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Differential expression of AURKA and AURKB genes in bone marrow stromal mesenchymal cells of myelodysplastic syndrome: correlation with G-banding analysis and FISH
2012; Elsevier BV; Volume: 41; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2012.10.009
ISSN1873-2399
AutoresFábio Morato de Oliveira, Antonio R. Lucena‐Araujo, Maria do Carmo Favarin, Patrícia Vianna Bonini Palma, Eduardo Magalhães Rego, Roberto Passetto Falcão, Dimas Tadeu Covas, Aparecida Maria Fontes,
Tópico(s)Sarcoma Diagnosis and Treatment
ResumoIt has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells. It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells. According to a multistep pathogenesis model proposed for leukemias, after the initial damage of the hematopoietic stem cell, additional genomic abnormalities may affect these cells, providing them a proliferative advantage [1Bernasconi P. Molecular pathways in myelodysplastic syndromes and acute myeloid leukemia: relationships and distinctions-a review.Br J Haematol. 2008; 142: 695-708Crossref PubMed Scopus (55) Google Scholar, 2Knudson A. Mutation and cancer: statistical study of retinoblastoma.Proc Natl Acad Sci U S A. 1971; 68: 820-823Crossref PubMed Scopus (5427) Google Scholar]. In addition to the genomic instability, the hematopoietic microenvironment is involved in the pathophysiology of myelodysplastic syndrome (MDS) [3Bejar R. Levine R. Ebert B.L. Unraveling the molecular pathophysiology of myelodysplastic syndromes.J Clin Oncol. 2011; 29: 504-515Crossref PubMed Scopus (249) Google Scholar, 4Podar K. Richardson P.G. Hideshima T. Chauhan D. Anderson K.C. The malignant clone and the bone-marrow environment.Best Pract Res Clin Haematol. 2007; 20: 597-612Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. Disruption of the architecture of hematopoiesis is a common finding in MDS. It involves both altered localization of hematopoietic elements within the bone marrow and alterations in components that compose the microenvironment. In this way, all hematopoietic lineages in MDS may be affected [4Podar K. Richardson P.G. Hideshima T. Chauhan D. Anderson K.C. The malignant clone and the bone-marrow environment.Best Pract Res Clin Haematol. 2007; 20: 597-612Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 5Nimer S.D. MDS: a stem cell disorder—but what exactly is wrong with the primitive hematopoietic cells in this disease?.Hematology Am Soc Hematol Educ Program. 2008; : 43-51Crossref PubMed Scopus (60) Google Scholar, 6Raaijmakers M.H. Myelodysplastic syndromes: revisiting the role of the bone marrow microenvironment in disease pathogenesis.Int J Hematol. 2012; 95: 17-25Crossref PubMed Scopus (50) Google Scholar]. Another important element of the hematopoietic microenvironment is the bone marrow stromal mesenchymal cells (MSCs). These cells have an important role supporting and regulating the proliferation and differentiation of hematopoietic stem cells; they also have an immunoregulatory function [7Friedenstein A.J. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.Exp Hematol. 1974; 2: 83-92PubMed Google Scholar, 8Meirelles Lda S. Fontes A.M. Covas D.T. Caplan A.I. Mechanisms involved in the therapeutic properties of mesenchymal stem cells.Cytokine Growth Factor Rev. 2009; 20: 419-427Abstract Full Text Full Text PDF PubMed Scopus (1002) Google Scholar]. Whether abnormalities associated with MSCs contribute to pathogenesis of MDS and leukemias, and subsequently disease progression, is still not totally clear. However, some differences have been observed between the MSCs of leukemia patients and those of healthy donors [9Flores-Figueroa E. Montesinos J.J. Flores-Guzmán P. et al.Functional analysis of myelodysplastic syndromes-derived mesenchymal stem cells.Leuk Res. 2008; 32: 1407-1416Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10Konopleva M. Konoplev S. Hu W. Zaritskey A.Y. Afanasiev B.V. Andreeff M. Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins.Leukemia. 2002; 16: 1713-1724Crossref PubMed Scopus (307) Google Scholar]. It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce MDS with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia (AML) [11Raaijmakers M.H. Mukherjee S. Guo S. et al.Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia.Nature. 2010; 464: 852-857Crossref PubMed Scopus (802) Google Scholar]. However, the presence of cytogenetic abnormalities in the MSCs of patients with hematologic disorders is considered a controversial finding by some groups [12Blau O. Hofmann W.K. Baldus C.D. et al.Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia.Exp Hematol. 2007; 35: 221-229Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 13Flores-Figueroa E. Arana-Trejo R.M. Gutiérrez-Espíndola G. Pérez-Cabrera A. Mayani H. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization.Leuk Res. 2005; 29: 215-224Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 14Lopez-Villar O. Garcia J.L. Sanchez-Guijo F.M. et al.Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q- syndrome.Leukemia. 2009; 23: 664-672Crossref PubMed Scopus (106) Google Scholar, 15Klaus M. Stavroulaki E. Kastrinaki M.C. et al.Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes.Stem Cells Dev. 2010; 19: 1043-1054Crossref PubMed Scopus (57) Google Scholar]. Classic cytogenetic and fluorescence in situ hybridization (FISH) aim to determine whether the MSCs of leukemia patients harbor genomic abnormalities that may act on the fate of hematopoietic stem cells [12Blau O. Hofmann W.K. Baldus C.D. et al.Chromosomal aberrations in bone marrow mesenchymal stroma cells from patients with myelodysplastic syndrome and acute myeloblastic leukemia.Exp Hematol. 2007; 35: 221-229Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 13Flores-Figueroa E. Arana-Trejo R.M. Gutiérrez-Espíndola G. Pérez-Cabrera A. Mayani H. Mesenchymal stem cells in myelodysplastic syndromes: phenotypic and cytogenetic characterization.Leuk Res. 2005; 29: 215-224Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 14Lopez-Villar O. Garcia J.L. Sanchez-Guijo F.M. et al.Both expanded and uncultured mesenchymal stem cells from MDS patients are genomically abnormal, showing a specific genetic profile for the 5q- syndrome.Leukemia. 2009; 23: 664-672Crossref PubMed Scopus (106) Google Scholar, 15Klaus M. Stavroulaki E. Kastrinaki M.C. et al.Reserves, functional, immunoregulatory, and cytogenetic properties of bone marrow mesenchymal stem cells in patients with myelodysplastic syndromes.Stem Cells Dev. 2010; 19: 1043-1054Crossref PubMed Scopus (57) Google Scholar]. Aurora kinases are mitotic kinases with an important role regulating the G2/M phase of the cell cycle and various mitotic events, including centrosome duplication, mitotic spindle assembly, chromosome segregation, and cytokinesis at the end of telophase [16Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints.Nat Rev Mol Cell Biol. 2001; 2: 21-32Crossref PubMed Scopus (1228) Google Scholar]. A correlation between overexpression of the AURKA gene and clinical aggressiveness has been described in esophageal cancer, gastric cancer, bladder cancer, and hepatocellular carcinoma [17Jeng Y.M. Peng S.Y. Lin C.Y. Hsu H.C. Overexpression and amplification of Aurora-A in hepatocellular carcinoma.Clin Cancer Res. 2004; 10: 2065-2071Crossref PubMed Scopus (270) Google Scholar, 18Kamada K. Yamada Y. Hirao