dalton′s lymphoma (DL was selected. Double staining, transmission electron microscope (TEM, fluorescence microscopy, Western blotting, Reverse Transcriptase-Polymerase Chain Reaction, and DNA fragmentation assay were used to detect heat shock factor 1 (HSF1 and hsp70 expression and PKC phosphorylation, and apoptotic characteristic of DL cells. Results: Chelerythrine exposure resulted in significant morphological alteration comparable to that of apoptosis. Furthermore, it was confirmed by fluorescence microscopy, TEM analysis, and DNA fragmentation assay that 10 ΅g/mL of chelerythrine is capable of inducing apoptosis in DL cells. The suppression in HSF1 expression and subsequent inhibition of hsp70 expression in chelerythrine-treated DL cells suggest that chelerythrine induces apoptosis in DL cells by inhibiting the expression of these cytoprotective proteins. Conclusion: Chelerythrine is capable of inducing apoptosis DL cells in vitro and therefore, it could be useful in combating tumor growth and progression.

  18. PC-407 inhibited proliferation and induced apoptosis in human colon cancer SW-1116 cells

    Institute of Scientific and Technical Information of China (English)

    Li-liHAO; Qi-bingMEI; Bang-leZHANG; MinJIA; Xiao-qiangLI; FengZHANG

    2004-01-01

    AIM: To study whether PC-407 [4-[5-naphthyl-3- (trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide] inhibits cell viability and induces apoptosis in human colon cancer SW-1116 cells. METHODS: Inhibition of SW-1116 proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with fluorescence microscope and electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis.The amount of apoptotic cells was measured by flow cytometry. RESULTS: PC-407 inhibited SW-1116 cell proliferation in a concentration-dependent manner after 3 d of treatment, and the IC5o for PC-407 inhibition of cell number was 16.67±0.17μmol/L. After incubation of SW-1116 cells with PC-407 20μmol/L for 24 h, morphological changes of typical apoptosis were observed by AO/EB staining or transmission electron microscopy. Flow cytometry analysis showed that PC-407 induced apoptosis in SW-1116 cells in a time- and concentration-dependant manner. The agarose gel electrophoresis of DNA revealed a "ladder" pattern 48 h later. CONCLUSION: PC-407 inhibited proliferation and induced apoptosis in the human colon cancer SW-1116 cell line.

  19. PC-407 inhibited proliferation and induced apoptosis in human colon cancer SW-1116 cells

    Institute of Scientific and Technical Information of China (English)

    Li-li HAO; Qi-bing MEI; Bang-le ZHANG; Min JIA; Xiao-qiang LI; Feng ZHANG

    2004-01-01

    AIM: To study whether PC-407 [4-[5-naphthyl-3- (trifluoromethyl)-1H-pyrazol-l-yl] benzenesulfonamide] inhibits cell viability and induces apoptosis in human colon cancer SW-1116 cells. METHODS: Inhibition of SW-1116proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with fluorescence microscope and electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis.The amount of apoptotic cells was measured by flow cytometry. RESULTS: PC-407 inhibited SW-1116 cell proliferation in a concentration-dependent manner after 3 d of treatment, and the ICs0 for PC-407 inhibition of cell number was 16.67±0.17 μmol/L. After incubation of SW-1116 cells with PC-407 20 μmol/L for 24 h, morphological changes of typical apoptosis were observed by AO/EB staining or transmission electron microscopy. Flow cytometry analysis showed that PC-407 induced apoptosis in SW-1116 cells in a time- and concentration-dependant manner. The agarose gel electrophoresis of DNA revealed a "ladder" pattern 48 h later. CONCLUSION: PC-407inhibited proliferation and induced apoptosis in the human colon cancer SW-1116 cell line.

  20. Apoptosis of human colon carcinoma HT-29 cells induced by ceramide

    Institute of Scientific and Technical Information of China (English)

    Xiao-Feng Zhang; Bai-Xiang Li; Chun-Yan Dong; Rui Ren

    2006-01-01

    AIM: To investigate the effect of exogenous ceramideinduced apoptosis on human colon carcinoma HT-29cells.METHODS: Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay. mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction (RT-PCR) assay.RESULTS: After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50 μmol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile,ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members.CONCLUSION: Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.

  1. Study on apoptosis of prostate cancer cell induced by 125I seed irradiation

    International Nuclear Information System (INIS)

    Objective: To explore the mechanism of apoptosis induced by 125I seed irradiation on PC3 cells. Methods: Human prostate cancer cell line PC3 was treated by irradiation of 125I (2.77 cGy/h) with various dose. Agarose gel electrophoresis of DNA and flows cytometry were used to detect the apoptosis of PC3 cells and indirect immunofluorescence assay was used to detect the expression of Bcl-2. The activity of Caspase-3 was measured by Caspase Colorimetric Assay Kits. Results: Apoptosis of PC3 cells could be efficiently induced by 125I seed irradiation. The apoptotic peaks were found by flow cytometry and DNA ladder appeared on 1.8% agarose gel. The activity of Caspase-3 on PC3 cells treated by 125I seed irradiation was not changed significantly. Bcl-2 gene expression was down-regulated with the sample concentration increased. Conclusion: 125I irradiation can induce the apoptosis of PC3 cells and the mechanism of apoptosis is related with down regulation of Bcl-2 gene expression and is not related with Caspase-3 activity. (authors)

  2. Growth Inhibition and Apoptosis Inducing Mechanisms of Curcumin on Human Ovarian Cancer Cell Line A2780

    Institute of Scientific and Technical Information of China (English)

    ZHENG Li-duan; TONG Qiang-song; WU Cui-huan

    2006-01-01

    Objective: To explore the growth inhibition effects and apoptosis inducing mechanisms of curcumin on human ovarian cancer cell line A2780. Methods: After treatment with 10-50 μmol/L curcumin for 6-24 h, the growth activity of A2780 cancer cells were studied by [ 4, 5-dimethylthiazol-2-yl]-2, 5-diphenyItetrazolium bromide (MTT) colorimetry. Cellular apoptosis was inspected by flow cytometery and acridine orange-ethidium bromide fluorescent staining methods. The fragmentation of cellular chromosome DNA was detected by DNA ladder, the ultrastructural change was observed under a transmission electron microscope,and the protein levels of nuclear factor-kappa B (NF-κB, P65) and cysteinyl aspartate specific protease-3 (Caspase-3) in ovarian cancer cells were measured by immunohistochemistry. Results: After treatment with various concentrations of curcumin, the growth inhibition rates of cancer cells reached 62.05%- 89.24%,with sub-G1 peaks appearing on histogram. Part of the cancer cells showed characteristic morphological changes of apoptosis under fluorescence and electron microscopes, and the rate of apoptosis was 21.5 % -33.5%. The protein expression of NF-κB was decreased, while that of Caspase-3 was increased in a timedependent manner. Conclusion: Curcumin could significantly inhibit the growth of human ovarian cancer cells;inducing apoptosis through up-regulating Caspase-3 and down-regulating gene expression of NF-κB is probably one of its molecular mechanisms.

