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Staurosporine-Induced Cell Death in Trypanosoma brucei and the Role of Endonuclease G during Apoptosis

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DOI: 10.4236/ojapo.2014.32003    3,860 Downloads   7,017 Views   Citations

ABSTRACT

Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Barth, T. , Bruges, G. , Meiwes, A. , Mogk, S. , Mudogo, C. and Duszenko, M. (2014) Staurosporine-Induced Cell Death in Trypanosoma brucei and the Role of Endonuclease G during Apoptosis. Open Journal of Apoptosis, 3, 16-31. doi: 10.4236/ojapo.2014.32003.

References

[1] Debrabant, A., Lee, N., Bertholet, S., Duncan, R. and Nakhasi, H.L. (2003) Programmed Cell Death in Trypanosomatids and Other Unicellular Organisms. International Journal for Parasitology, 33, 257-267.
http://dx.doi.org/10.1016/S0020-7519(03)00008-0
[2] Figarella, K., Rawer, M., Uzcategui, N.L., Kubata, B.K., Lauber, K., et al. (2005) Prostaglandin D2 Induces Programmed Cell Death in Trypanosoma brucei Bloodstream Form. Cell Death & Differentiation, 12, 335-346.
http://dx.doi.org/10.1038/sj.cdd.4401564
[3] Figarella, K., Uzcategui, N.L., Beck, A., Schoenfeld, C., Kubata, B.K., et al. (2006) Prostaglandin-Induced Programmed Cell Death in Trypanosoma brucei Involves Oxidative Stress. Cell Death & Differentiation, 13, 1802-1814.
http://dx.doi.org/10.1038/sj.cdd.4401862
[4] Lee, N., Bertholet, S., Debrabant, A., Muller, J., Duncan, R., et al. (2002) Programmed Cell Death in the Unicellular Protozoan Parasite Leishmania. Cell Death & Differentiation, 9, 53-64.
http://dx.doi.org/10.1038/sj.cdd.4400952
[5] Desjeux, P. (2004) Leishmaniasis: Current Situation and New Perspectives. Comparative Immunology Microbiology and Infectious Diseases, 27, 305-318.
http://dx.doi.org/10.1016/j.cimid.2004.03.004
[6] Kubata, B.K., Duszenko, M., Kabututu, Z., Rawer, M., Szallies, A., et al. (2000) Identification of a Novel Prostaglandin f(2alpha) Synthase in Trypanosoma brucei. Journal of Experimental Medicine, 192, 1327-1338.
http://dx.doi.org/10.1084/jem.192.9.1327
[7] Madeo, F., Frohlich, E., Ligr, M., Grey, M., Sigrist, S.J., et al. (1999) Oxygen Stress: A Regulator of Apoptosis in Yeast. Journal of Cell Biology, 145, 757-767.
http://dx.doi.org/10.1083/jcb.145.4.757
[8] Frohlich, K.U. and Madeo, F. (2000) Apoptosis in Yeast—A Monocellular Organism Exhibits Altruistic Behaviour. FEBS Letters, 473, 6-9.
http://dx.doi.org/10.1016/S0014-5793(00)01474-5
[9] Christensen, S.T., Kemp, K., Quie, H. and Rasmussen, L. (1996) Cell Death, Survival and Proliferation in Tetrahymena thermophila. Effects of Insulin, Sodium Nitroprusside, 8-Bromo Cyclic GMP, NG-Methyl-L-arginine and Methylene Blue. Cell Biology International, 20, 653-666.
http://dx.doi.org/10.1006/cbir.1996.0087
[10] Arnoult, D., Tatischeff, I., Estaquier, J., Girard, M., Sureau, F., et al. (2001) On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-Inducing Factor during Dictyostelium discoideum Cell Death. Molecular Biology of the Cell, 12, 3016-3030.
http://dx.doi.org/10.1091/mbc.12.10.3016
[11] Engelberg-Kulka, H., Amitai, S., Kolodkin-Gal, I. and Hazan, R. (2006) Bacterial Programmed Cell Death and Multi-cellular Behavior in Bacteria. PLoS Genet, 2, Article ID: e135.
http://dx.doi.org/10.1371/journal.pgen.0020135
[12] Bayles, K.W. (2003) Are the Molecular Strategies That Control Apoptosis Conserved in Bacteria? Trends in Microbiology, 11, 306-311.
http://dx.doi.org/10.1016/S0966-842X(03)00144-6
[13] Welburn, S.C., Macleod, E., Figarella, K. and Duzensko, M. (2006) Programmed Cell Death in African Trypanosomes. Parasitology, 132, S7-S18.