T. et al.Amplification/overexpression of Aurora-A in human gastric carcinoma: potential role in differentiated type gastric carcinogenesis.Oncol Rep. 2004; 12: 593-599PubMed Google Scholar, 19Tanaka E. Hashimoto Y. Ito T. et al.The clinical significance of Aurora-A/STK15/BTAK expression in human esophageal squamous cell carcinoma.Clin Cancer Res. 2005; 11: 1827-1834Crossref PubMed Scopus (110) Google Scholar, 20Sen S. Zhou H. White R.A. A putative serine/threonine kinase encoding gene BTAK on chromosome 20q13 is amplified and overexpressed in human breast cancer cell lines.Oncogene. 1997; 14: 2195-2200Crossref PubMed Scopus (415) Google Scholar]. Recently, our group demonstrated a significant association between high expression of AURKA and cytogenetic profile in AML. According to our findings, AURKA expression was independently associated with high white blood cell counts, and the majority of AML patients who overexpressed AURKA and AURKB exhibited unfavorable cytogenetic abnormalities [21Lucena-Araujo A.R. de Oliveira F.M. Leite-Cueva S.D. dos Santos G.A. Falcao R.P. Rego E.M. High expression of AURKA and AURKB is associated with unfavorable cytogenetic abnormalities and high white blood cell count in patients with acute myeloid leukemia.Leuk Res. 2011; 35: 260-264Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. In the present study, we compared the expression profile of AURKA and AURKB in hematopoietic cells and MSCs of 60 MDS patients and 20 healthy donors. We also performed G-banding, spectral karyotype (SKY) analysis, and FISH on MSCs by using the most typical panel of FISH probes for MDS [inv(3q)(q21;q26.2), t(3;3)(q21;q26.2), −5/del(5q), −7/del(7q), +8 and del(20q)] and AURKA/B. Our results showed that significant differences were observed between the levels of expression of aurora AURKA and AURKB in MSCs compared with hematopoietic cells, healthy donors, and cytogenetic analysis. Bone marrow samples of 60 untreated MDS patients were selected for this study (22 men, 38 women; median age of 54 years; range, 28–85 years old). All samples were collected between May 2009 and June 2012. The patients selected for this study had no previous history of any other hematologic disorder and had a confirmed diagnosis of MDS by clinical and laboratory parameters. According to the World Health Organization classification [22Vardiman 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 (1776) Google Scholar], the majority of patients studied were classified as refractory cytopenia with multilineage dysplasia 34/60 (56%), followed by refractory anemia 11/60 (18%), refractory anemia with excess blasts I 11/60 (18%), and four cases of 5q syndrome (8%). The clinical and biological characteristics of the 60 MDS patients included in the present investigation are presented in Table 1. Normal bone marrow samples were obtained from 20 healthy donors (10 men, 10 women) with a median age of 52 years (Table 2). The MDS and healthy donor samples have been obtained from the University Hospital, Medical School of Ribeirão Preto, University of São Paulo, Brazil, and from other hematologic centers in the State of São Paulo, Brazil. The study was approved by the University Hospital Ethics Committee, and all individuals gave their written, informed consent before entering the study.Table 1Clinical and cytogenetic characteristics of MDS and MSC MDS-derived samplesMDSMXCWHO classificationAge/SexHematopoietic cells: G-banding analysisIPSSMSC: G-banding analysisMSC: SKY analysisMSC - FISH analysist(3;3)/inv(3q)del(5q)del(7q)trissomy 