Mice heterozygous for CREB binding protein are hypersensitive to γ-radiation and invariably develop myelodysplastic/myeloproliferative neoplasm
2011; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês
10.1016/j.exphem.2011.12.004
ISSN1873-2399
AutoresStephanie N. Zimmer, Madeleine E. Lemieux, Bijal Karia, Claudia Day, Ting Zhou, Qing Zhou, Andrew L. Kung, Uthra Suresh, Yidong Chen, Marsha C. Kinney, Alexander J. R. Bishop, Vivienne I. Rebel,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoMyelodysplastic syndrome is a complex family of preleukemic diseases in which hematopoietic stem cell defects lead to abnormal differentiation in one or more blood lineages. Disease progression is associated with increasing genomic instability and a large proportion of patients go on to develop acute myeloid leukemia. Primarily a disease of the elderly, it can also develop after chemotherapy. We have previously reported that CREB binding protein (Crebbp) heterozygous mice have an increased incidence of hematological malignancies, and others have shown that CREBBP is one of the genes altered by chromosomal translocations found in patients suffering from therapy-related myelodysplastic syndrome. This led us to investigate whether hematopoietic tumor development in Crebbp+/− mice is preceded by a myelodysplastic phase and whether we could uncover molecular mechanisms that might contribute to its development. We report here that Crebbp+/− mice invariably develop myelodysplastic/myeloproliferative neoplasm within 9 to 12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in poly(ADP-ribose) polymerase-1 activity after irradiation. In addition, protein levels of XRCC1 and APEX1, key components of base excision repair machinery, are reduced in unirradiated Crebbp+/− cells or upon targeted knockdown of CREBBP levels. Our results provide validation of a novel myelodysplastic/myeloproliferative neoplasm mouse model and, more importantly, point to defective repair of DNA damage as a contributing factor to the pathogenesis of this currently incurable disease. Myelodysplastic syndrome is a complex family of preleukemic diseases in which hematopoietic stem cell defects lead to abnormal differentiation in one or more blood lineages. Disease progression is associated with increasing genomic instability and a large proportion of patients go on to develop acute myeloid leukemia. Primarily a disease of the elderly, it can also develop after chemotherapy. We have previously reported that CREB binding protein (Crebbp) heterozygous mice have an increased incidence of hematological malignancies, and others have shown that CREBBP is one of the genes altered by chromosomal translocations found in patients suffering from therapy-related myelodysplastic syndrome. This led us to investigate whether hematopoietic tumor development in Crebbp+/− mice is preceded by a myelodysplastic phase and whether we could uncover molecular mechanisms that might contribute to its development. We report here that Crebbp+/− mice invariably develop myelodysplastic/myeloproliferative neoplasm within 9 to 12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in poly(ADP-ribose) polymerase-1 activity after irradiation. In addition, protein levels of XRCC1 and APEX1, key components of base excision repair machinery, are reduced in unirradiated Crebbp+/− cells or upon targeted knockdown of CREBBP levels. Our results provide validation of a novel myelodysplastic/myeloproliferative neoplasm mouse model and, more importantly, point to defective repair of DNA damage as a contributing factor to the pathogenesis of this currently incurable disease. Myelodysplastic syndromes (MDS) is a complex family of preleukemic diseases in which hematopoietic stem cell (HSC) defects lead to abnormal differentiation in one or more blood lineages. Disease progression is associated with increasing genomic instability and a large proportion of patients go on to develop acute myeloid leukemia (AML) (reviewed in [1Corey S.J. Minden M.D. Barber D.L. Kantarjian H. Wang J.C. Schimmer A.D. Myelodysplastic syndromes: the complexity of stem-cell diseases.Nat Rev Cancer. 