  3. 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.

  4. The novel NF-κB inhibitor IMD-0354 induces apoptosis in chronic lymphocytic leukemia.

    Science.gov (United States)

    Kanduri, M; Tobin, G; Aleskog, A; Nilsson, K; Rosenquist, R

    2011-03-01

    Nuclear factor-κB (NF-κB) is an important regulator of cell survival and has been shown to be constitutively active in chronic lymphocytic leukemia (CLL) cells. Recently, a novel NF-κB inhibitor, IMD-0354 (N-(3, 5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide), was shown to specifically inhibit the phosphorylation of IκBα by IkB kinases, thus preventing NF-κB release. In this study, we investigated if IMD-0354 can inhibit NF-κB activation and induce apoptosis in CLL cells in vitro. The rate of increase in apoptosis, drug sensitivity and DNA-binding activity of NF-κB were studied using Annexin V stainings, the fluorometric microculture cytotoxicity assay and electrophoretic mobility shift assay, respectively. Finally, the impact of IMD-0354 treatment on the expression of a set of apoptosis-related genes was investigated. The results clearly show that IMD-0354 induced apoptosis (mean 26%, range 8-48%) in CLL cells, independent of immunoglobulin heavy variable (IGHV) gene mutational status, and showed a dose-dependent cytotoxic effect. IMD-0354 treatment also significantly lowered the DNA-binding activity of NF-κB in CLL cells. In addition, we identified differences in expression levels of pro- and antiapoptotic genes following IMD-0354 treatment. In summary, our novel findings show that IMD-0354 can induce apoptosis in CLL cells, and thus merits further investigation as an anticancer agent in vivo. PMID:22829125

  5. Apoptosis in irradiated murine tumors.

    Science.gov (United States)

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E

    1991-09-01

    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors.

  6. Apoptosis in irradiated murine tumors.

    Science.gov (United States)

    Stephens, L C; Ang, K K; Schultheiss, T E; Milas, L; Meyn, R E

    1991-09-01

    Early radiation responses of transplantable murine ovarian (OCaI) and hepatocellular (HCaI) carcinomas were examined at 6, 24, 48, 96, and 144 h after single photon doses of 25, 35, or 45 Gy. Previous studies using tumor growth delay and tumor radiocurability assays had shown OCaI tumors to be relatively radiosensitive and HCaI tumors to be radioresistant. At 6 h, approximately 20% of nuclei in OCaI tumors showed aberrations characteristic of cell death by apoptosis. This contrasted to an incidence of 3% in HCaI tumors. Mitotic activity was eliminated in OCaI tumors but was only transiently suppressed in HCaI tumors. At 24-96 h, OCaI tumors continued to display apoptosis and progressive necrosis, whereas HCaI tumors responded by exhibiting marked pleomorphism. Factors other than mitotic activity may influence tumor radiosensitivity, and one of these may be susceptibility to induction of apoptosis (programmed cell death), because this was a prominent early radiation response by the radiosensitive OCaI tumors. PMID:1886987

  7. Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Radhika Kapoor

    Full Text Available Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor & Endo-G (endonuclease-G from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

  8. Inhibitory effect of picroside Ⅱ on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hua GAO; Ya-wei ZHOU

    2005-01-01

    Aim: To investigate the influence of picroside Ⅱ on hepatocyte apoptosis and its mechanism. Methods: Morphological changes and quantification of apoptotic cells were determined under transmission electron microscopy and flow cytometry respectively. DNA fragmentation was visualized by agarose gel electrophoresis.Semi-quantitative reverse transcription-PCR (RT-PCR) was used to analyze the expression of bcl-2 and bax genes. The content of manganese-superoxide dismutase (SOD) in liver mitochondria was detected by the Marland method. The content of malonic aldehyde (MDA) and the protein level in liver tissue were determined by thiobarbituric acid colorimetry and Lowry method. Results:Picroside Ⅱ decreased the levels of alanine aminotransferase and aspartate aminotransferase in the serum resulting from acute-liver injured mice induced with D-GalN and LPS; it also reduced the content of MDA, and thus, enhanced the activity of SOD. Picroside Ⅱ 10 mg/kg was found to protect hepatocytes against apoptosis in a dose-dependent manner; it up-regulated the expression of bcl-2 genes,thus increased the bcl-2/bax ratio. Conclusion: Picroside Ⅱ can protect hepatocytes against injury and prevent hepatocytes from apoptosis. It might by upregulating the bcl-2 gene expression and antioxidation.

  9. The apoptosis in various stages of infantile hemangioma

    Institute of Scientific and Technical Information of China (English)

    YUAN Si-ming; XING Xin; OUYANG Tian-xiang; NI Can-rong; YANG Zhi-yong

    2005-01-01

    Objective: To detect the apoptosis in various stages of infantile hemangioma. Methods:Total 52 samples of infantile hemangioma (including 8 fresh samples) were included in this study. Agarose gel electrophoresis, transmission electron microscopy(TEM) and in situ TdT mediated dUTP-biotin nick end labeling(TUNEL) staining were used to observe the apoptosis. H-E staining was used to analyze the number of cells,the number and area of microvessels in hemangiomas. Results: The typical "ladder" occurred in the DNA electrophoresis of the hemangioma tissue in the late proferating stage. Many apoptotic cells were found in infantile hemangiomas with TEM. TUNEL staining identified that there were apoptotic cells througout the pathologic evolution of infantile hemangioma and the AI( % ) was the highest in the late proferating stage. There existed close relationship between the AI(%) and the total number of cells in hemangioma. Conclusion: The decrease of cells resulted from the apoptosis may be the major cause of the spontaneous involution of infantile hemangioma.

