| Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic stem cells-derived myeloid neoplasms, dysplastic MDS hematopoietic stem/progenitor cells(HSC/HPC) representing aberration in both of proliferation and differentiation. Colony-forming cells(CFC) assay is the classic method for studing HPC in vitro and evaluating the capacity of hematopoietic cells.The Working Conferrence in Vienna(2006) made concesus statements, regarding "Markedly and persistently reduced colony-formation (±cluster formation) of bone marrow or/and circulating progenitor cells (CFU-assay)" as one of co-criterias in MDS diagnosis, nevertheless without regarding specific type or degree of CFC reduction. In this study, CFC-assay results in MDS patients were analyzed, and compared with non-severe aplastic anemia (NSAA) patients. Thus we may generalize the characters of CFC in MDS, and some biological features of this bone marrow failure disease could be revealed as well.Data of in vitro CFC and correlation with other related laboratory tests in155newly diagnosed MDS pateints were analyzed retrospectively, and to compare with data of in vitro CFC in122newly diagnosed NSAA. Results as follows:①Median number of BFU-E was9(0-157)/105BMNC, CFU-E30(0-425)/105BMNC and CFU-GM14(0-125)/105BMNC in patients with MDS, being significantly lower than those in healthy control; number of BFU-E and/or CFU-E was lower than the lower limit of normal in66cases (43.3%), CFU-GM lower in3cases (1.6%)and BFU-E and/or CFU-E with CFU-GM lower in70cases (43.9%).②Cluster/CFU-GM ratio was significantly lower (0.65vs1.0,p=0.049) in low blast group (MDS<5%blast in bone marrow smear)than in high blast group (MDS≥5%blast).There was a positive correlation between BFU-E and CFU-E (rblast<5%=0.728,p=0.000and rbiast≥5%-0.696,p=0.000) in both groups, with a higher correlation coefficient in low blast group(p<0.05). In all MDS patients, cluster had positive correlation with each type of CFC (r=0.415,0.338,0.642for BFU-E, CFU-E, CFU-GM, respectively. p=0.000), but had negative correlation with neutrophil alkaline phosphatase(N-ALP)positive rate and scores (rrate=-0.315,p=0.001and rscores=-2575,p=0.006).③The median number of each type of CFC was significantly higher in MDS group than in NSAA group(BFU-E9vs5,p=0.017;CFU-E30vs19.5,p=0.023;CFU-GM14vs10, p=0.003, respectively). Positive correlation between BFU-E and CFU-E were revealed in both MDS and NSAA group(rMDS=0.712,p=0.000and rNSAA=0.757,p=0.000),with a lower correlation coefficient in MDS(p<0.05).In this study we found early onset MDS already presented markedly decreased HPC, and particularly in erythroid progenitors extensively and severely. These results also proved number of BFU-E, CFU-E and CFU-GM can reflect capacity of HPC in vivo but not stand for normal hematopoietic clones, the number of clusters represent pathologic HPC clones but not exactly leukemic blasts; and indicated pathologic hematopoietic clones in patients with>5%blast onset have different biological characteristics from those with<5%blast. Human MLFIIP gene locus is at chromosome4q35.1, encoding a protein with some classic functional domains; recently some research groups proved MLFIIP protein is a constitutive kinetochore component and regulates cell-cycle status by recruitment of function-specific proteins. Homologous gene in murine hematopoiesis system represents early erythroblasts-specific expression. MLF1IP expression screen in our patients bone marrow monuclear cells (BMNCs) showed, mRNA level in Polycythemia Vera (PV) was significantly higher than controls. PV is a subtype of Myeloproliferative neoplasmas with characteristics of significantly increased hemoglobin in peripheral blood. Therefor, we hypothesized MLF1IP is probably involved in PV pathogenesis, and we proved human K562erythroleukemic cells with reduced MLFIIP expression represented decreased colonies, G2/M-phase block in cell cycle and increased VP16-induced apoptosis.To investigate a potential function and mechanism of MLF1IP in PV, absolute quantity real-time PCR was used to examine MLF1IP mRNA level of various in vitro colony-forming units (CFUs) of patients and controls BMNCs; MLF1IP transgenic mice (Tg) was generated and kept, hematological features of which were analysed and probable molecular mechanisms were studied. We report major results as follows:1. Various in vitro CFUs of BMNCs were examined by absolute quantitation real-time PCR:85CFUs from22PV,19CFUs from6cord blood (CB) and16CFUs from8MDS. Results showed, MLF1IP mRNA level in erythroblast-colonies of7days cultured (EU1) from PV was apparently higher than MDS; among all PV-CFUs collected from culture medium with EPO, EU1represented higher expression level than EU2(erythroblast-colonies of14days cultured) and GM-colonies (GMU).2. The entire coding region of human MLF1IP was cloned into the HS21/45-vav plasmid to generate transgenic mice, which were crossed with wild-tpe C57BL/6J mice, negative siblings of transgenic mice were used as controls. The hematological features of F1to F3were analysed:HB concentration and PLT count of Tg were apparently higher than controls in10to20weeks, achieving highest in around20weeks; then fell slightly after40weeks, PLT count fell to the some level of controls, whereas HB concentration was still hihger than controls; ANC count of Tg were quite similar to controls during their whole life-span. 3. Fresh BM cells from mouse femur were isolated and cultured in semi-solid medium with recombinant cytokines (M3434), scoring colonies after7,10,14days for BFU-E, CFUGM, CFUGEMM, respectively. We observed BM cells from Tg produced significantly increased number of BFU-E and theses colonies represented larger size with more scattered cell-clusters, whereas neither CFUGM nor CFUGEMM differed from controls.4. Plasma EPO level in Tg was proved significantly reduced than in controls by use of ELISA.5. Murine BM cells were stained with anti-Lineage specific surface antigens (B220/GR1/TER119/CD41) and detected by flow cytometry, representing similar proportion of GR1+or CD41+cells between Tg and controls. To assess proportions of different stages erythroblast populations, BM cells were stained with anti-CD71and anti-TER119and flow cytometric analysed combined with FSC value. Higher proportions of early erythroblasts (CD71highTER119med/CD71highTER119highFSChish) from Tgthan from controls were proved, as mice aged (over40weeks) even intensified meanwhile total erythroblasts in BM ruduced.6. Fresh isolated BM cells were fixed and stained with PI for cell cycle assessment by flow cytometry. A higher proportion of BM cells in active cell-cycle status (S/G2/M) was found in Tg mice.7. Relative quantitation real-time PCR was used to detect mRNA level in BM cells:mRNA level of cyclinD2and CDK1was significantly increased in Tg.8. Level of CKI, anti-apoptosis and STAT proteins was determined by immunoblot analysis. Expression of cell-cycle inhibitor p21and p27reduced markedly, meanwhile bcl-2and p-STAT5increased.Thus, we proved expression pattern of MLF1IP in human hematopoiesis system, especially in PV, follows early erythroblast-specific high level meanwhile mature blood cells and other lineages very little or no expression; data from in vivo studies indicate MLF1IP compromises early erythroblast-specific inhibitory effect on cell cycle, resulting in increased active cell-cycle status committed erythroprogenitors, eventuell promotes early erythroblasts exansion prior to terminal erythroid differentiation. Consequently, we conclude MLF1IP involved in pathogenesis of PV via specific expression pattern and function in cell-cycle regulation. |
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