Abstract
Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.
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References
Seita J, Weissman IL (2010) Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev 2:640–653
Wilson A, Trumpp A (2006) Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 6:93–106
Sugiyama T, Nagasawa T (2012) Bone marrow niches for hematopoietic stem cells and immune cells. Inflamm Allergy Drug Targets 11:201–206
Takubo K (2014) Homeostatic regulation of hematopoiesis by the hematopoietic stem cell niche Seikagaku. J Jpn Biochem Soc 86:755–765
Kiel MJ, Yilmaz ÖH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121
Sugiyama T, Kohara H, Noda M, Nagasawa T (2006) Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 25:977–988
Law S, Chaudhuri S (2007) Niche theory stem-stromal imbalance and Aplastic anaemia. J Stem Cells 2:227–235
Basak P, Chatterjee S, Das P, Das M, Pereira JA, Dutta RK, Chaklader M, Chaudhuri S, Law S (2010) Leukemic stromal hematopoietic microenvironment negatively regulates the normal hematopoiesis in mouse model of leukemia. Chin J Cancer 29:969–979
Basak P, Chatterjee S, Das M, Das P, Pereira JA, Dutta RK, Chaklader M, Chaudhuri S, Law S (2010) Phenotypic alteration of bone marrow HSC and microenvironmental association in experimentally induced leukemia. Curr Stem Cell Res Ther 5:379–386
Das M, Chaudhuri S, Law S (2013) Unveiling the paradoxical nature of myelodysplastic syndromes (MDS): why hypercellular marrow strongly favors accelerated apoptosis. Biochem Cell Biol 91:303–308
Lin FC, Karwan M, Saleh B, Hodge DL, Chan T, Boelte KC, Keller JR, Young HA (2014) IFN-γ causes aplastic anemia by altering hematopoietic stem/progenitor cell composition and disrupting lineage differentiation. Blood 124:3699–3708
Ogawa S (2014) MDS-related mutations in aplastic anemia. Blood 124(17):2619–2620
Evans AG, Calvi LM (2015) Notch signaling in the malignant bone marrow microenvironment: implications for a niche-based model of oncogenesis. Ann N Y Acad Sci 1335:63–77
Young NS, Maciejewski J (1997) The pathophysiology of acquired aplastic anemia. N Engl J Med 336:1365–1372
Brodsky RA, Jones RJ (2005) Aplastic anaemia. Lancet 365:1647–1656
Fleming LE, Timmeny W (1993) Aplastic anemia and pesticides: an etiologic association? J Occup Environ Med 35:1106–1116
Langnas AN, Markin RS, Cattral MS, Naides SJ (1995) Parvovirus B19 as a possible causative agent of fulminant liver failure and associated aplastic anemia. Hepatology 22:1661–1665
Young NS (2002) Acquired aplastic anemia. Ann Intern Med 136:534–546
Young NS, Calado RT, Scheinberg P (2006) Current concepts in the pathophysiology and treatment of aplastic anemia. Blood 108:2509–2519
Chatterjee S, Dutta RK, Basak P, Das P, Das M, Pereira JA, Chaklader M, Chaudhuri S, Law S (2010) Alteration in marrow stromal microenvironment and apoptosis mechanisms involved in aplastic anemia: an animal model to study the possible disease pathology. Stem Cells Int. doi:10.4061/2010/932354
Botnick LE, Hannon EC, Vigneulle R, Hellman S (1981) Differential effects of cytotoxic agents on hematopoietic progenitors. Cancer Res 41:2338–2342
Vincent PC (1986) Drug-induced aplastic anaemia and agranulocytosis. Drugs 31:52–63
Paci E, Buiatti E, Costantini AS, Miligi L, Pucci N, Scarpelli A, Petrioli G, Simonato L, Winkelmann R, Kaldor JM (1989) Aplastic anemia leukemia and other cancer mortality in a cohort of shoe workers exposed to benzene. Scand J Work Environ Health 15:313–318
Law S, Basu K, Banerjee S, Begum B, Chaudhuri S (2006) Cord blood-derived plasma factor (CBPF) potentiates the low cytokinetic and immunokinetic profile of bone marrow cells in pesticide victims suffering from acquired aplastic anaemia (AAA): an in vitro correlate. Immunol Invest 35:209–225
Chatterjee S, Chaklader M, Basak P, Das P, Das M, Pereira JA, Dutta RK, Chaudhuri S, Law S (2010) An animal model of chronic aplastic bone marrow failure following pesticide exposure in mice. Int J Stem Cells 3:54–62
Chen YF, Wu ZM, Xie C, Bai S, Zhao LD (2013) Expression level of IL-6 secreted by bone marrow stromal cells in mice with aplastic anemia. ISRN Hematol. doi:10.1155/2013/986219
Chaklader M, Law S (2015) Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia. Mol Cell Biochem 401:115–131