  10. High glucose augments stress-induced apoptosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Hui Tian

    2009-01-01

    Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

  11. Marijuana smoke condensate induces p53-mediated apoptosis in human lung epithelial cells.

    Science.gov (United States)

    Kim, Ha Ryong; Jung, Mi Hyun; Lee, Soo Yeun; Oh, Seung Min; Chung, Kyu Hyuck

    2013-01-01

    Since the largely abused worldwide used of marijuana, there have been many ongoing debates regarding the adverse health effects of marijuana smoking. Marijuana smoking was recently proved to cause pulmonary toxicity by inducing genotoxic effects or generating reactive oxygen species. Because p53, a tumor suppressor gene, has an important pathophysiologic role in the regulation of lung epithelial cell DNA damage responses, we hypothesized that p53 may be involved in the oxidative stress-mediated apoptosis induced by marijuana smoking. First, we confirmed that marijuana smoke condensate (MSC) induces oxidative stress in BEAS-2B cells. We observed that reactive oxygen species (ROS) generation was increased by MSC in the DCFH-DA assay. Also, antioxidant enzyme (superoxide dismutase, catalase) activity and their mRNA expressions were up-regulated by MSC. Second, we investigated p53 involvement in the MSC-induced apoptotic pathway in BEAS-2B cells. The results showed that MSC increased caspase-3 activation and DNA fragmentation as markers of apoptosis. In addition, the mRNA levels of apoptosis-related genes (p53 and Bax) were increased by MSC and phospho-p53, along with the increase of Bax protein expression by MSC. Apoptosis and apoptosis-related gene expression were partially blocked by an inhibitor of p53-dependent transcriptional activation (pifithrin-α). The results indicate that p53 plays a role in MSC-induced apoptosis. Taken together, the findings of the present study suggest that MSC partially induces p53-mediated apoptosis through ROS generation in human lung epithelial cells and this may have broader implications for our understanding of pulmonary diseases. PMID:23665932

  12. Canine distemper virus induces apoptosis in cervical tumor derived cell lines

    Directory of Open Access Journals (Sweden)

    Rajão Daniela S

    2011-06-01

    Full Text Available Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi, by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

  13. Induction of Apoptosis by Functionalized Fullerene-based Sonodynamic Therapy in HL-60 cells.

    Science.gov (United States)

    Yumita, Nagahiko; Watanabe, Takahiro; Chen, Fu-Shih; Momose, Yasunori; Umemura, Shin-Ichiro

    2016-06-01

    Ultrasound has been widely utilized for medical diagnosis and therapy due to its ability to penetrate deep-seated tissue with less attenuation of energy and minimal undesirable side-effects. Functionalized fullerenes, such as polyhydroxy fullerene (PHF), have attracted particular attention due to their water solubility and potential application in tumor imaging and therapy as carbon nanomaterials. The present study investigated sonodynamically-induced apoptosis using PHF. Cell suspensions were treated with 2-MHz continuous ultrasound in the presence of PHF for 3 min and apoptosis was assessed by cell morphology using confocal microscopy, fragmentation of DNA (ladder pattern after agarose-gel electrophoresis) and caspase-3 activation. Cells were ultrasound-irradiated from the bottom of the culture dishes under the following condition: frequency, 2 MHz; output power, 3 W/cm(2) Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells after sonodynamic exposure (ultrasound and PHF) was significantly higher than produced from other treatments, such as ultrasound alone and PHF alone. Furthermore, DNA fragmentation, caspase-3 activation and enhanced 2,2,6,6-tetramethyl-4-piperidinyloxy (4oxoTEMPO) formation were observed in the sonodynamically-treated cells. Histidine, a well-known reactive oxygen scavenger, significantly inhibited sonodynamically-induced apoptosis, caspase-3 activation and 4oxoTEMPO formation. Sonodynamic therapy with PHF induced apoptosis that was characterized by a series of typical morphological features, such as shrinkage of the cell and fragmentation into membrane-bound apoptotic bodies, in HL-60 cells. The significant inhibition of sonodynamically-induced apoptosis, caspase-3 activation, and 4oxoTEMPO formation due to histidine and tryptophan suggests that reactive oxygen species, such as singlet oxygen, are involved in the sonodynamic induction of apoptosis. These findings indicate that PHF

  14. Chloride channel protein 2 prevents glutamate-induced apoptosis in retinal ganglion cells

    Science.gov (United States)

    Bi, Miao-Miao; Hong, Sen; Ma, Ling-Jun; Zhou, Hong-Yan; Lu, Jia; Zhao, Jing; Zheng, Ya-Juan

    2016-01-01

    Objective(s): The purpose of this study was to investigate the role of chloride channel protein 2 (ClC-2) in glutamate-induced apoptosis in the retinal ganglion cell line (RGC-5). Materials and Methods: RGC-5 cells were treated with 1 mM glutamate for 24 hr. The expression of ClC-2, Bax, and Bcl-2 was detected by western blot analysis. Cell survival and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Caspase-3 and -9 activities were determined by a colorimetric assay. The roles of ClC-2 in glutamate-induced apoptosis were examined by using ClC-2 complementary deoxyribonucleic acid (cDNA) and small inference ribonucleic acid (RNA) transfection technology. Results: Overexpression of ClC-2 in RGC-5 cells significantly decreased glutamate-induced apoptosis and increased cell viability, whereas silencing of ClC-2 with short hairpin (sh) RNA produced opposite effects. ClC-2 overexpression increased the expression of Bcl-2, decreased the expression of Bax, and decreased caspase-3 and -9 activation in RGC-5 cells treated with glutamate, but silencing of ClC-2 produced opposite effects. Conclusion: Our data suggest that ClC-2 chloride channels might play a protective role in glutamate-induced apoptosis in retinal ganglion cells via the mitochondria-dependent apoptosis pathway. PMID:27635193

  15. A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

    Science.gov (United States)

    Shu, Benshui; Wang, Wenxiang; Hu, Qingbo; Huang, Jingfei; Hu, Meiying; Zhong, Guohua

    2015-07-01

    The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds. PMID:25828604

  16. Octreotide induces caspase activation and apoptosis inhuman hepatoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Nikos J Tsagarakis; Ioannis Drygiannakis; Antonis G Batistakis; George Kolios; Elias A Kouroumalis

    2011-01-01

    AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells.METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinomacells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method.RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependentlydecreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 andcaspase-2 activity. TNF-α significantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation onlyat concentrations of 10-8 mol/L, while lower concentrations increased proliferation.CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurementsof serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

  17. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells.