2007; 7: 118-129Crossref PubMed Scopus (268) Google Scholar]). Primarily a disease of the elderly, MDS/AML can also develop after treatment with alkylating agents, radiation, and topoisomerase II inhibitors [2Godley L.A. Larson R.A. Therapy-related myeloid leukemia.Semin Oncol. 2008; 35: 418-429Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 3Vardiman J.W. Harris N.L. Brunning R.D. The World Health Organization (WHO) classification of the myeloid neoplasms.Blood. 2002; 100: 2292-2302Crossref PubMed Scopus (1786) Google Scholar]. The poor outcomes and increasing incidence of MDS, due to an aging population and increasing numbers of cancer survivors, motivated our efforts to better understand the pathogenesis of this disease.Studies in marrow or blood cells from patients suffering from AML or myeloproliferative neoplasms (MPNs) suggest that inadequate DNA repair may play an important role in the etiology of these diseases. It has been shown that some of the frequently observed genomic aberrations in these diseases [4Sallmyr A. Fan J. Datta K. et al.Internal tandem duplication of FLT3 (FLT3/ITD) induces increased ROS production, DNA damage, and misrepair: implications for poor prognosis in AML.Blood. 2008; 111: 3173-3182Crossref PubMed Scopus (213) Google Scholar, 5Koptyra M. Falinski R. Nowicki M.O. et al.BCR/ABL kinase induces self-mutagenesis via reactive oxygen species to encode imatinib resistance.Blood. 2006; 108: 319-327Crossref PubMed Scopus (256) Google Scholar, 6Nowicki M.O. Falinski R. Koptyra M. et al.BCR/ABL oncogenic kinase promotes unfaithful repair of the reactive oxygen species-dependent DNA double-strand breaks.Blood. 2004; 104: 3746-3753Crossref PubMed Scopus (198) Google Scholar, 7Plo I. Nakatake M. Malivert L. et al.JAK2 stimulates homologous recombination and genetic instability: potential implication in the heterogeneity of myeloproliferative disorders.Blood. 2008; 112: 1402-1412Crossref PubMed Scopus (140) Google Scholar] cause excessive DNA damage by increasing the production of reactive oxygen species and/or usage of alternative, error-prone DNA repair pathways. This mechanism of genomic instability, or mutator phenotype, as proposed by Loeb [8Loeb L.A. A mutator phenotype in cancer.Cancer Res. 2001; 61: 3230-3239PubMed Google Scholar], explains why progression of many of these diseases is associated with increasing genetic abnormalities. MDS patient samples have been less extensively investigated in this context; however, increased oxidative DNA damage has been observed in blood cells from MDS patients [9Jankowska A.M. Gondek L.P. Szpurka H. Nearman Z.P. Tiu R.V. Maciejewski J.P. Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome.Leukemia. 2008; 22: 551-558Crossref PubMed Scopus (40) Google Scholar, 10Novotna B. Bagryantseva Y. Siskova M. Neuwirtova R. Oxidative DNA damage in bone marrow cells of patients with low-risk myelodysplastic syndrome.Leuk Res. 2009; 33: 340-343Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar] and DNA repair deficiencies have been demonstrated in MDS patients with a high risk of progressing toward leukemia [9Jankowska A.M. Gondek L.P. Szpurka H. Nearman Z.P. Tiu R.V. Maciejewski J.P. Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome.Leukemia. 2008; 22: 551-558Crossref PubMed Scopus (40) Google Scholar, 11Kuramoto K. Ban S. Oda K. Tanaka H. Kimura A. Suzuki G. Chromosomal instability and radiosensitivity in myelodysplastic syndrome cells.Leukemia. 2002; 16: 2253-2258Crossref PubMed Scopus (18) Google Scholar]. Moreover, children suffering from diseases due to mutated genes essential for DNA repair, such as Fanconi anemia [12Alter B.P. Giri N. Savage S.A. et al.Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study.Br J Haematol. 2010; 150: 179-188PubMed Google Scholar], Bloom disease [13Iwahara Y. Ishii K. Watanabe S. Taguchi H. Hara H. Miyoshi I. Bloom's syndrome complicated by myelodysplastic syndrome and multiple neoplasia.Intern Med. 1993; 32: 399-402Crossref PubMed Scopus (9) Google Scholar, 14Poppe B. Van Limbergen H. Van Roy N. et al.Chromosomal aberrations in Bloom syndrome patients with myeloid malignancies.Cancer Genet Cytogenet. 