8del(20q)1RA67/M46,XY[20]Low44,XY,−9[5],−12[6]/46,XY[13]44,XY,−9[3],−12[3]/46,XY[7]negnegnegnegneg258/F46,XX[20]Low46,XX[20]46,XX[10]negnegnegnegneg357/F46,XX[20]Low49,XX,+7[4],+15[6],+17[4]/46,XX[14]49,XX,+7[2],+15[3],+17[4]/46,XX[5]negnegnegnegneg443/F46,XX[20]Low46,XX[20]46,XX[10]negnegnegnegneg547/F46,XX,del(7)(q22q36)[3]/46,XX[17]INT-146,XX[20]46,XX[10]negnegnegnegneg680/F47,XX,+15[4]/46,XX[16]INT-146,XX[20]46,XX[10]negnegnegnegneg745/F46,XX[20]Low45,XX,−5[4]/46,XX[16]45,XX,−5[3]/46,XX[7]negposnegnegneg871/M46,XY[20]Low46,XY[20]46,XY[10]negnegnegnegneg967/M46,XY[20]Low46,XY[20]46,XY[10]negnegnegnegneg1054/M47,XY,+8[6]/46,XY[14]INT-146,XY[20]46,XY[10]negnegnegnegneg1164/M46,XY[20]Low46,XY[20]46,XY[10]negnegnegnegneg12RCMD66/F46,XX,inv(3)(q21q26)[7]/46,XX[13]INT-146,XY[20]46,XY[10]negnegnegnegneg1377/F46,XX,del(20)(q11)[7]/46,XX[13]INT-146,XX[20]46,XX[10]negnegnegnegneg1485/F46,XX,del(20)(q11)[6]/46,XX[14]INT-145,X,−X[3]/46,XX[17]45,X,−X[3]/46,XX[7]negnegnegnegneg1553/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg1659/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg1757/F47,XX,+8[20]INT-143,XX,−13,−19,−22[6]/46,XX[14]43,XX,−13,−19,−22[4]/46,XX[6]negnegnegnegneg1872/F46,XX,dup(1)(q32q21)[5]/46,XX[15]INT-146,XX[20]46,XX[10]negnegnegnegneg1959/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg2055/F46,XX,del(20)(q11)[8]/46,XX[12]INT-145,XX,−7[4]/46,XX[16]45,XX,−7[4]/46,XX[6]negnegposnegneg2152/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg2261/F46,XX[20]INT-147,XX,+15[2]/46,XX[18]47,XX,+15[2]/46,XX[8]negnegnegnegneg2371/F46,XX[20]INT-144,XX,−6[3],−19[3]/46,XX[14]44,XX,−6[2],−19[3]/46,XX[5]negnegnegnegneg2459/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg2566/F46,XX,del(7)(q22)[8]/46,XX[12]INT-246,XX[20]46,XX[10]negnegnegnegneg2653/F46,XX[20]Low46,XX[20]46,XX[10]negnegnegnegneg2757/F47,XX,+i(17)(q10)[6]/46,XX[14]INT-247,XX,+12,t(14;15)(q24;q22)[7]/46,XX[13]47,XX,+12,t(14;15)(q24;q22)[2]/46,XX[8]negnegnegnegneg2852/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg2965/F44,XX,−4[3],del(7)(p15)[2],del(12)(p12)[3]/46,XX[10]INT-245,XX,−6[5]/46,XX[15]45,XX,−6[3]/46,XX[7]negnegnegnegneg3071/M46,XY,del(20)(q11)[6]/46,XY[14]INT-144,XY,−4[6],−5[7]/46,XY[7]44,XY,−4[3],−5[2]/46,XY[5]negposnegnegneg3172/M45–46,X,−Y[6],del(17)(p12)[4]/46,XY[16]INT-146,XY[20]46,XY[10]negnegnegnegneg3269/M46,XY[20]INT-146,XY,i(12)(q10)[3]/46,XY[17]46,XY,i(12)(q10)[3]/46,XY[7]negnegnegnegneg3372/M47,XY,+8[7]/46,XY[13]INT-146,XY[20]46,XY[10]negnegnegnegneg3467/M46–47,XY,add(17)(p12)[4],+8[6]/46,XY[14]INT-246,XY[20]46,XY[10]negnegnegnegneg3567/M47,XY,+7[8]/46,XY[12]INT-246,XY[20]46,XY[10]negnegnegnegneg3662/M46,XY,del(13)(q12q14)[9]/46,XY[9]INT-146,XY[20]46,XY[10]negnegnegnegneg3765/M46,XY[20]INT-146,XY[20]46,XY[10]negnegnegnegneg3861/M47,XY,+8[8]/46,XY[11]INT-146,XY,del(13)(q24)[6]/46,XY[14]46,XY,del(13)(q24)[3]/46,XY[7]negnegnegnegneg3962/M47–48,XY,+5[3],+del(8)(q21)[4]/46,XY[16]INT-244,X,−Y[4],−19[5]/46,XY[12]44,X,−Y[3],−19[6]/46,XY[6]negnegnegnegneg4044/M47,XY,+8[20]INT-146,XY[20]46,XY[10]negnegnegnegneg4139/M46,XY[20]INT-146,XY[20]46,XY[10]negnegnegnegneg4227/M48,XY,+8,+9[14]/46,XY[6]INT-146,XY[20]46,XY[10]negnegnegnegneg4333/M46,XY[20]INT-145,XY−6[5]/46,XY[15]45,XY−6[2]/46,XY[8]negnegnegnegneg4442/F46,XX,del(20)(q10)[8]/46,XX[12]INT-146,XX[20]46,XX[10]negnegnegnegneg4536/F47,XX,+8[12]/46,XX[8]INT-146,XX[20]46,XX[10]negnegnegnegneg46RAEB I62/F47,XX,+9[6]/46,XX[14]INT-246,XX[20]46,XX[10]negnegnegnegneg4783/F47,XX,+4[5]/46,XY[15]INT-246,XX[20]46,XX[10]negnegnegnegneg4847/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg4965/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg5059/F46,XX[20]INT-146,XX[20]46,XX[10]negnegnegnegneg5174/M45,X,−Y[8]/46,XY[12]INT-146,XY[20]46,XY[10]negnegnegnegneg5258/M46,XY[20]INT-146,XY[20]46,XY[10]negnegnegnegneg5359/M46,XY,dup(1)(q21q32)[9]/46,XY[11]INT-245,XY,−Y[4]/46,XY[16]45,XY,−Y[3]/46,XY[7]negnegnegnegneg5461/F47,XX,+8,del(20)(q13q13)[14]/46,XX[6]INT-246,XX[20]46,XX[10]negnegnegnegneg5547/F46,XX,del(7)(q11)[6]/46,XX[14]INT-246,XX[20]46,XX[10]negnegnegnegneg5628/F47,XX,inv(3)(p21q25),+21[20]INT-246,XX[20]46,XX[10]negnegnegnegneg575q