    Science.gov (United States)

    Sen, Nilkantha; Das, Benu Brata; Ganguly, Agneyo; Banerjee, Bijoylaxhmi; Sen, Tanusree; Majumder, Hemanta K

    2006-11-01

    Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells. PMID:16707127

  18. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

    Full Text Available Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF. SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B followed by irreversible DNA degradation by a nuclease(s. Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

  19. THE STUDY OF ELEMENE OF INDUCTION APOPTOSIS ON ASCITES HEPATOMA CELL LINE Hca-F25/CL-16A3

    Institute of Scientific and Technical Information of China (English)

    Zuo Yunfei; Zhang Yaozheng; Zhang Hong

    1998-01-01

    Objective: To investigate the effect of inducing apoptosis of Elemene on ascites hepatoma cell line HcaF25/cL-16A3. By using immunhistochemistry and DNA electrophoresis, the mechanism of Elemene antitumor was studied. Results: The results showed that the Elemene can inhibit expression of bcl-2 in ascites hepatoma cell line Hca-F25/CL-16A3, and the Eiemene also can make DNA fragmentation in this cell line in vitro and in vivo.Conclusion: The data suggest that Elemene can inhibit the growth of tumor by inducing apoptosis.

  20. Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

    OpenAIRE

    Chao, Connie; Herr, Deron; Chun, Jerold; Xu, Yang

    2006-01-01

    Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and syner...

  1. Apoptosis of HL—60 cells induced by anti—tumor fraction from China cobra venom

    Institute of Scientific and Technical Information of China (English)

    LinZT; ZengYY

    2002-01-01

    An anti-tumor fraction from Chinese cobra venom can induce typical morphological changes and flows-cytometrical plots of apoptosis and DNA fragmentation in HL-60 cells in relation to the concentration of the fraction and its treatment time.When the dose is over 20mg·L-1,however,the fraction mainly caused the necrosis in HL-60 cells.By using RT-PCR method,it was found that the apoptosis was related to the inhibition of bcl-2 gene expression in HL-60 cells.

  2. Molecular signal transduction in vascular cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Apoptosis is a form of genetically programmed cell death, which plays a key role in regulation of cellularity in a variety of tissue and cell types including the cardiovascular tissues. Under both physiological and pathophysiological conditions, various biophysiological and biochemical factors, including mechanical forces, reactive oxygen and nitrogen species, cytokines, growth factors, oxidized lipoproteins, etc., may influence apoptosis of vascular cells. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. Abnormal expression and dysfunction of these apoptosis-regulating genes may attenuate or accelerate vascular cell apoptosis and affect the integrity and stability of atherosclerotic plaques. Clarification of the molecular mechanism that regulates apoptosis may help design a new strategy for treatment of atherosclerosis and its major complication, the acute vascular syndromes.

  3. Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique.

    Science.gov (United States)

    Ehemann, Volker; Sykora, Jaromir; Vera-Delgado, Jorge; Lange, Adelheid; Otto, Herwart F

    2003-05-01

    Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase. PMID:12706866

  4. Estrogen withdrawal from osteoblasts and osteocytes causes increased mineralization and apoptosis.

    Science.gov (United States)

    Brennan, M Á; Haugh, M G; O'Brien, F J; McNamara, L M

    2014-07-01

    Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

  5. Estrogen withdrawal from osteoblasts and osteocytes causes increased mineralization and apoptosis.

    Science.gov (United States)

    Brennan, M Á; Haugh, M G; O'Brien, F J; McNamara, L M

    2014-07-01

    Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17β-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis. PMID:24446157

  6. 15-lipoxygenase-1 mediates cyclooxygenase-2 inhibitor induced apoptosis in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    It has been found that expression of 15-lipoxygenasc-1(15-LOX-1) and its main product,13-C-hydroxyoctadecadienoic acid (13-S-HODE),are decreased in human colorectal and esophageal cancers and that nonsteroidal anti-inflammatory drugs(NSAIDs) can therspeutically induce 15-LOC-1 expression to trigger apoptosis in those cancer cells independently COX-2.We found that a specific COX-2 inhibitor SC-236 similarly induce apoptosis in gastric cancer cells,although the mechanisms of these effects remain to be defined.In the present study,we tested whether SC-236 induced apoptosis through up-regulation of 15-LOX-1 in gastric cancer cells.We found that,(a) SC-236 inhibited growth of gastric cancer cells mainly by apoptosis induced;(b) SC-236 induced 15-LOX-1 expression and increased endogenous 13-S-HODE product,instead of 15-S-HETE during apoptosis in gastric cancer cells without 15-LOX-1 expression before treatment by SC-236;(c)sc-236 didn't effect expression of COX-1,COX-2,5-LOX and 12-LOX;and (d)15-LOX-1 inhibition suppressed SC-236 induced apoptosis.These findings demonstrated that SC-236 induced apoptosis in gastric cancer cells via up-regulation of 25-LOX-1.They also support the concept that the loss of the proapopotic role of 15-LOX-1 in epithelial cancers is not limited to human colorectal and esophageal cancers.