2001; 128: 39-42Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar], and Rothmund-Thomson syndrome [15Narayan S. Fleming C. Trainer A.H. Craig J.A. Rothmund-Thomson syndrome with myelodysplasia.Pediatr Dermatol. 2001; 18: 210-212Crossref PubMed Scopus (19) Google Scholar, 16Pianigiani E. De Aloe G. Andreassi A. Rubegni P. Fimiani M. Rothmund-Thomson syndrome (Thomson-type) and myelodysplasia.Pediatr Dermatol. 2001; 18: 422-425Crossref PubMed Scopus (39) Google Scholar], have an increased risk of developing MDS.CREB binding protein (CREBBP) interacts with DNA damage response/repair proteins, such as TP53 [17Gu W. Roeder R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 18Gu W. Shi X.L. Roeder R.G. Synergistic activation of transcription by CBP and p53.Nature. 1997; 387: 819-823Crossref PubMed Scopus (520) Google Scholar] and BRCA1 [19Pao G.M. Janknecht R. Ruffner H. Hunter T. Verma I.M. CBP/p300 interact with and function as transcriptional coactivators of BRCA1.Proc Natl Acad Sci U S A. 2000; 97: 1020-1025Crossref PubMed Scopus (182) Google Scholar], among others, to enhance their function. CREBBP also helps remodel chromatin through its histone acetyltransferase activity, thereby facilitating DNA repair [20Karagiannis T.C. Harikrishnan K.N. El-Osta A. Disparity of histone deacetylase inhibition on repair of radiation-induced DNA damage on euchromatin and constitutive heterochromatin compartments.Oncogene. 2007; 26: 3963-3971Crossref PubMed Scopus (58) Google Scholar, 21Masumoto H. Hawke D. Kobayashi R. Verreault A. A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response.Nature. 2005; 436: 294-298Crossref PubMed Scopus (480) Google Scholar]. Finally, CREBBP modulates the activity of poly(ADP-ribose) polymerase-1 (PARP1), an accessory factor in transcriptional regulation and base-excision repair (BER) (reviewed in [22Krishnakumar R. Kraus W.L. The PARP side of the nucleus: molecular actions, physiological outcomes, and clinical targets.Mol Cell. 2010; 39: 8-24Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar]). Because the amount of CREBBP is dose-limiting within the cell [23Horvai A.E. Xu L. Korzus E. et al.Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300.Proc Natl Acad Sci U S A. 1997; 94: 1074-1079Crossref PubMed Scopus (388) Google Scholar, 24Kamei Y. Xu L. Heinzel T. et al.A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar], a decrease in its availability is likely to impair its ability to enhance DNA repair.We previously reported that ∼40% of Crebbp heterozygous mice develop hematological malignancies [25Kung A.L. Rebel V.I. Bronson R.T. et al.Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP.Genes Dev. 2000; 14: 272-277PubMed Google Scholar] and others have shown that CREBBP is one of the genes involved in chromosomal translocations found in patients suffering from therapy-related MDS [26Bedford D.C. Kasper L.H. Fukuyama T. Brindle P.K. Target gene context influences the transcriptional requirement for the KAT3 family of CBP and p300 histone acetyltransferases.Epigenetics. 2010; 5: 9-15Crossref PubMed Scopus (209) Google Scholar]. We now report that Crebbp+/− mice invariably develop MDS/MPN within 9 to 12 months of age and are hypersensitive to γ-radiation. Mechanistically, we find a marked decrease in PARP1 activity upon exposure to ionizing radiation and a reduction of key BER proteins in progenitor and stem cell-enriched bone marrow (BM), suggesting deficient DNA repair as a contributing factor to their disease.Material and methodsMiceCrebbp+/− mice [25Kung A.L. Rebel V.I. Bronson R.T. et al.Gene dose-dependent control of hematopoiesis and hematologic tumor suppression by CBP.Genes Dev. 2000; 14: 272-277PubMed Google Scholar] were fully backcrossed onto a C57BL/6 background. Wild-type (WT) littermates served as controls. Mice were bred and maintained under microisolator conditions at the animal facility of University of Texas Health Science Center at San Antonio. All animal