http://dx.doi.org/10.1017/S0031182006000825
[14] Li, L.Y., Luo, X. and Wang, X. (2001) Endonuclease G Is an Apoptotic DNase When Released from Mitochondria. Nature, 412, 95-99.
http://dx.doi.org/10.1038/35083620
[15] Ohsato, T., Ishihara, N., Muta, T., Umeda, S., Ikeda, S., et al. (2002) Mammalian Mitochondrial Endonuclease G. Digestion of R-Loops and Localization in Intermembrane Space. European Journal of Biochemistry, 269, 5765-5770.
http://dx.doi.org/10.1046/j.1432-1033.2002.03238.x
[16] BoseDasgupta, S., Das, B.B., Sengupta, S., Ganguly, A., Roy, A., Dey, S., Tripathi, G., Dinda, B. and Majumder, H.K. (2008) The Caspase-Independent Algorithm of Programmed Cell Death in Leishmania Induced by Baicalein: The Role of LdEndoG, LdFEN-1 and LdTatD as a DNA “Degradesome”. Cell Death and Differentiation, 15, 1629-1640.
http://dx.doi.org/10.1038/cdd.2008.85
[17] Rico, E., Alzate, J.F., Arias, A.A., Moreno, D., Clos, J., Gagod, F., Morenoe, I., Domíngueze, M. and Jiménez-Ruiz, A. (2009) Leishmania infantum Expresses a Mitochondrial Nuclease Homologous to EndoG that Migrates to the Nucleus in Response to an Apoptotic Stimulus. Molecular and Biochemical Parasitology, 163, 28-38.
http://dx.doi.org/10.1016/j.molbiopara.2008.09.007
[18] Cote, J. and Ruiz-Carrillo, A. (1993) Primers for Mitochondrial DNA Replication Generated by Endonuclease G. Sci- ence, 261, 765-769.
http://dx.doi.org/10.1126/science.7688144
[19] Low, R.L. (2003) Mitochondrial Endonuclease G Function in Apoptosis and mtDNA Metabolism: A Historical Perspective. Mitochondrion, 2, 225-236.
http://dx.doi.org/10.1016/S1567-7249(02)00104-6
[20] Schafer, P., Scholz, S.R., Gimadutdinow, O., Cymerman, I.A., Bujnicki, J.M., Ruiz-Carrillo, A., Pingoud, A. and Meiss, G. (2004) Structural and Functional Characterization of Mitochondrial EndoG, a Sugar Non-Specific Nuclease Which Plays an Important Role during Apoptosis. Journal of Molecular Biology, 338, 217-228.
http://dx.doi.org/10.1016/j.jmb.2004.02.069
[21] Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X.D. and Xue, D. (2001) Mitochondrial Endonuclease G Is Important for Apoptosis in C. elegans. Nature, 412, 90-94.
http://dx.doi.org/10.1038/35083608
[22] Wolburg, H., Mogk, S., Acker, S., Frey, C., Meinert, M., Sch?nfeld, C., Lazarus, M., Urade, Y., Kubata, B.K. and Duszenko, M. (2012) Late Stage Infection in Sleeping Sickness. PLoS ONE, 7, Article ID: e34304.
http://dx.doi.org/10.1371/journal.pone.0034304
[23] Arnoult, D., Akarid, K., Grodet, A., Petit, P.X., Estaquier, J. and Ameisen, J.C. (2002) On the Evolution of Programmed Cell Death: Apoptosis of the Unicellular Eukaryote Leishmana Major Involves Cysteine Proteinase Activation and Mitochondrion Permeabilization. Cell Death and Differentiation, 9, 65-81.
http://dx.doi.org/10.1038/sj.cdd.4400951
[24] Bruno, S., Ardelt, B., Skierski, J.S., Traganos, F. and Darzynkiewicz, Z. (1992) Different Effects of Staurosporine, an Inhibitor of Protein Kinases, on the Cell Cycle and Chromatin Structure of Normal and Leukemic Lymphocytes. Cancer Research, 52, 470-473.
[25] Belmokhtar, C.A., Torriglia, A., Counis, M.F., Courtois, Y., Jacquemin-Sablon, A. and Ségal-Bendirdjian, E. (2000) Nuclear Translocation of a Leukocyte Elastase Inhibitor/Elastase Complex during Staurosporine-Induced Apoptosis: Role in the Generation of Nuclear L-DNase II Activity. Experimental Cell Research, 254, 99-109.
http://dx.doi.org/10.1006/excr.1999.4737
[26] Belmokhtar, C.A., Hillion, J. and Segal-Bendirdjian, E. (2001) Staurosporine Induces Apoptosis through both Caspase-Dependent and Caspase-Independent Mechanisms. Oncogene, 20, 3354-3362.
http://dx.doi.org/10.1038/sj.onc.1204436