  7. Enhancement of therapeutic drug and DNA delivery into cells by electroporation* Enhancement of therapeutic drug and DNA delivery into cells by electroporation

    Science.gov (United States)

    Rabussay, Dietmar; Dev, Nagendu B.; Fewell, Jason; Smith, Louis C.; Widera, Georg; Zhang, Lei

    2003-02-01

    The effectiveness of potentially powerful therapeutics, including DNA, is often limited by their inability to permeate the cell membrane efficiently. Electroporation (EP) also referred to as `electropermeabilization' of the outer cell membrane renders this barrier temporarily permeable by inducing `pores' across the lipid bilayer. For in vivo EP, the drug or DNA is delivered into the interstitial space of the target tissue by conventional means, followed by local EP. EP pulses of micro- to millisecond duration and field strengths of 100-1500 V cm-1 generally enhance the delivery of certain chemotherapeutic drugs by three to four orders of magnitude and intracellular delivery of DNA several hundred-fold. We have used EP in clinical studies for human cancer therapy and in animals for gene therapy and DNA vaccination. Late stage squamous cell carcinomas of the head and neck were treated with intratumoural injection of bleomycin and subsequent EP. Of the 69 tumours treated, 25% disappeared completely and another 32% were reduced in volume by more than half. Residence time of bleomycin in electroporated tumours was significantly greater than in non-electroporated lesions. Histological findings and gene expression patterns after bleomycin-EP treatment indicated rapid apoptosis of the majority of tumour cells. In animals, we demonstrated the usefulness of EP for enhanced DNA delivery by achieving normalization of blood clotting times in haemophilic dogs, and by substantially increasing transgene expression in smooth muscle cells of arterial walls using a novel porous balloon EP catheter. Finally, we have found in animal experiments that the immune response to DNA vaccines can be dramatically enhanced and accelerated by EP and co-injection of micron-sized particles. We conclude that EP represents an effective, economical and safe approach to enhance the intracellular delivery, and thus potency, of important drugs and genes for therapeutic purposes. The safety and pharmaco

  8. Apoptosis in normal oral tissues and odontogenesis

    OpenAIRE

    Ruchita Bali; Akhilesh Chandra; Renuka Verma

    2013-01-01

    Programmed cell death or apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development, and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders, and many types of cancers. The process of apoptosis is generally ch...

  9. Apoptosis: A Review of Programmed Cell Death

    OpenAIRE

    Elmore, Susan

    2007-01-01

    The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions incl...

  10. On limit and limit setting.

    Science.gov (United States)

    Gorney, J E

    1994-01-01

    This article investigates the role of limit and limit setting within the psychoanalytic situation. Limit is understood to be a boundary between self and others, established as an interactional dimension of experience. Disorders of limit are here understood within the context of Winnicott's conception of the "anti-social tendency." Limit setting is proposed as a necessary and authentic response to the patient's acting out via holding and empathic responsiveness, viewed here as a form of boundary delineation. It is proposed that the patient attempts to repair his or her boundary problem through a seeking of secure limits within the analyst. The setting of secure and appropriate limits must arise from a working through of the analyst's own countertransference response to the patient. It is critical that this response be evoked by, and arise from, the immediate therapeutic interaction so that the patient can experience limit setting as simultaneously personal and authentic. PMID:7972580

  11. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.

  12. The cellular decision between apoptosis and autophagy

    Institute of Scientific and Technical Information of China (English)

    Yong-Jun Fan; Wei-Xing Zong

    2013-01-01

    Apoptosis and autophagy are important molecular processes that maintain organismal and cellular homeostasis,respectively.While apoptosis fulfills its role through dismantling damaged or unwanted cells,autophagy maintains cellular homeostasis through recycling selective intracellular organelles and molecules.Yet in some conditions,autophagy can lead to cell death.Apoptosis and autophagy can be stimulated by the same stresses.Emerging evidence indicates an interplay between the core proteins in both pathways,which underlies the molecular mechanism of the crosstalk between apoptosis and autophagy.This review summarizes recent literature on molecules that regulate both the apoptotic and autophagic processes.

  13. Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls.

    Science.gov (United States)

    Mnich, Christian D; Hoek, Keith S; Virkki, Leila V; Farkas, Arpad; Dudli, Christa; Laine, Elisabeth; Urosevic, Mirjana; Dummer, Reinhard

    2009-01-01

    Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV-induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low-dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm(2) of UVB light skin patches which were pretreated with either OM24-containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un-irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24 treatment did not significantly affect UV-induced erythema and thymidine dimer formation, OM24 treatment significantly reduced UV-induced p53 expression in keratinocytes. We also found that OM24 treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL-positive cells). Carefully controlled DNA microarray analyses showed that OM24 treatment does not induce off-target changes in gene expression, reducing the likelihood of unwanted side-effects. Topical GTE (OM24) reduces UVB-mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.

  14. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  15. Selenium, apoptosis, and colorectal adenomas.

    OpenAIRE

    Connelly-Frost, Alexandra; Poole, Charles; Satia, Jessie A.; Kupper, Lawrence L.; Millikan, Robert C.; Sandler, Robert S.

    2006-01-01

    Dietary modulation of carcinogenesis-related pathwaysDietary item or component studied:seleniumPathways studied:apoptosisStudy type (in vitro, animals, humans): humansStudy design (if human):cross-sectional studyStudy size (if human):803 participantsTissue/biological material/sample size: serum, 2 colon biopsiesMode of exposure (if in vivo) (acute, chronic, root of exposure):dietary & lifestyle questionnairesImpact on pathway (including dose-response):for 50-120 μgr/l selenium Pe~0.5-0.3For ...

  16. Pulse mode of laser photodynamic treatment induced cell apoptosis.

    Science.gov (United States)

    Klimenko, Vladimir V; Knyazev, Nickolay A; Moiseenko, Fedor V; Rusanov, Anatoliy A; Bogdanov, Alexey A; Dubina, Michael V

    2016-03-01

    One of the factors limiting photodynamic therapy (PDT) is hypoxia in tumor cells during photodynamic action. PDT with pulse mode irradiation and appropriate irradiation parameters could be more effective in the singlet oxygen generation and tissue re-oxygenation than continuous wave (CW) mode. We theoretically demonstrate differences between the cumulative singlet oxygen concentration in PDT using pulse mode and CW mode of laser irradiation. In vitro experimental results show that photodynamic treatment with pulse mode irradiation has similar cytotoxicity to CW mode and induces mainly cell apoptosis, whereas CW mode induces necrotic cell death. We assume that the cumulative singlet oxygen concentration and the temporal distribution of singlet oxygen are important in photodynamic cytotoxicity and apoptosis initiation. We expect our research may improve irradiation protocols and photodynamic therapy efficiency. PMID:26790610

  17. Quench limits

    International Nuclear Information System (INIS)

    With thirteen beam induced quenches and numerous Machine Development tests, the current knowledge of LHC magnets quench limits still contains a lot of unknowns. Various approaches to determine the quench limits are reviewed and results of the tests are presented. Attempt to reconstruct a coherent picture emerging from these results is taken. The available methods of computation of the quench levels are presented together with dedicated particle shower simulations which are necessary to understand the tests. The future experiments, needed to reach better understanding of quench limits as well as limits for the machine operation are investigated. The possible strategies to set BLM (Beam Loss Monitor) thresholds are discussed. (author)