Chattopadhyay S, Chatterjee R, Law S (2015) Noncanonical Wnt5a-Ca2+-NFAT signaling axis in pesticide induced bone marrow aplasia mouse model: a study to explore the novel mechanism of pesticide toxicity. Environ Toxicol. doi:10.1002/tox.22123
Chatterjee S, Basak P, Das M, Das P, Pereira JA, Dutta RK, Chaklader M, Chaudhuri S, Law S (2008) Kinetic impairment of haemopoietic stem cells in experimentally induced leukemia and aplastic anemia: an inverse correlation. J Stem Cells 4:179–189
Schmitt E, Paquet C, Beauchemin M, Bertrand R (2007) DNA-damage response network at the crossroads of cell-cycle checkpoints cellular senescence and apoptosis. J Zhejiang Univ Sci B 8:377–397
Park MR, Choi YJ, Kwon DN, Park C, Bui HT, Gurunathan S, Cho SG, Song H, Seo HG, Min G, Kim JH (2013) Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries. Sci Rep 3. doi:10.1038/srep01384
Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995) Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789
Allard D, Figg N, Bennett MR, Littlewood TD (2008) Akt regulates the survival of vascular smooth muscle cells via inhibition of FoxO3a and GSK3. J Biol Chem 283:19739–19747
Nowsheen S, Yang ES (2012) The intersection between DNA damage response and cell death pathways. Experimental Oncology 34:243–254
Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G (1993) Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev 7:812–821
Diehl JA, Cheng M, Roussel MF, Sherr CJ (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12:3499–3511
Margolis SS, Perry JA, Forester CM, Nutt LK, Guo Y, Jardim MJ, Kornbluth S (2006) Role for the PP2A/B56δ phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127:759–773
Sallman DA, Wei S, List A (2014) PP2A: the achilles heal in MDS with 5q deletion. Front Oncol. doi:10.3389/fonc.2014.00264
Xiu-Qing Y, Xiao-Xue Z, Yang-Yang Y et al (2011) Glycogen synthase kinase-3beta regulates Tyr307 phosphorylation of protein phosphatase-2A via protein tyrosine phosphatase 1B but not Src. Biochem J 437:335–344
Yao XQ, Li XC, Zhang XX, Yin YY, Liu B, Luo DJ, Wang Q, Wang JZ, Liu GP (2012) Glycogen synthase kinase-3β regulates leucine-309 demethylation of protein phosphatase-2A via PPMT1 and PME-1. FEBS Lett 586:2522–2528
Beurel E, Jope RS (2006) The paradoxical pro-and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog Neurobiol 79:173–189
Toyoshima-Morimoto F, Taniguchi E, Nishida E (2002) Plk1 promotes nuclear translocation of human Cdc25C during prophase. EMBO Rep 3:341–348
Yuan JH, Feng Y, Fisher RH, Maloid S, Longo DL, Ferris DK (2004) Polo-Like Kinase 1 inactivation following mitotic DNA damaging treatments is independent of ataxia telangiectasia mutated kinase. Mol Cancer Res 2:417–426
Kollareddy M, Dzubak P, Zheleva D, Hajduch M (2008) Aurora kinases: structure functions and their association with cancer. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 152:27–33
Kimura M, Yoshioka T, Saio M, Banno Y, Nagaoka H, Okano Y (2013) Mitotic catastrophe and cell death induced by depletion of centrosomal proteins. Cell Death Dis. doi:10.1038/cddis.2013.108
Krystyniak A, Garcia-Echeverria C, Prigent C, Ferrari S (2006) Inhibition of Aurora A in response to DNA damage. Oncogene 25:338–348
Bhatia P, Menigatti M, Brocard M, Morley SJ, Ferrari S (2010) Mitotic DNA damage targets the Aurora A/TPX2 complex. Cell Cycle 9:4592–4599
Li J, Zhu BD, Chen YF, Chen ZW (2006) Effect of rhodiola polysaccharide on peripheral blood cells and bone marrow cell cycle of myelosuppressed anemia mice. Sich J Anat 3:2
Chatterjee S, Basak P, Das P, Das M, Pereira JA, Dutta RK, Chaklader M, Chaudhuri S, Law S (2009) Primitive SCA-1 positive bone marrow HSC in mouse model of aplastic anemia: a comparative study through flowcytometric analysis and scanning electron microscopy. Stem Cells Int. doi:10.4061/2010/614395
Chen YF, Zhao ZQ, Wu ZM, Zou ZY, Luo XJ, Li J, Xie C, Liang Y (2014) The role of RIP1 and RIP3 in the development of aplastic anemia induced by cyclophosphamide and busulphan in mice. Int J Clin Exp Pathol 7:8411–8420
Chatterjee R, Chattopadhyay S, Sanyal S, Daw S, Law S (2016) Pathophysiological scenario of hematopoietic disorders: a comparative study of aplastic anemia myelodysplastic syndrome and leukemia in experimental animals. Proc Zool Soc 69:114–124
