Role of tumor suppressor p53 in megakaryopoiesis and platelet function
2011; Elsevier BV; Volume: 40; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2011.10.006
ISSN1873-2399
AutoresPani A. Apostolidis, Donna S. Woulfe, Massiel Chavez, William M. Miller, Eleftherios T. Papoutsakis,
Tópico(s)Blood groups and transfusion
ResumoThe pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53−/− megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53−/− marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53−/− megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53−/− mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53−/− mice. Although their platelet counts were normal, p53−/− mice exhibited significantly longer bleeding times and p53−/− platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies. The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53−/− megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53−/− marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53+/+ megakaryocytes. This was due to 30% greater DNA synthesis in p53−/− megakaryocytes and 31% greater apoptosis in p53+/+ megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53+/+ and p53−/− mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53−/− mice. Although their platelet counts were normal, p53−/− mice exhibited significantly longer bleeding times and p53−/− platelets were less sensitive than p53+/+ platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies. Activation of the p53 tumor suppressor generally results in cell cycle arrest and/or induction of apoptosis [1Vousden K.H. Lane D.P. p53 in health and disease.Nat Rev Mol Cell Biol. 2007; 8: 275-283Crossref PubMed Scopus (1687) Google Scholar]. Mounting evidence has implicated p53 as a critical regulator of hematopoietic stem cell proliferation, stress hematopoiesis following lethal irradiation, and hematopoietic stem cell aging [2Dumble M. Moore L. Chambers S.M. et al.The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging.Blood. 2007; 109: 1736-1742Crossref PubMed Scopus (203) Google Scholar, 3Liu Y. Elf S.E. Miyata Y. et al.p53 regulates hematopoietic stem cell quiescence.Cell Stem Cell. 2009; 4: 37-48Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 4TeKippe M. Harrison D.E. Chen J. Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice.Exp Hematol. 2003; 31: 521-527Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar] (reviewed in [5Liu Y. Elf S.E. Asai T. et al.The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior.Cell Cycle. 2009; 8: 3120-3124Crossref PubMed Scopus (52) Google Scholar]). Our recent studies [6Chen C. Fuhrken P.G. Huang L.T. et al.A systems-biology analysis of isogenic megakaryocytic and granulocytic cultures identifies new molecular components of megakaryocytic apoptosis.BMC Genomics. 2007; 8: 384Crossref PubMed Scopus (16) Google Scholar, 7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar, 8Fuhrken P.G. Chen C. Apostolidis P.A. Wang M. Miller W.M. Papoutsakis E.T. Gene Ontology-driven transcriptional analysis of CD34+ cell-initiated megakaryocytic cultures identifies new transcriptional regulators of megakaryopoiesis.Physiol Genomics. 2008; 33: 159-169Crossref PubMed Scopus (21) Google Scholar, 9Fuhrken P.G. Chen C. Miller W.M. Papoutsakis E.T. Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation.Exp Hematol. 2007; 35: 476-489Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 10Giammona L.M. Panuganti S. Kemper J.M. et al.Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition.Exp Hematol. 2009; 37 (1340–1352, e3)Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar] and earlier work by others [11Ritchie A. Braun S.E. He J. Broxmeyer H.E. Thrombopoietin-induced conformational change in p53 lies downstream of the p44/p42 mitogen activated protein kinase cascade in the human growth factor-dependent cell line M07e.Oncogene. 1999; 18: 1465-1477Crossref PubMed Scopus (19) Google Scholar, 12Ritchie A. Vadhan-Raj S. Broxmeyer H.E. Thrombopoietin suppresses apoptosis and behaves as a survival factor for the human growth factor-dependent cell line, M07e.Stem Cells. 