  18. Effects of amifostine on radiation-induced apoptosis in mouse ovary

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Lee, Kyoung Hee; Yoon Yong Dal [Hanyang Univ., Seoul (Korea, Republic of)

    2002-07-01

    The present study was designed to assess the radioprotective effects of amifostine on ovarian follicles. Three week-old female mice with or without pretreatment of amifostine were irradiated with 6.42 Gy of {gamma} -ray. Ovaries were collected 0 and 6h after irradiation. DNA fragmentation pattern and expression of genes and activity of proteins related with apoptosis were investigated by means of RT-PCR and Western blot. Proliferation of granulosa cells was reduced and incidence rate of follicular atresia was increased in ovarian follicles in {gamma} -ray irradiated mice compared to those in control or amifostine-treated group. DNA fragmentation was increased in time-dependent manner in granulosa cells of all irradiated groups. However, no difference between amifostine pre-treated group and irradiated groups was found and the expression of p53 as tumor suppressor gene and Bax as one of pro-apoptotic Bcl-2 family was increased in irradiated mice ovaries. PARP as DNA damage sensor was cleaved and its upstream regulator, Caspase-3 was activated. Fas (CD95), one of membrane bound receptor which initiated apoptosis pathway was expressed by {gamma} -ray. Pretreatment of amifostine before irradiation, however, inhibited the expression of p53 and Bax, the cleavage of PARP and the activation of Caspase-3. In conclusion, amifostine has an inhibitory effect on gonadal cell apoptosis induced by {gamma} -ray irradiation, through lowering the expression of apoptotic signaling peptide and the activation of DNA repair enzymes.

  19. SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis.

    Science.gov (United States)

    Ozturk, Sait; Papageorgis, Panagiotis; Wong, Chen Khuan; Lambert, Arthur W; Abdolmaleky, Hamid M; Thiagalingam, Arunthathi; Cohen, Herbert T; Thiagalingam, Sam

    2016-01-19

    Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis-free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications. PMID:26739564

  20. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup;

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  1. DNA vaccines against influenza.

    Science.gov (United States)

    Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka

    2014-01-01

    Genetic vaccine technology has been considerably developed within the last two decades. This cost effective and promising strategy can be applied for therapy of cancers and for curing allergy, chronic and infectious diseases, such as a seasonal and pandemic influenza. Despite numerous advantages, several limitations of this technology reduce its performance and can retard its commercial exploitation in humans and its veterinary applications. Inefficient delivery of the DNA vaccine into cells of immunized individuals results in low intracellular supply of suitable expression cassettes encoding an antigen, in its low expression level and, in turn, in reduced immune responses against the antigen. Improvement of DNA delivery into the host cells might significantly increase effectiveness of the DNA vaccine. A vast array of innovative methods and various experimental strategies have been applied in order to enhance the effectiveness of DNA vaccines. They include various strategies improving DNA delivery as well as expression and immunogenic potential of the proteins encoded by the DNA vaccines. Researchers focusing on DNA vaccines against influenza have applied many of these strategies. Recent examples of the most successful modern approaches are discussed in this review.

  2. Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells.

    Science.gov (United States)

    Kwon, H J; Hong, Y K; Kim, K H; Han, C H; Cho, S H; Choi, J S; Kim, Byung-Woo

    2006-04-21

    Ptercarpus santalinus (Fabaceae) has been used as a folk remedy in Korea, and it has been shown to exhibit antiinflammations, antiulcers and anticancer effects. In this study, therefore, we report the cytotoxic activity and the mechanism of cell death exhibited by the methanol extract of Ptercarpus santalinus (MEPS) against human cervical adenocarcinoma cell line, HeLa. Treatment of HeLa cells with various concentrations of MEPS resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as determined by cell viability, chromatin condensation, DNA fragmentation and sub-G1 phase accumulation. In Western blot analysis, apoptosis in the HeLa cells was associated with the release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, -9 and proteolytic cleavage of PARP. These results suggest that MEPS exhibits antiproliferative effect on HeLa cells via apoptosis, and it may be a potential candidate in field of anticancer drug discovery. PMID:16326057

  3. Effect of Uncaria tomentosa Extract on Apoptosis Triggered by Oxaliplatin Exposure on HT29 Cells

    Directory of Open Access Journals (Sweden)

    Liliane Z. de Oliveira

    2014-01-01

    Full Text Available Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy. Uncaria tomentosa extract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whether Uncaria tomentosa extract modulates apoptosis induced by chemotherapy exposure. Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells were grown in the presence of oxaliplatin and/or Uncaria tomentosa extract. Results. The hydroalcoholic extract of Uncaria tomentosa enhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis. Conclusion. Uncaria tomentosa extract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended.

  4. CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53

    DEFF Research Database (Denmark)

    Hollmann, Annette C; Gong, Qiaoke; Owens, Trevor

    2002-01-01

    Crosslinking CD40 induces normal B-cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. As p53 is required for many apoptotic pathways, we analyzed the effects of CD40 ligation and their correlation with p53 function in four murine B-lymphoma lines. A20...... of detectable p21 mRNA in A20 and M12 cells. P21 mRNA was increased to detectable levels in M12 cells upon CD40 ligation; however, blocking this effect with the p53 inhibitor pifithrin had no effect on CD40-mediated apoptosis. Sequencing showed that p53 in A20 and M12 cells contained point mutations leading...... to amino acid substitutions in DNA binding regions, but was unmutated in WEHI231 and WEHI 279. These results suggest that CD40-mediated apoptosis can occur in the absence of functional p53....

  5. Effect of Uncaria tomentosa Extract on Apoptosis Triggered by Oxaliplatin Exposure on HT29 Cells.

    Science.gov (United States)

    de Oliveira, Liliane Z; Farias, Iria Luiza G; Rigo, Melânia L; Glanzner, Werner G; Gonçalves, Paulo Bayard D; Cadoná, Francine C; Cruz, Ivana B; Farias, Júlia G; Duarte, Marta M M F; Franco, Luzia; Bertol, Gustavo; Colpo, Elisangela; Brites, Patricia C; Rocha, João Batista T; Leal, Daniela B R

    2014-01-01

    Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy. Uncaria tomentosa extract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whether Uncaria tomentosa extract modulates apoptosis induced by chemotherapy exposure. Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells) were grown in the presence of oxaliplatin and/or Uncaria tomentosa extract. Results. The hydroalcoholic extract of Uncaria tomentosa enhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis. Conclusion. Uncaria tomentosa extract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended.