Syndrome58/F46,XX,del(5)(q31)[4]/46,XX[16]Low47,XX,+9[6]/46,XX[14]47,XX,+9[2]/46,XX[8]negnegnegnegneg5834/F46,X,i(X)(p10),del(5)(q13q33)[16]/46,XX[4]INT-146,XX[20]46,XX[10]negnegnegnegneg5928/F46,XX,del(5)(q13q33)[20]Low46,XX[20]46,XX[10]negnegnegnegneg6045/F47,XX,del(5)(q22q33),+8[15]/46,XX[5]INT-146,XX[20]46,XX[10]negnegnegnegnegFISH = fluorescence in situ hybridization; IPSS = International Prognostic Score System; MSC = mesenchymal stem cell; RA = refractory anemia; RAEB I = refractory anemia with excess blasts-1; RCMD = refractory cytopenia with multilineage dysplasia; SKY = spectral karyotype; WHO = World Health Organization. Open table in a new tab Table 2Cytogenetic characteristics of health donors (control group)Health donorsAge (years)SexHematopoietic cells: G-banding analysisMSC: G-banding analysis157F46,XX[20]46,XX[20]258F46,XX[20]46,XX[20]347F46,XX[20]46,XX[20]443F46,XX[20]46,XX[20]557F46,XX[20]46,XX[20]650F46,XX[20]46,XX[20]745F46,XX[20]46,XX[20]861F46,XX[20]46,XX[20]957F46,XX[20]46,XX[20]1044F46,XX[20]46,XX[20]1154M46,XY[20]46,XY[20]1256M46,XY[20]46,XY[20]1347M46,XY[20]46,XY[20]1445M46,XY[20]46,XY[20]1553M46,XY[20]46,XY[20]1649M46,XY[20]46,XY[20]1737M46,XY[20]46,XY[20]1852M46,XY[20]46,XY[20]1959M46,XY[20]46,XY[20]2055M46,XY[20]46,XY[20]F = Female; M = male. Open table in a new tab FISH = fluorescence in situ hybridization; IPSS = International Prognostic Score System; MSC = mesenchymal stem cell; RA = refractory anemia; RAEB I = refractory anemia with excess blasts-1; RCMD = refractory cytopenia with multilineage dysplasia; SKY = spectral karyotype; WHO = World Health Organization. F = Female; M = male. Mononuclear cells (MNCs) were isolated from bone marrow samples using Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) during initial diagnosis. Eight MNCs were cultured in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 15% heat-inactivated standard fetal bovine serum (HyClone, Logan, UT, USA), L-glutamine (2 mmol/L; Invitrogen), and 1% penicillin-streptomycin (Invitrogen). All MSCs (MDS and healthy donors) were collected after a third passage for subsequent cytogenetic analyses, FISH studies, and RNA isolation. The following monoclonal antibodies were used to characterize both MSCs: CD105-phycoerythrin (PE) (Serothec, Oxford, UK), CD73-PE, CD31-fluorescein isothiocyanate (FITC), CD45-FITC, CD14-PE, CD34-PE, HLA-Dr-FITC, CD90-PE, CD13-PE, CD140-PE, and CD146-PE (Becton Dickinson, San Jose, CA, USA). Adipogenic and osteogenic differentiation was evaluated as described previously [23Covas D.T. Siufi J.L. Silva A.R. Orellana M.D. Isolation and culture of umbilical vein mesenchymal stem cells.Braz J Med Biol Res. 2003; 36: 1179-1183Crossref PubMed Scopus (152) Google Scholar]. MSCs were cultured at 3 × 104/cm2 in either osteogenic or adipogenic medium (Dulbecco modified Eagle medium [DMEM] with 10 mmol/LM β-glycerophosphate, 10−7 mol/L dexamethasone, and 0.2 mmol/L ascorbic acid and DMEM with 10% FCS, 10−6 mol/L dexamethasone, 50 μg/mL ascorbic acid, and 100 μg/mL 1-methyl-3-isobutyl-xanthine, respectively) for up to 3 weeks. After 22 days, differentiation to osteoblasts was observed by Von Kossa stain for calcium phosphate. Evidence of adipocytes differentiation was shown by oil-red staining for lipid. The doubling time of MSCs from healthy donors (n = 6) was compared with those of aneuploidy MSCs from MDS (n = 14). Doubling time was calculated at each passage as described previously [24Stenderup K. Justesen J. Clausen C. Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.Bone. 2003; 33: 919-926Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar]. Cytogenetic analysis of hematopoietic cells and MSCs was performed as described in standard protocols. MNCs were cultured for 72 hours in α-MEM (Invitrogen) supplemented with 15% heat-inactivated standard fetal bovine serum (HyClone, Logan, UT, USA) supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN, 2009) [25Shaffer L.G. Slovak M.L. Campbell L.J. ISCN 2009: an international system for human cytogenetic nomenclature 2009. S. Karger, Basel2009Google Scholar]. The metaphase images were acquired by using an Axio Imager M2 microscope (Zeiss, Jena, Germany) equipped with the BandView software, version 5.5 (ASI, Carlsbad, CA, USA). To confirm chromosomal abnormalities identified by G-banding in both hematopoietic cells and MSCs, SKY analysis was performed. We also applied a panel of "MDS FISH probe set" [inv(3q)(q21;q26.2), t(3;3)(q21;q26.2), −5/del(5q), −7/del(7q), +8 and del(20q)] on MSCs to search for specific abnormalities of MDS. The hybridization spots were evaluated using an Axio Imager M2 microscope (Zeiss) equipped with the FISHView software, version 5.5 (ASI). The cutoff levels for t(3;3)/inv(3q) (>2.5%), del(5q) (>3%), del(7q) (>2.4%), trisomy 8 (>2.5%), and del(20q) (>2.0%) were established according to the interphase FISH patterns observed in a group of 30 age- and sex-matched normal control peripheral blood samples studied with the same probes. The FISH probes used were purchased from Kreatech Diagnostics (Amsterdam, The Netherlands). For each sample, 300 interphase cells were viewed and counted. For SKY analysis, chromosome labeling was performed with the SKY fluorescent labeling kit (Applied Spectral Imaging, Migdal HaEmek, Israel) according to the manufacturer's protocol. A minimum of 20 metaphases were analyzed using the SkyView 5.5 software (ASI). In addition, FISH for AURKA and AURKB amplifications was also performed according to the manufacturer's instructions, on interphase nuclei of both hematopoietic cells and MSCs using commercial probes (AURKA: on AURKA (20q13)/20q11; and AURKB: AURKB (17/p13)/SE17; Kreatech Diagnostics). AURKA and AURKB probes are designed as a dual-color assay to detect amplification at 20q13 and 17p13, respectively. Amplification involving these genes regions will show multiple red signals, whereas the controls (MPARE1 for AURKA and SE17 for AURKB), both located in the centromeric region of their chromosomes, will provide two green signals. The criteria used for AURKA and AURKB gene amplifications were based on the number of spots presented during analysis. Genomic RNA was isolated from both MNCs and MSCs using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. Complementary DNA (cDNA) was synthesized from ∼1 μg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and following the manufacturer's instructions. For analysis of aurora kinase genes, primes and probes developed by Assay on Demand were used (AURKA: Hs00269212_m1; and AURKB: Hs00177782_m1; Applied Biosystems). The AURKA and AURKB genes and GAPDH messenger RNA, used as endogenous internal control for each sample, were analyzed in duplicate on the same MicroAmp optical 96-well plates using a 7500 Real-Time PCR System (Applied Biosystems). Real-time quantitative polymerase chain reaction (PCR) assays were performed in a final reaction volume of 20 μL. The comparative cycle threshold (Ct) method was used to determine the relative expression level of AURKA and AURKB genes. On a comparative analysis of MDS, MSCs samples, and healthy donors (MNCs), AURKA and AURKB gene expression