1996; 14: 330-336Crossref PubMed Scopus (50) Google Scholar, 13Sigurjonsson O.E. Gudmundsson K.O. Haraldsdottir V. Rafnar T. Agnarsson B.A. Gudmundsson S. Flt3/Flk-2 ligand in combination with thrombopoietin decreases apoptosis in megakaryocyte development.Stem Cells Dev. 2004; 13: 183-191Crossref PubMed Scopus (15) Google Scholar] provided evidence that the p53 pathway, largely unexplored in megakaryopoiesis, plays an important role in megakaryocytic (Mk) differentiation. We have recently shown that knock-down of p53 increased ploidy in Mk cultures [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar]. There are relatively few studies that specifically examine megakaryopoiesis or platelet production in the absence of p53. One such study indicated faster recovery of colony-forming unit Mk progenitors (CFU-Mk) after 5-fluorouracil treatment in p53−/− vs p53+/+ mice and a threefold increased bone marrow (BM) content of Lin−Sca-1+c-Kit+ cells in p53−/− mice following 5-fluorouracil treatment [14Wlodarski P. Wasik M. Ratajczak M.Z. et al.Role of p53 in hematopoietic recovery after cytotoxic treatment.Blood. 1998; 91: 2998-3006Crossref PubMed Google Scholar]. Platelet recovery after irradiation of p53−/− mice was three times faster [15Horie K. Kubo K. Yonezawa M. p53 dependency of radio-adaptive responses in endogenous spleen colonies and peripheral blood-cell counts in C57BL mice.J Radiat Res (Tokyo). 2002; 43: 353-360Crossref PubMed Scopus (24) Google Scholar], suggesting that p53 loss can result in faster platelet production. Despite the known propensity of p53−/− mice to succumb to cancer, mainly lymphoma and sarcoma [16Jacks T. Remington L. Williams B.O. et al.Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar], p53 loss has not been linked to abnormal megakaryopoiesis. On the contrary, a recent study demonstrated that intercrossing of p53−/− mice with mice engineered to contain an equivalent to the human deletion of the q arm of chromosome 5 (5q- syndrome) and whose BM cells express high levels of the p53 protein, reversed the low Mk/erythroid progenitor levels and low platelet levels encountered in the 5q- syndrome [17Barlow J.L. Drynan L.F. Hewett D.R. et al.A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q- syndrome.Nat Med. 2010; 16: 59-66Crossref PubMed Scopus (274) Google Scholar]. This study suggests that p53 loss can favor megakaryopoiesis and platelet production. Additionally, a study examining platelet recovery following myelosuppression in p53−/− and p53+/+ mice hypothesized that loss of p53 confers to hematopoietic stem cells and committed Mk precursors the ability to resist apoptosis to the same extent that administration of thrombopoietin (Tpo) supports survival [18Pestina T.I. Cleveland J.L. Yang C. Zambetti G.P. Jackson C.W. Mpl ligand prevents lethal myelosuppression by inhibiting p53-dependent apoptosis.Blood. 2001; 98: 2084-2090Crossref PubMed Scopus (34) Google Scholar]. However, none of these studies examined Mk differentiation in response to Tpo administration in the absence of p53, or Mk/platelet recovery in response to induced thrombocytopenia. The first aim of this study was to characterize Mk polyploidization in the BM of p53+/+ and p53−/− mice at steady state together with de novo platelet production. Additionally, we aimed to examine the regulation of Mk polyploidization and platelet production in response to (1) treatment of mice with Tpo to promote Mk differentiation and increased thrombopoiesis; and (2) administration of an antiplatelet antibody to specifically target the platelets and elicit an Mk differentiation and platelet production cascade to replenish normal platelet levels in p53+/+ and p53−/− mice. The next question we aimed to address was the impact of p53 on platelet functional responses. The presence of the p53 protein and the p53 inhibitor MDM2 in platelets has been previously documented [19Eidelman O. Jozwik C. Huang W. et al.Gender dependence for a subset of the low-abundance signaling proteome in human platelets.Hum Genomics Proteomics. 