  6. INDUCTION OF APOPTOSIS IN HUMAN GASTRIC CARCINOMA CELL LINE MGC-803 BY MONOCLONAL ANTIBODY PD4

    Institute of Scientific and Technical Information of China (English)

    Xiao Hai; Dong Zhiwei; Shou Chengchao

    1998-01-01

    Objective: To study the effect of McAb PD4 on the cell cycle and on cell injury in the gastric carcinoma cell line,MGC-803. Methods: The effects of McAb PD4 on cell proliferation cycle and cell injury of MGC-803 cells were examined by flow cytometry analysis, DNA electrophoresis and terminal deoxynucle otidyl transferase assay. Fas antigen was investigated by ELISA.Results: McAb PD4 inhibited tumor-growth of MGC-803cells in nude mice by inducing apoptosis. Conclusion:P40 is a tumor-associated antigen distinct from the Fas antigen. Molecular cloning of P40 will define the pathway and mechanism of apoptosis induced by McAb PD4.Induction of apoptosis by McAb PD4 may be a useful therapeutic approach in treating cancer.

  7. Pinpointing differences in cisplatin-induced apoptosis in adherent and non-adherent cancer cells

    DEFF Research Database (Denmark)

    Tastesen, Hanne Sørup; Holm, Jacob Bak; Poulsen, Kristian Arild;

    2010-01-01

    ascites tumour cells (ELA). Loss of KCl and cell shrinkage are hallmarks in apoptosis and has been shown in EATC. However, we find no reduction in cell volume and only a minor loss of K(+) which is accompanied by net uptake of Na(+) following 18 hours cisplatin exposure in ELA. Glutathione and taurine...... have previously been demonstrated to protect cells from apoptosis. We find, however, that increase or decrease in the cellular content of glutathione and taurine has no effect on cisplatin-induced cell death in EATC and ELA. Nevertheless, knock-down of the taurine transporter TauT leads...... to a significant increase in apoptosis in ELA following cisplatin exposure. We find that cytosolic accumulation of cisplatin is similar in EATC and ELA. However, the nuclear accumulation and DNA-binding of cisplatin is significant lower in ELA compared to EATC. We suggest three putative reasons for the observed...

  8. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

    Directory of Open Access Journals (Sweden)

    Tzuu-Yuan Huang

    2012-01-01

    Full Text Available Demethoxycurcumin (DMC; a curcumin-related demethoxy compound has been recently shown to display antioxidant and antitumor activities. It has also produced a potent chemopreventive action against cancer. In the present study, the antiproliferation (using the MTT assay, DMC was found to have cytotoxic activities against GBM 8401 cell with IC50 values at 22.71 μM and induced apoptosis effects of DMC have been investigated in human brain malignant glioma GBM 8401 cells. We have studied the mitochondrial membrane potential (MMP, DNA fragmentation, caspase activation, and NF-κB transcriptional factor activity. By these approaches, our results indicated that DMC has produced an inhibition of cell proliferation as well as the activation of apoptosis in GBM 8401 cells. Both effects were observed to increase in proportion with the dosage of DMC treatment, and the apoptosis was induced by DMC in human brain malignant glioma GBM 8401 cells via mitochondria- and caspase-dependent pathways.

  9. Effect of Uncaria tomentosa Extract on Apoptosis Triggered by Oxaliplatin Exposure on HT29 Cells.

    Science.gov (United States)

    de Oliveira, Liliane Z; Farias, Iria Luiza G; Rigo, Melânia L; Glanzner, Werner G; Gonçalves, Paulo Bayard D; Cadoná, Francine C; Cruz, Ivana B; Farias, Júlia G; Duarte, Marta M M F; Franco, Luzia; Bertol, Gustavo; Colpo, Elisangela; Brites, Patricia C; Rocha, João Batista T; Leal, Daniela B R

    2014-01-01

    Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy. Uncaria tomentosa extract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whether Uncaria tomentosa extract modulates apoptosis induced by chemotherapy exposure. Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells) were grown in the presence of oxaliplatin and/or Uncaria tomentosa extract. Results. The hydroalcoholic extract of Uncaria tomentosa enhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis. Conclusion. Uncaria tomentosa extract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended. PMID:25505920

  10. Dose limits

    International Nuclear Information System (INIS)

    The dose limit is defined to be the level of harmfulness which must not be exceeded, so that an activity can be exercised in a regular manner without running a risk unacceptable to man and the society. The paper examines the effects of radiation categorised into stochastic and non-stochastic. Dose limits for workers and the public are discussed

  11. Limited Neutrality

    DEFF Research Database (Denmark)

    Nielsen, Morten Ebbe Juul

    2006-01-01

    Article Concerning the prospect of a kind of limited neutrality in place of the standard liberal egalitarian "neutrality of justification."......Article Concerning the prospect of a kind of limited neutrality in place of the standard liberal egalitarian "neutrality of justification."...

  12. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

    Science.gov (United States)

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-04-02

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

  13. 78 FR 22269 - International Conference on Harmonisation; Draft Guidance on M7 Assessment and Control of DNA...

    Science.gov (United States)

    2013-04-15

    ... M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To Limit... ``M7 Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit... and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential...

  14. Crosstalk between apoptosis and inflammation in atherosclerosis

    NARCIS (Netherlands)

    Westra, Marijke Marianne

    2010-01-01

    In this thesis the role of several apoptosis regulating proteins in the development of atherosclerosis and atherosclerotic plaque stability is investigated. Apoptosis of different cell types in atherosclerotic plaques, such as macrophages and smooth muscle cells may inhibit or promote plaque develop

  15. Apoptosis in odontogenesis - a brief review

    OpenAIRE

    Nair, Bindu J

    2010-01-01

    Tooth formation is an excellent example of epithelial mesenchymal interaction As the developingtooth passes through the various morphologic stages it is observed that apoptosis occurs selectively incertain locations. Here a review is done to throw light into the role of apoptosis and the factorsgoverning the same during odontogenesis .