was calculated as a relative quantification to the GAPDH housekeeping gene. The gene expression of AURKA and AURKB from MDS and MSCs samples was calculated as relative quantification to normal controls (ΔΔCt = ΔCtpatient – ΔCthealthy donors+) and expressed as 2−ΔΔCt. MSCs from MDS-derived lysates and from healthy subjects were used for Western blot analysis to determine the AURKA and AURKB protein concentration. Protein electrophoresis was performed using 10% of sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred to Hybond-P polyvinylidene diflouride membranes (Amersham Biosciences). The membranes were probed with rabbit anti-human Aurora-A antibody and Aurora-B antibody (Abcam, Cambridge, MA, USA; 1:500 dilution) and then probed again with anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Ambion, Austin, TX, USA; 1:8000 dilution). Comparisons between different groups were made using the Student t test and the two sided exact Fisher test (dichotomous variables); p < 0.05 was considered significant. Clonal chromosomal abnormalities were seen in 34 of 60 patients (57%), and normal karyotype was observed in 26 patients (43%). In all samples studied, the metaphase number was considered good, with more than 15 metaphases per slide (Table 1). Complex karyotype (≥3 abnormalities) was observed in only one patient (Fig. 1A, B), and most of the patients studied presented a single cytogenetic abnormality (24 of 60; 40%). Numerical abnormalities were seen in 18 of 60 (30%), with the extra copy of chromosome 8 being the most common abnormality. However, structural abnormalities were also present in 23 of 60 (38%) of the patients (Table 1). According to the ISCN criteria all abnormalities were clonal and confirmed by FISH. Among the MSC samples, 42 of 60 (70%) presented a normal karyotype, and 18 patients (30%) had chromosomal abnormalities (Table 1). As also seen in hematopoietic cells, the metaphase number in MSCs was considered satisfactory, with >10 metaphases per slide. Twenty metaphase cells were analyzed for each patient, with an estimated resolution of 450 bands per haploid set. Most of the chromosomal abnormalities seen in MSCs samples were numeric (14 of 18; 78%). In three patients, we found structural chromosomal abnormalities (Table 1). SKY analysis confirmed all alterations detected in MSCs (Table 1; Fig. 2). In addition, FISH analysis using a panel of probes for common abnormalities seen in MDS patients showed loss of chromosome 7 in only one MSC sample (Table 1). Considering the MSC group with abnormal karyotype, previous chromosomal abnormalities were seen in hematopoietic cells of 10 patients. However, the profile of cytogenetic abnormalities found was different. On the other hand, the six MDS patients with normal karyotype presented chromosomal abnormalities in MSCs (Table 1). Classic cytogenetic analysis (G-banding) was performed in 20 MSC samples of healthy donors (median age, 52 years). The median number of passages was three, and all karyotypes were described according to the ISCN (2009) [25Shaffer L.G. Slovak M.L. Campbell L.J. ISCN 2009: an international system for human cytogenetic nomenclature 2009. S. Karger, Basel2009Google Scholar]. For each patient, 20 metaphase cells were analyzed, at an average resolution of 450 bands per haploid set. All samples displayed a normal karyotype (Table 2). The average doubling times for MSCs from healthy donors (n = 6) and aneuploidy MSCs from MDS (n = 7) groups were 47.6 and 78.6 hours, respectively. We compared the expression profile of AURKA and AURKB in hematopoietic cells, MSCs, and healthy donors. We stratified the patients according to the ka
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