Chaklader M, Das P, Pereira JA, Chaudhuri S, Law S (2012) Altered canonical hedgehog-gli signalling axis in pesticide-induced bone marrow aplasia mouse model. Archi Ind Hyg Toxicol 63:271–282
Erenpreiss J, Bars J, Lipatnikova V, Ernapreisa J (2001) Comparative study of cytochemical tests for sperm chromatin integrity. J Androl 22:45–53
Sanyal S, Das P, Law S (2016) Effect of chronic pesticide exposure on murine cornea: a histopathological cytological and flow cytometric approach to study ocular damage by xenobiotics. Cell Biol Toxicol 32:7–22
Schuurhuis GJ, Meel MH, Wouters F, Min LA, Terwijn M, de Jonge NA, Kelder A, Snel AN, Zweegman S, Ossenkoppele GJ, Smit L (2013) Normal hematopoietic stem cells within the AML bone marrow have a distinct and higher ALDH activity level than co-existing leukemic stem cells. PloS one. doi:10.1371/journal.pone.0078897
Chatterjee R, Chattopadhyay S, Law S (2016) Alteration of classical and hematopoiesis specific p53 pathway in the bone marrow hematopoietic stem/progenitor compartment facilitates leukemia progression in experimental mice. Leuk Res 47:70–77
Gajkowska A, Oldak T, Jastrzewska M, Machaj EK, Walewski J, Kraszewska E, Pojda Z (2006) Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods. Folia Histochem Cytobiol 44:53–60
Papadaki HA, Tsagournisakis M, Mastorodemos V, Pontikoglou C, Damianaki A, Pyrovolaki K, Stamatopoulos K, Fassas A, Plaitakis A, Eliopoulos GD (2005) Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis. Bone Marrow Transpl 36:1053–1063
Okada S, Nakauchi H, Nagayoshi K, Nishikawa SL, Miura Y, Suda T (1992) In vivo and in vitro stem cell function of c-kit-and Sca-1-positive murine hematopoietic cells. Blood 80:3044–3050
Morley A, Trainor K, Seshadri R, Sorrell J (1978) Is aplastic anaemia due to abnormality of DNA? Lancet 312:9–12
Kohn KW, Hartley JA, Mattes WB (1987) Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res 15:10531–10549
Siddik ZH (2002) Mechanisms of action of cancer chemotherapeutic agents: dna-interactive alkylating agents and antitumour platinum-based drugs. Cancer Handb. doi:10.1002/0470025077.chap84b
Beletti ME, Costa LDF, Guardieiro MM (2005) Morphometric features and chromatin condensation abnormalities evaluated by toluidine blue staining in bull spermatozoa. Braz J Morphol Sci 22:85–90
Kim HS, Kang MJ, Kim SA, Oh SK, Kim H, Ku SY, Kim SG, Moon SY, Choi YM (2013) The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis. Clin Exp Reprod Med 40:23–28
Philpott NJ, Scopes J, Marsh JC, Gordon-Smith EC, Gibson FM (1995) Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance. Exp Hematol 23:1642–1648
Maciejewski JP, Selleri C, Sato T, Anderson S, Young NS (1995) Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia. Br J Haematol 91:245–252
Killick SB, Cox CV, Marsh JCW, Gordon-Smith EC, Gibson FM (2000) Mechanisms of bone marrow progenitor cell apoptosis in aplastic anaemia and the effect of anti-thymocyte globulin: examination of the role of the Fas–Fas-L interaction. Br J Haematol 111:1164–1169
Hui SP, Dutta A, Ghosh S (2010) Cellular response after crush injury in adult zebrafish spinal cord. Dev Dyn 239:2962–2979
Chen MS, Ryan CE, Piwnica-Worms H (2003) Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol Cell Biol 23:7488–7497
Bryant C, Scriven K, Massey AJ (2014) Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human leukemia and lymphoma cells. Mol Cancer. doi:10.1186/1476-4598-13-147
Acknowledgments
We are thankful to the Director of Calcutta School of Tropical Medicine for her support in successful completion of the study and to the Department of Biotechnology, Government of West Bengal for the sponsorship (Sanction No.124 (A)-BT (Estt)/RD-3/12 dt.27.2.13). We are also thankful to the Director of Center for Research in Nanoscience and Nanotechnology for helping us with scanning electron microscopy facility.
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Chatterjee, R., Chattopadhyay, S. & Law, S. Deregulation of vital mitotic kinase–phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia. Mol Cell Biochem 422, 121–134 (2016). https://doi.org/10.1007/s11010-016-2811-1
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DOI: https://doi.org/10.1007/s11010-016-2811-1