  16. Apoptosis in odontogenesis - a brief review

    Directory of Open Access Journals (Sweden)

    Bindu J. Nair

    2010-01-01

    Full Text Available Tooth formation is an excellent example of epithelial mesenchymal interaction As the developingtooth passes through the various morphologic stages it is observed that apoptosis occurs selectively incertain locations. Here a review is done to throw light into the role of apoptosis and the factorsgoverning the same during odontogenesis .

  17. Induction of apoptosis by homoharringtonine in G1 phase human chronic myeloid leukemic cells

    Institute of Scientific and Technical Information of China (English)

    MAI Wen-yuan; LIN Mao-fang

    2005-01-01

    Background Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. For the sake of illustrating the mechanisms of HHT in the treatment of leukemia, we assessed the effect of HHT on the apoptosis of human chronic myeloid leukemic cell line K562.Methods The apoptosis of K562 cells induced by HHT was analyzed by transmission electron microscopy, agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling.Results Characteristic apoptosis-related features emerged in K562 cells after exposed to HHT at a concentration 0.05-100 μg/ml. Transmission electron microscopy of HHT treated K562 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation. Typical DNA ladder in agarose gel electrophoresis was observed in the cells exposed to HHT. The cell cycle analysis measured by flow cytometry showed G1 phase cells decreased with the increase of S phase cells while apoptosis was induced by HHT in K562 cells. The percentage of apoptotic cells in K562 cells treated with 50 μg/ml of HHT decreased significantly when pretreated with 1 μg/ml of cycloheximide, 0.05 μg/ml of Actinomycin D respectively.Conclusions HHT has apoptotic effects on K562 cells. The HHT induced apoptosis mainly of the cells in G1 phase and this process required RNA transcription and protein synthesis.

  18. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shi-Wei [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Wu, Chun-Ying [Division of Gastroenterology and Hepatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, Yen-Ting [Department of Medical Research and Education, Cheng Hsin General Hospital, Taipei, Taiwan (China); Kao, Jun-Kai [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Pediatrics, Children' s Hospital, Changhua Christian Hospital, Changhua, Taiwan (China); Lin, Chi-Chen; Chang, Chia-Che; Mu, Szu-Wei; Chen, Yu-Yu [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chiu, Husan-Wen [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chang, Chuan-Hsun [Department of Surgical Oncology, Cheng Hsin General Hospital, Taipei, Taiwan (China); Department of Nutrition Therapy, Cheng Hsin General Hospital, Taipei, Taiwan (China); School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan (China); Liang, Shu-Mei [Institute of Biotechnology, National Cheng-Kung University, Tainan, Taiwan (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan (China); Chen, Yi-Ju [Department of Dermatology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Huang, Jau-Ling [Department of Bioscience Technology, Chang Jung Christian University, Tainan, Taiwan (China); Shieh, Jeng-Jer, E-mail: shiehjj@vghtc.gov.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan (China)

    2013-02-15

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status.

  19. Phenotype-dependent apoptosis signalling in mesothelioma cells after selenite exposure

    Directory of Open Access Journals (Sweden)

    Rundlöf Anna-Klara

    2009-06-01

    Full Text Available Abstract Background Selenite is a promising anticancer agent which has been shown to induce apoptosis in malignant mesothelioma cells in a phenotype-dependent manner, where cells of the chemoresistant sarcomatoid phenotype are more sensitive. Methods In this paper, we investigate the apoptosis signalling mechanisms in sarcomatoid and epithelioid mesothelioma cells after selenite treatment. Apoptosis was measured with the Annexin-PI assay. The mitochondrial membrane potential, the expression of Bax, Bcl-XL, and the activation of caspase-3 were assayed with flow cytometry and a cytokeratin 18 cleavage assay. Signalling through JNK, p38, p53, and cathepsins B, D, and E was investigated with chemical inhibitors. Furthermore, the expression, nuclear translocation and DNA-binding activity of p53 was investigated using ICC, EMSA and the monitoring of p21 expression as a downstream event. Levels of thioredoxin (Trx were measured by ELISA. Results In both cell lines, 10 μM selenite caused apoptosis and a marked loss of mitochondrial membrane potential. Bax was up-regulated only in the sarcomatoid cell line, while the epithelioid cell line down-regulated Bcl-XL and showed greater caspase-3 activation. Nuclear translocation of p53 was seen in both cell lines, but very little p21 expression was induced. Chemical inhibition of p53 did not protect the cells from apoptosis. p53 lost its DNA binding ability after selenite treatment and was enriched in an inactive form. Levels of thioredoxin decreased after selenite treatment. Chemical inhibition of MAP kinases and cathepsins showed that p38 and cathepsin B had some mediatory effect while JNK had an anti-apoptotic role. Conclusion We delineate pathways of apoptosis signalling in response to selenite, showing differences between epithelioid and sarcomatoid mesothelioma cells. These differences may partly explain why sarcomatoid cells are more sensitive to selenite.

  20. p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells

    International Nuclear Information System (INIS)

    Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status. - Highlights: ► Compound C caused more apoptosis in p53 wildtype than p53-mutant skin cancer cells. ► Compound C can upregulate p53 expression and induce p53 activation. ► Compound C induced p53 effects were dependent on ROS induced DNA damage pathway. ► p53-knockdown attenuated compound C-induced apoptosis but not autophagy. ► Compound C-induced apoptosis in skin cancer cells was dependent on p53 status

  1. Nitrofen suppresses cell proliferation and promotes mitochondriamediated apoptosis in type Ⅱ pneumocytes

    Institute of Scientific and Technical Information of China (English)

    Qiang-song TONG; Li-duan ZHENG; Shao-tao TANG; Guo-song JIANG; Qing-lan R UAN; Fu-qing ZENC; Ji-hua DONG

    2007-01-01

    Aim: To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia. Methods: After administration of nitrofen to cultured type H A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony forma-tion assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot. Results: Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompa-nied by downregulation of PCNA. As a result, the DNA synthesis of nitrofen-treated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover,nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Aia-Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-XL, but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 μmol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells. Conclusion: Nitrofen suppresses the proliferation of cultured type Ⅱ pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involv-ing the activation of p38-MAPK.

  2. Effects of Selenium and Zinc on Renal Oxidative Stress and Apoptosis Induced by Fluoride in Rats