2010; 2010: 164906Crossref PubMed Scopus (31) Google Scholar]. However, the effect of the loss or functional inactivation of p53 on hemostasis is unknown. Here, we present findings postulating a reduced hemostatic response in p53−/− mice. Our study suggests a novel role for p53 in regulating hemostasis, perhaps by affecting components of the platelet granules or regulating platelet surface integrin activation. All procedures involving mice were approved by the University of Delaware Institutional Animal Care and Use Committee. Male p53−/− mice and p53+/+ (wild-type) littermates (B6.129S2.Trp53tm1Tyj/J colony) [16Jacks T. Remington L. Williams B.O. et al.Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar] were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed with free access to food and water. p53−/− mice and age-matched p53+/+ littermates less than 3 months of age were injected once subcutaneously with 1.2 μg recombinant murine (rm) Tpo (Peprotech, Rocky Hill, NJ, USA) diluted in saline or saline as control [20Larson M.K. Watson S.P. Regulation of proplatelet formation and platelet release by integrin alpha IIb beta3.Blood. 2006; 108: 1509-1514Crossref PubMed Scopus (113) Google Scholar, 21Arnold J.T. Daw N.C. Stenberg P.E. Jayawardene D. Srivastava D.K. Jackson C.W. A single injection of pegylated murine megakaryocyte growth and development factor (MGDF) into mice is sufficient to produce a profound stimulation of megakaryocyte frequency, size, and ploidization.Blood. 1997; 89: 823-833Crossref PubMed Google Scholar]. On days 2 and 5 after Tpo treatment, the mice were bled retro-orbitally and sacrificed. Platelets were enumerated using the Unopette reservoirs (Becton-Dickinson, Franklin Lakes, NJ, USA) and a hemacytometer (Hausser, Horsham, PA, USA). p53−/− mice and age-matched p53+/+ littermates less than 4 months of age were injected once intraperitoneally with 0.5 μg rat anti-mouse CD41 (clone: MWReg30; BD-Pharmingen, San Diego, CA, USA) per gram of body weight [22Hitchcock I.S. Fox N.E. Prevost N. Sear K. Shattil S.J. Kaushansky K. Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage specific FAK knockout.Blood. 2008; 111: 596-604Crossref PubMed Scopus (65) Google Scholar]. The antibody had been dialyzed before injection for 4 hours in 1 L ice-cold phosphate-buffered saline (PBS) to remove traces of sodium azide using the Slide-A-Lyzer mini dialysis units (Pierce, Rockford, IL, USA). Daily blood counts were conducted for 5 days using a Hemavet (Drew Scientific, Waterbury, CT, USA). Some of the mice were sacrificed on day 2 after anti-CD41 injection, while the rest were sacrificed on day 5 in order to harvest BM. Reticulated platelets were measured as described previously [23Lindsey S. Papoutsakis E.T. The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization.Br J Haematol. 2011; 152: 469-484Crossref PubMed Scopus (32) Google Scholar]. Briefly, 1 μL EDTA-anticoagulated blood was incubated in a total volume of 60 μL with 10 μg/mL thiazole orange (Sigma-Aldrich, St Louis, MO, USA) and 1 μL anti–CD41-phycoerythrin (clone MWReg30; BD-Pharmingen) for 15 minutes in the dark at room temperature. Samples were then fixed by addition of 1% paraformaldehyde for no more than 30 minutes at room temperature and acquired on a FACSAria. Measurement of CD41 mean fluorescence using flow cytometry was used to estimate αIIbβ3 expression on the platelet surface. Blood was collected from the retro-orbital vein using plain Natelson capillaries and left to clot at room temperature in the absence of anticoagulant. Serum was then collected by high-speed centrifugation. Tpo levels in the serum were measured using the Quantikine mouse Tpo ELISA (R&D Systems, Minneapolis, MN, USA) [22Hitchcock I.S. Fox N.E. Prevost N. Sear K. Shattil S.J. Kaushansky K. Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage specific FAK knockout.Blood. 