procedures were in accordance with University policies regarding animal care and use.Total body irradiation (TBI) and survival analysisWT and Crebbp+/− mice (3- to 6-month-old) received a total dose of 10 or 11 Gy (90–100 cGy/min from a Co60 source (Theratron T-780 unit; Atomic Energy of Canada Limited, Chalk River, Ontario, Canada), delivered as two equal doses of 5 or 5.5 Gy, respectively, 5 hours apart. Kaplan-Meier curves and log rank survival statistics were generated using the R-project survival package [27R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria2009Google Scholar, 28Harrington D.P. Fleming T.R. A class of rank test procedures for sensored survival data.Biometrika. 1982; 69: 553-566Crossref Scopus (775) Google Scholar].Blood analysis, histology, flow cytometry, and in vitro methylcellulose assaysStandard techniques were used. See Supplementary Materials and Methods for details (online only, available at www.exphem.org).Short hairpin RNA (shRNA) knockdown of CREBBP in EML1 cellsA lentivirus-encoded shRNA targeting the sequence 5′-CAAGCACTGGGAATTCTCT-3′ from mouse Crebbp was created by cloning oligonucleotides into the FSIPPW vector as described previously [29Kanellopoulou C. Muljo S.A. Kung A.L. et al.Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.Genes Dev. 2005; 19: 489-501Crossref PubMed Scopus (1048) Google Scholar]. A lentivirus targeting enhanced green fluorescent protein (5′-AAGAACGGCATCAAGGTGAACTT-3′) was used as a control. Both were packaged as described previously [30Lois C. Hong E.J. Pease S. Brown E.J. Baltimore D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors.Science. 2002; 295: 868-872Crossref PubMed Scopus (1594) Google Scholar]. Cotransfection of 293TD cells was performed using lipofectamine 2000 as per manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).EML1 cells (CRL-11691; ATCC, Manassas, VA, USA) [31Tsai S. Bartelmez S. Sitnicka E. Collins S. Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development.Genes Dev. 1994; 8: 2831-2841Crossref PubMed Scopus (239) Google Scholar], were cultured in Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum (StemCell Technologies, Vancouver, BC, Canada) and recombinant murine stem cell factor (100 ng/mL; R&D Systems, Minneapolis, MN, USA) and were never carried for more than 3 months. Undifferentiated EML1 cells were split 1 day before infection. Virus-containing supernatant supplemented with 8 μg/mL protamine was added to the cells and left until a complete medium change the next morning. At the end of day 2, another round of infection was performed using a flow-through infection protocol, as described previously [32Chuck A.S. Palsson B.O. Consistent and high rates of gene transfer can be obtained using flow-through transduction over a wide range of retroviral titers.Hum Gene Ther. 1996; 7: 743-750Crossref PubMed Scopus (93) Google Scholar]. On day 3, infected cells were selected in puromycin (3 μg/mL). EML1 cells were cloned in methylcellulose-based medium (M3234, StemCell Technologies) and expanded in liquid medium.Gene expression and network analysisTotal RNA was isolated in two independent experiments from HSCs sorted from WT and Crebbp+/− day 14.5 fetal livers as described in the Supplementary Material and Methods (online only, available at www.exphem.org). For each, 12 ng were amplified, in duplicate, using the Ovation RNA Amplification Kit (NuGen Technologies, Inc., San Carlos, CA, USA) and hybridized to Affymetrix Gene Chip Mouse Genome 430 2.0. Data files are MIAME-compliant and available from the Gene Expression Omnibus (accession GSE18061). Arrays were normalized, corrected for background and analyzed using R and the Bioconductor gcrma package [27R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria2009Google Scholar, 33Gentleman R.C. Carey V.J. Bates D.M. et al.Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar]. After averaging technical replicates, litter-paired t-tests with p values 1.5 were used as the cutoff for calling significant change. Quantitative reverse transcription polymerase chain reaction for two genes on three independent samples were consistent with the microarray results (Klf6 average ± standard deviation: on microarray = 1.8 ± 0.0, by quantitative reverse transcription polymerase chain reaction = 2.8 ± 1.4; Tcf4 average ± standard deviation on microarray = 1.1 ± 0.03, by quantitative reverse transcription polymerase chain reaction = 1.1 ± 0.09). To generate protein interaction networks (PINs), murine genes were mapped via the National Center for Biotechnology Information HomoloGene database (May 2009) to their human homologs (Supplementary Table E1; online only, available at www.exphem.org). The human proteins were used to retrieve direct binding partners from the human interactome [34Rual J.F. Venkatesan K. Hao T. et al.Towards a proteome-scale map of the human protein-protein interaction network.Nature. 2005; 437: 1173-1178Crossref PubMed Scopus (2260) Google Scholar], where both binding partners were called "present" by Affymetrix MAS5 (Bioconductor affy package implementation [35Gautier L. Cope L. Bolstad B.M. Irizarry R.A. affy–analysis of Affymetrix GeneChip data at the probe level.Bioinformatics. 2004; 20: 307-315Crossref PubMed Scopus (3788) Google Scholar]) in at least one sample, resulting in a reference "HSC PIN" of 4,237 proteins and 14,704 interactions. Similarly, the 93 distinct genes we found significantly altered in Crebbp+/− HSCs relative to WT corresponded to 39 human homologs that were represented in the HSC PIN. Together, these 39 proteins and their direct interactors comprise the Crebbp-target PIN of 258 proteins and 257 interactions. Cytoscape [36Cline M.S. Smoot M. Cerami E. et al.Integration of biological networks and gene expression data using Cytoscape.Nat Protoc. 2007; 2: 2366-2382Crossref PubMed Scopus (1791) Google Scholar] was used to visualize the resulting networks and its BinGO plugin [37Maere S. Heymans K. Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks.Bioinformatics. 2005; 21: 3448-3449Crossref PubMed Scopus (3030) Google Scholar] to determine Gene Ontology (GO) annotation enrichment. We used our HSC PIN as the background for enrichment with a p value <0.01 as cutoff.Protein extractsPeripheral blood (PB) cells were obtained from more than three mice and pooled. Leukocytes were irradiated with 6 Gy (1 Gy/min) using a Gammacell 40 Cesium Unit (Atomic Energy of Canada Limited). Postirradiation cells were either put on ice directly or incubated for various times at 37°C to allow DNA repair to occur. Cells were lyzed in RIPA buffer for CREBBP Westerns or in NaCl lysis buffer (0.1 M NaCl, 50 mM Tris-HCl [pH 7.2], 1 mM dithiothreitol) containing phosphatase and protease inhibitors in other cases. After lysis, CREBBP and PARP1 samples were centrifuged at 14,000 rpm for 10 minutes at 4°C and supernatant protein concentrations determined by BCA protein assays (Pierce, Rockford, IL, USA). For BER protein Westerns, lysates were passed five times through a QIAshredder homogenizer (Qiagen, Valencia, CA, USA), then centrifuged at 16,000 rcf for 10 minutes at 4°C and the supernatants concentrated for 40 minutes at 14,800 rcf in Amicon Ultra-0.5 filter devices (Millipore, Billerica, MA, USA). After concentration, the protein lysates were diluted 1:1 with a 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol solution, and protein concentrations determined by Bradford assays (Sigma, St Louis, MO, USA).Western blotsEqual amounts of protein extract were separated on 12% Bis-Tris gels and transferred to nitrocellulose membranes (Invitrogen). Primary antibodies used: CREBBP (AC26 [38Yao T.P. Oh S.P. Fuchs M. et al.Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300.Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (813) Google Scholar]), XRCC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and from Abcam, Cambridge, MA, USA: ACTB, LIG1, POLB, APEX1, TRP53, phospho-TRP53 (Ser15), PARP1. They were visualized with horseradish peroxidase–coupled secondary antibodies (Cell Signaling, Danvers, MA, USA) and ECL Plus solution (Amersham, Piscataway, NJ, USA) and quantified with densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA).PARP1 activity assayPARP1 activity in protein extracts from PB and BM (200 ng/25 μL), or EML1 cells (400 ng/25 μL) was measured using the HT Colorimetric PARP1/Apoptosis Assay Kit (Trevigen, Gaithersburg, MD, USA) following manufacturer's instructions. The absorbance of the colorimetric substrate was read at 450 nm on a Spectramax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).Statistical analysisUnless otherwise indicated, Excel (Microsoft, Redmond, WA, USA) was used to perform t-tests. The R stats package [27R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria2009Google Scholar] was used for the paired condition and time-series t-test and the Kolmogorov-Smirnov distribution test. In all cases, p values <0.05 were considered statistically significant.ResultsMyelodysplastic features of Crebbp+/− miceTo determine whether Crebbp+/− mice might harbor previously undetected MDS, we compared the hematopoietic system of 3- to 4-month-old and 9- to 12-month-old Crebbp+/− mice with that of age-matched WT controls. The number of cells harvested from two femurs was similar for both age groups and genotypes (Fig. 1A ), but Crebbp+/− mice were significantly smaller by weight than their WT counterpart (Fig. 1B). When BM cellularity was corrected for weight, we found that the marrow of Crebbp+/− mice was significantly more cellular than WT (Fig. 1C). At both ages, a mild but significant splenomegaly was also observed in Crebbp+/− mice (Fig. 1D).This marrow hypercellularity was not accompanied by an increase in the number of colony-forming cells (CFCs). On the contrary, 9- to 12-month-old Crebbp+/− mice had significantly fewer CFCs in their marrow than WT mice, most notably granulocytic and monocytic CFCs (Fig. 1E). No significant differences were detectable between young Crebbp+/− and WT mice (data not shown). A decrease in the numbers of myeloid CFCs in the context of an overall increase in BM cellularity and splenomegaly is indicative of abnormal myeloid differentiation.Histological examination of blood smears and BM preparations revealed distinct dysplastic features [39Beachy S.H. Aplan P.D. Mouse models of myelodysplastic syndromes.Hematol Oncol Clin North Am. 2010; 24: 361-375Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 40Brunning R.D. Orazi A. Germing U. Le Beau M.M. Myelodysplastic syndromes/neoplasms, overview.in: Swerdlow S.H. Campo E. Harris N.L. WHO classification of tumours of haematopoietic and lymphoid tissues. International Agency for Research on Cancer (IARC), Lyon2008: 88-93Google Scholar] of Crebbp+/− blood cell differentiation (Fig. 2), including hypersegmented granulocytes (Fig. 2B; 55% of 9- to 12-month-old mice) and leukocytes with a pseudo Pelger-Huët anomaly (Fig. 2C; 22%). Crebbp+/− BM preparations confirmed the hypercellularity and showed an increased myeloid to erythroid ratio, mostly due to an excess of mature granulocytes (Fig. 2E). More than half of the 9- to 12-month-old Crebbp+/− mice exhibited either increased numbers of megakaryocytes or abnormal forms such as hyperlobulated cells (Fig. 2F) or naked nuclei (Fig. 2G).Figure 2Histopathology and Annexin V staining of WT and Crebbp+/− animals. (A–C) Wright-stained PB cells from 9- to 12-month-old mice. (A) WT control. (B, C) Crebbp+/− blood smears showing hypersegmented granulocytes (i.e., granulocytes with more than six segments of irregular size) (B, inset) and (C) a pseudo Pelger-Huët anomaly. (D–H) Bone sections from 9- to 12-month-old mice. (D) WT control. (E–H) Crebbp+/− sections. (E) Hypercellular marrow and an increased myeloid to erythroid ratio due to increased mature granulocytes, particularly near the bony trabeculae. (F, G) Abnormal megakaryocytic differentiation with hyperlobulated megakaryocytes (F, arrow and inset) and naked megakaryocytic nuclei (G, arrow and inset). (H, and inset) Clusters of immature precursor cells present in the middle of the marrow cavity. Magnification: 40× (A, B, D–H), 60× (C). (I) Quantification of apoptosis by Annexin V staining of whole (WBM) and lineage-depleted (Lin−) bone marrow isolated