[27] Johansson, A.C., Steen, H., Ollinger, K. and Roberg, K. (2003) Cathepsin D Mediates Cytochrome c Release and Caspase Activation in Human Fibroblast Apoptosis Induced by Staurosporine. Cell Death and Differentiation, 10, 1253-1259. http://dx.doi.org/10.1038/sj.cdd.4401290
[28] Pimentel-Elardo, S.M., Kozytska, S., Bugni, T.S., Ireland, C.M., Moll, H. and Hentschel, U. (2010) Anti-Parasitic Compounds from Streptomyces sp. Strains Isolated from Mediterranean Sponges. Marine Drugs, 8, 373-380.
http://dx.doi.org/10.3390/md8020373
[29] Gale Jr., M., Carter, V. and Parsons, M. (1994) Cell Cycle-Specific Induction of an 89 kDa Serine/Threonine Protein Kinase Activity in Trypanosoma Brucei. Journal of Cell Science, 107, 1825-1832.
[30] Malaquias, A.T. and Oliveira, M.M. (1999) Phospholipid Signalling Pathways in Trypanosoma cruzi Growth Control. Acta Tropica, 73, 93-108.
http://dx.doi.org/10.1016/S0001-706X(99)00016-9
[31] Duszenko, M., Figarella, K., Macleod, E.T. and Welburn, S.C. (2006) Death of a Trypanosome: A Selfish Altruism. Trends in Parasitology, 22, 536-542.
http://dx.doi.org/10.1016/j.pt.2006.08.010
[32] Debrabant, A. and Nakhasi, H. (2003) Programmed Cell Death in Trypanosomatids: Is It an Altruistic Mechanism for Survival of the Fittest? Kinetoplastid Biology and Disease, 2, 7.
http://dx.doi.org/10.1186/1475-9292-2-7
[33] Wirtz, E., Leal, S., Ochatt, C. and Cross, G.A. (1999) A Tightly Regulated Inducible Expression System for Conditional Gene Knock-Outs and Dominant-Negative Genetics in Trypanosoma brucei. Molecular and Biochemical Parasitology, 99, 89-101.
http://dx.doi.org/10.1016/S0166-6851(99)00002-X
[34] Glauert, A.M., Butterworth, A.E., Sturrock, R.F. and Houba, V. (1978) The Mechansim of Antibody-Dependent, Eo- sinophil-Mediated Damage to Schistosomula of Schistosoma Mansoni in Vitro: A Study by Phase-Contrast and Electron Microscopy. Journal of Cell Science, 34, 173-192.
[35] Widlak, P., Li, L.Y., Wang, X. and Garrard, W.T. (2001) Action of Recombinant Human Apoptotic Endonuclease G on Naked DNA and Chromatin Substrates: Cooperation with Exonuclease and DNase I. Journal of Biological Chemistry, 276, 48404-48409.
[36] Gannavaram, S., Vedvyas, C. and Debrabant, A. (2008) Conservation of the Pro-Apoptotic Nuclease Activity of Endonuclease G in Unicellular Trypanosomatid Parasites. Journal of Cell Science, 121, 99-109.
http://dx.doi.org/10.1242/jcs.014050
[37] Steensma, D.P., Timm, M. and Witzig, T.E. (2003) Flow Cytometric Methods for Detection and Quantification of Apoptosis. Methods in Molecular Medicine, 85, 323-332.
[38] Elmore, S. (2007) Apoptosis: A Review of Programmed Cell Death. Toxicologic Pathology, 35, 495-516.
http://dx.doi.org/10.1080/01926230701320337
[39] Riccardi, C. and Nicoletti, I. (2006) Analysis of Apoptosis by Propidium Iodide Staining and Flow Cytometry. Nature Protocols, 1, 1458-1461.
http://dx.doi.org/10.1038/nprot.2006.238
[40] Jimenez-Ruiz, A., Alzate, J.F., Macleod, E.T., Luder, C.G., Fasel, N. and Hurd, H. (2010) Apoptotic Markers in Protozoan Parasites. Parasites & Vectors, 3, 104.
[41] Kroemer, G. and Martin, S.J. (2005) Caspase-Independent Cell Death. Nature Medicine, 11, 725-730.
http://dx.doi.org/10.1038/nm1263
[42] Piacenza, L., Irigoin, F., Alvarez, M.N., Peluffo, G., Taylor, M.C., Kelly, J.M., Wilkinson, S.R. and Radi, R. (2007) Mitochondrial Superoxide Radicals Mediate Programmed Cell Death in Trypanosoma cruzi: Cytoprotective Action of Mitochondrial Iron Superoxide Dismutase Overexpression. Biochemical Journal, 403, 323-334.
http://dx.doi.org/10.1042/BJ20061281

  
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