2008; 111: 596-604Crossref PubMed Scopus (65) Google Scholar]. Absorbance was read at 450 nm with the correction set to 595 nm using a DTX880 microplate reader (Beckman-Coulter, Brea, CA, USA). Red blood cell–depleted BM cells were processed as described in our previous study [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar] and cultured in Iscove's modified Dulbecco's medium/10% fetal bovine serum/1% penstrep media containing 50 ng/mL rmTpo [24Kostyak J.C. Naik U.P. Calcium- and integrin-binding protein 1 regulates endomitosis and its interaction with Polo-like kinase 3 is enhanced in endomitotic Dami cells.PLoS One. 2011; 6: e14513Crossref PubMed Scopus (12) Google Scholar]. Culture flasks were maintained in a fully humidified incubator at 37°C and 5% CO2. To detect ploidy among Mk cells, red blood cell–depleted BM cells or cultured Mk cells were stained with anti–CD41-fluorescein isothiocyanate (FITC; BD Pharmingen), fixed with 0.5% paraformaldehyde in PBS, permeabilized with 70% methanol, treated with 10 mg/mL RNAse and counterstained with propidium iodide [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar, 25Giammona L.M. Fuhrken P.G. Papoutsakis E.T. Miller W.M. Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes.Br J Haematol. 2006; 135: 554-566Crossref PubMed Scopus (62) Google Scholar]. For DNA synthesis, p53+/+ and p53−/− Mk cell cultures were incubated for 12 hours with 10 μM bromo-deoxy-uridine (BD Pharmingen) at 37°C, then stained with anti–CD41-FITC, fixed and permeabilized per the manufacturer's protocol, treated with 10 mg/mL RNAse and stained with PI [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar]. For detection of Mk apoptosis, Hoechst 33342 was directly added at 0.01 mM into the Mk cell cultures and incubated for 2 hours at 37°C/5% CO2 [26Gilles L. Guieze R. Bluteau D. et al.P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1.Blood. 2008; 111: 4081-4091Crossref PubMed Scopus (42) Google Scholar]. At the end of the incubation, Mk cells were washed with phosphate-buffered saline, stained with anti–CD41-FITC for 30 minutes at 4°C, washed again with PBS, resuspended in 1× Annexin binding buffer (BD Pharmingen), stained with Annexin V-PE (BD-Pharmingen) and acquired. Mice were anesthetized by isoflurane inhalation, tails were prewarmed at 37°C for 5 minutes, 3-mm of the distal end was cut off using a scalpel, and the tail was immersed into fresh saline prewarmed at 37°C. Primary bleeding time is defined as the time until bleeding cessation for a minimum of 10 seconds. If bleeding resumed, the duration of the primary and subsequent bleeds were added up and reported as the total bleeding time. If the primary or total bleeding time exceeded 10 minutes, the assay was stopped and the tail was taken out of the saline and pressure was applied to stop the bleeding in order to avoid losing too much blood [27Tucker K.L. Sage T. Stevens J.M. et al.A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion.Blood. 2008; 112: 4523-4531Crossref PubMed Scopus (50) Google Scholar]. Heparinized blood was collected from the retro-orbital veins and platelet-rich plasma was isolated by low-speed centrifugation. Platelet-rich plasma was passed through a Sepharose column as described previously [28Prevost N. Woulfe D.S. Jiang H. et al.Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets.Proc Natl Acad Sci U S A. 2005; 102: 9820-9825Crossref PubMed Scopus (119) Google Scholar], gel-filtered platelets were isolated and calcium was added to a final concentration of 1 mM. For flow cytometric assays, Alexa-Fluor-488–conjugated fibrinogen (Molecular Probes, Eugene, OR, USA) or FITC-conjugated anti–P-selectin (BD Pharmingen) were added at 12.5 μg/mL or 1:100 dilution, respectively, together with a range of concentrations of AYPGKF (Kimmel Cancer Center of the Thomas Jefferson University, Philadelphia, PA, USA), a PAR-4 agonist peptide. The platelets were stimulated for 10 minutes at 37°C, then fixed for 10 minutes with 2% paraformaldehyde at room temperature. For microscopy, gel-filtered platelets were stimulated for 5, 15, or 30 minutes at 37°C with 50 μM AYPGKF or 10 μM phorbol 12-myristate 13-acetate (PMA) on glass slides precoated with 100 μg/mL human fibrinogen (Enzyme Research Laboratories, South Bend, IN, USA). Platelets were fixed and permeabilized as described [28Prevost N. Woulfe D.S. Jiang H. et al.Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets.Proc Natl Acad Sci U S A. 2005; 102: 9820-9825Crossref PubMed Scopus (119) Google Scholar], stained with Alexa Fluor-568–phalloidin and mounted. Slides were examined using an LSM 5 Live High Speed Confocal microscope (Zeiss, Oberkochen, Germany) equipped with a 100× oil objective. CHRF is a wild-type p53+ human CD41+CD34+ megakaryoblastic cell line, which responds to PMA and undergoes polyploidization coupled with marked apoptosis and extension of proplatelet-like cytoplasmic protrusions. P53-KD and control human megakaryoblastic CHRF cells were cultured in Iscove's modified Dulbecco's medium/10% fetal bovine serum and stimulated with 10 ng/mL PMA to induce terminal Mk differentiation, as described previously [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar, 9Fuhrken P.G. Chen C. Miller W.M. Papoutsakis E.T. Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation.Exp Hematol. 2007; 35: 476-489Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 29Jiang F. Jia Y. Cohen I. Fibronectin- and protein kinase C-mediated activation of ERK/MAPK are essential for proplateletlike formation.Blood. 2002; 99: 3579-3584Crossref PubMed Scopus (53) Google Scholar]. Cell samples taken from two independent biological replicate sets of p53-KD and control CHRF cell cultures either unstimulated (day 0) or on days 1, 3, 5, and 7 following stimulation with PMA were flash-frozen in liquid nitrogen to be used later for microarray analysis. Experimental methods for CHRF cell culture, RNA isolation, labeling, and hybridization for the microarray analysis in CHRF cells and data normalization have been described [9Fuhrken P.G. Chen C. Miller W.M. Papoutsakis E.T. Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation.Exp Hematol. 2007; 35: 476-489Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30Yang H. Haddad H. Tomas C. Alsaker K. Papoutsakis E.T. A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis.Proc Natl Acad Sci U S A. 2003; 100: 1122-1127Crossref PubMed Scopus (51) Google Scholar]. All data have been deposited in Gene Expression Omnibus as mandated by the Minimum Information about Microarray Experiment (MIAME) standards and have been assigned series record GSE30984. A list of platelet activation-related genes was curated based on the literature [6Chen C. Fuhrken P.G. Huang L.T. et al.A systems-biology analysis of isogenic megakaryocytic and granulocytic cultures identifies new molecular components of megakaryocytic apoptosis.BMC Genomics. 2007; 8: 384Crossref PubMed Scopus (16) Google Scholar, 9Fuhrken P.G. Chen C. Miller W.M. Papoutsakis E.T. Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation.Exp Hematol. 2007; 35: 476-489Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 31Mercher T. Cornejo M.G. Sears C. et al.Notch signaling specifies megakaryocyte development from hematopoietic stem cells.Cell Stem Cell. 2008; 3: 314-326Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 32Chang Y. Bluteau D. Debili N. Vainchenker W. From hematopoietic stem cells to platelets.J Thromb Haemost. 2007; 5: 318-327Crossref PubMed Scopus (90) Google Scholar, 33Chen Z. Hu M. Shivdasani R.A. Expression analysis of primary mouse megakaryocyte differentiation and its application in identifying stage-specific molecular markers and a novel transcriptional target of NF-E2.Blood. 2007; 109: 1451-1459Crossref PubMed Scopus (48) Google Scholar]. Differentially expressed genes were identified using statistical analysis of microarrays as implemented in MeV with a false discovery rate <0.05 [34Saeed A.I. Sharov V. White J. et al.TM4: a free, open-source system for microarray data management and analysis.Biotechniques. 