from WT and Crebbp+/− mice (n = 7). p value indicated where significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Unlike older mice, 3- to 4-month-old Crebbp+/− mice displayed none of these characteristics; interestingly, however, 2 of 14 young animals examined had small clusters of immature cells in the center of the marrow space (Fig. 2H and inset). This atypical localization of immature precursors is indicative of very early stages of myelodysplastic hematopoiesis and possibly the onset of leukemia. Another common finding in MDS, particularly in its early stages, is an increase in apoptosis in marrow progenitors (reviewed in [1Corey S.J. Minden M.D. Barber D.L. Kantarjian H. Wang J.C. Schimmer A.D. Myelodysplastic syndromes: the complexity of stem-cell diseases.Nat Rev Cancer. 2007; 7: 118-129Crossref PubMed Scopus (268) Google Scholar]). Consistent with this, Figure 2I shows a significant increase in Annexin V+ cells in the lineage-depleted (Lin−) fraction of marrow enriched for stem and progenitor cells but not in whole BM.Altered numbers of HSCs and myeloid progenitors in Crebbp+/− miceFluorescence-activated cell sorting analysis at 3 to 4 months of age showed that the only significant, albeit small, difference in hematopoietic cell populations between Crebbp+/− and WT mice was an increased proportion of Gr-1loMac-1++ myeloid cells in the BM (15.3% ± 2.4% vs 12.7% ± 1.3%; p = 0.015). By 9 to 12 months of age, however, the frequency of long-term HSCs was significantly lower in Crebbp+/− BM compared to control (Fig. 3A ), resulting in ∼2-fold fewer long-term HSCs per femur (2300 ± 1100 vs 4400 ± 1900, respectively; p = 0.007). These mice also showed a decrease in common myeloid progenitors and an expansion of granulocyte/macrophage progenitors (Fig. 3B). No differences between Crebbp+/− and WT mice were found with respect to megakaryocyte/erythroid progenitors (Fig. 3B) or common lymphoid progenitors (Fig. 3C).Figure 3Abnormal numbers of HSCs, common myeloid progenitors, and granulocyte/macrophage progenitors in Crebbp+/− mice. (A–C) Left and middle panels depict the sorting strategy used for each BM population, and bar graphs on the right show average percentages (±standard deviation) for the corresponding cell population(s). (A) Long-term HSCs (LT-HSCs) are selected from mature lineage marker–negative (Lin−) c-Kit++Sca-1+ cells (rightmost sorting gate of the dot plot) that are also CD34− (histogram). (B) Immature myeloid progenitors are identified by separating Lin−Sca-1−c-Kit++ BM cells on the basis of CD16/32 and CD34 expression as shown. (C) Common lymphoid progenitors (CLPs) are purified from whole BM (WBM) based first on expression of interleukin-7R but not of other mature lineage markers. Cells in the sorting gate of the dot plot are then further analyzed for c-Kit and Sca-1 expression (contour plot). CLPs are c-Kit+ Sca-1(intermediate) cells. Significant differences are indicated by p values with 9 to 12 mice used in each analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because primitive BM progenitors represent only a very small proportion of the total BM content, the expansion of granulocyte/macrophage progenitors alone cannot explain the greater marrow cellularity observed in the older Crebbp+/− mice relative to controls. Moreover, histological analysis suggested that the difference was due to an increase in mature myeloid cells in the marrow (Fig. 2E). Indeed, relative to controls, Crebbp+/− marrow contained significantly more Gr-1loMac-1+/+ and Gr-1+/+Mac-1+/+ myeloid cells (Fig. 4A ). Concurrent PB cell analysis of 9- to 12-month-old mice by fluorescence-activated cell sorting (Fig. 4B) and complete blood count (Fig. 4C) showed a significant increase in granulocytes, while the lymphoid cell compartment contracted. In contrast, total leukocyte, erythrocyte, and platelet numbers measured by complete blood count were similar to age-matched controls (data not shown). By 1 year of age, the relative number of myeloid cells had therefore significantly increased
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