2003; 34: 374-378Crossref PubMed Scopus (3944) Google Scholar]. Validation of microarray findings was done with real-time polymerase chain reaction, as described in our previous work [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar] using Taqman probes for ITGA2B (Hs01116228_m1), SELP (Hs00356351_m1), and MYH9 (Hs00159522_m1) from Applied Biosystems (Foster City, CA, USA). Data were normalized against housekeeping gene GUSB (Hs99999908_m1). Unless otherwise noted, all statistical comparisons were conducted using an unpaired two-tailed Student's t-test. We have previously shown that knock-down of p53 in human megakaryoblastic CHRF cells led to increased Mk polyploidization in part due to greater DNA synthesis upon induction of Mk differentiation [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar]. Additionally, we have shown that p53−/− culture-derived mouse Mk cells reach higher ploidy classes than p53+/+ Mk cells [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar]. Here, we aimed to better characterize this ex vivo–observed hyperploid murine p53−/− Mk phenotype. BM cells harvested from p53−/− mice and age-matched p53+/+ littermates were cultured ex vivo in the presence of 50 ng/mL Tpo for 6 days to induce Mk differentiation. Cell size analysis of p53+/+ and p53−/− cultured CD41+ cells was performed using the forward scatter area signal [35Muntean A.G. Pang L. Poncz M. Dowdy S.F. Blobel G.A. Crispino J.D. Cyclin D-Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization.Blood. 2007; 109: 5199-5207Crossref PubMed Scopus (71) Google Scholar]. On day 6 of culture, the mean ± standard error of mean (SEM) forward scatter signal for p53+/+ and p53−/− CD41+ cells was 121 ± 4 and 151 ± 9 (n = 7, p = 0.01), indicating that cell size was significantly increased in the highly polyploid p53−/− Mk cells. As we have previously observed [7Fuhrken P.G. Apostolidis P.A. Lindsey S. Miller W.M. Papoutsakis E.T. Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.J Biol Chem. 2008; 283: 15589-15600Crossref PubMed Scopus (34) Google Scholar], Mk cells derived from p53−/− BM progenitor cells always reached higher ploidy classes and exhibited higher percentage of highly polyploid cells. By day 6 of culture, there was a 1.7-fold increase in total CD41+ Mk cells for p53−/− Mk cells (n = 7, p < 0.02; Fig. 1A ) and substantially increased polyploidy: 26.1% ± 3.6% of p53−/− vs 8.2% ± 0.9% of p53+/+ Mk cells reached ploidy classes ≥64N (n = 7, p = 0.0004; Fig. 1B). Increased polyploidization observed in culture was partially due to 30% enhanced DNA synthesis among p53−/− Mk cells on day 4 of culture (n = 3–4; p < 0.03; Fig. 1C, D). Moreover, there was a tendency for increased polyploidization in ≥32N ploidy classes among actively cycling Mk cells (bromo-deoxy-uridine+) in p53−/− Mk cells on day 4 (n = 3–4; Fig. 1E, F). This finding indicated that increased DNA synthesis largely occurred in the highly polyploid (≥32 N) population of p53−/− Mk cells. Increased polyploidization in ex vivo–cultured p53−/− Mk cells can also be ascribed to diminished apoptosis in the absence of p53. Indeed, apoptosis, measured among nucleated CD41+ cells by assessing binding of Annexin V, was increased by an average of 31% in p53+/+ Mk cells on day 4 of culture with Tpo (Fig. 2A, B ). Mk cells in the BM were measured as the percent of nucleated CD41+ cells. The numbers of BM-resident Mk cells were similar in p53+/+ and p53−/− mice (0.21% ± 0.03% for both genotypes, n = 7), while among p53+/+ Mk cells, 29.7% ± 3.1% reached ploidy of 16 N or higher vs 30.2% ± 3.9% among p53−/− Mk cells. Sternum BM and spleen histology sections did not reveal any abnormalities in the morphology or content of p53−/− Mk cells (data not shown). Additionally, p53−/− mice have normal serum Tpo levels and platelet synthesis and only slightly lower platelet counts compared to their p53+/+ counterparts (Table 1).Table 1Complete blood counts in untreated p53+/+ and p53−/− mice (N = 11–14), reticulated platelets (N = 19–22) and serum thrombopoietin (Tpo; N = 4–6)p53+/+p53−/−WBC (×103/mm3)7.81 ± 0.648.85 ± 1.09NE (×103/mm3)1.89 ± 0.302.30 ± 0.33LY (×103/mm3)5.42 ± 0.395.73 ± 0.64MO (×103/mm3)0.41 ± 0.060.62 ± 0.12EO (×103/mm3)0.08 ± 0.020.15 ± 0.06BA (×103/mm3)0.02 ± 0.010.04 ± 0.02RBC (×106/mm3)8.56 ± 0.228.45 ± 0.28Hb (g/dL)12.1 ± 0.311.7 ± 0.4PLT (×103/mm3)907 ± 54789 ± 49MPV (fL)4.6 ± 0.14.6 ± 0.0% Retic14.0 ± 0.6%13.1 ± 0.6Tpo (pg/ml)3,494 ± 2303,226 ± 110WBC = white blood cells; NE = neutrophils; LY = lymphocytes; MO = monocytes; EO = eosinophils; BA = basophils; RBC = red blood cells; Hb = hemoglobin; PLT = platelets; MPV = mean platelet volume; %Retic = % reticula
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