| Acut megakaryoblastic leukemia (AMKL), also known as M7 in AML (acute myeloid leukemia), is associated with at least two distinct chromosome abnormalities: Down's syndrome (DS) and t (1;22)(p13;q13) chromosomal translocation which are characterized by an expansion of megakaryoblasts in bone marrow, myelofibrosis and thrombocytopenia. t (1;22) (p13;q13) translocation is the unique character of AML and results in the fusion of RBM15 and MKL1 genes on chromosomes 1 and 22, respectively. So far, the role of RBM15-MKL1 fusion protein remains poor investigated. To understand the role of RBM15-MKL1 fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL1 in hematopoiesis. However no role for MKL1 has been defined in hematopoietic differentiation.Our previous data indicated that the ectopic MKL1 expression promoted the differentiation and polyploidization of megakaryocytes induced from human erythroleukiea (HEL) cells with TPA. To further investigate the role of MKL1 in megakaryopoiesis, a two-phase megakaryocyte differentiation system of mobilized human peripheral blood CD34+ cells was developed; real-time quantitative PCR was used to detect relative MKL1 gene expression during megakaryocyte differentiation; lentivirus was constructed to study the effect of ectopic MKL1 expression on megakaryocyte differentiation and maturation; MKL1 gene knockout mice were produced to investigate the effect of MKL1 deficiency on platelet volume in peripheral blood and megakaryopoiesis in bone marrow.The main experiments and results are as follows:1. The development of two-phase megakaryocyte differentiation model of PB CD34+ cells in vitroA two-phase megakaryocyte differentiation model of PB CD34+ cells was developed and optimized. The homemade Cocktail medium and CC100 medium purchased from Stem Cell Technologies were used to induce expansion of PB CD34+ cells for 3, 4 or 5 days, respectively. And then the cells were tranfered into differentiation medium containing TPO and SCF for 7, 8 or 9 days. Culture conditions were optimized by comparing the relative fold expansion of CD41+ cells and polyploid cells to the initial CD34+ cells. After cultured in Cocktail medium for 3 days and in differentiation medium for additional 7 days, PB CD34+ cells obtained 16-fold expansion of CD41+ cells and 3-fold increasing of polyploid cells, which was the optimal condition we studied. With the optimized two-phase condition, PB CD34+ cells can be expanded and differentiate into more CD41+ and polyploid cells than only with one-phase culture protocol, which provides a new higher efficiency megakaryocyte differentiation model for our further researches.2. MKL1 expression during megakaryocyte differentiationMegakaryocytes at different mature stage of differentiation were selected by flow cytometry and BSA gradient and real time quantitative PCR was used to investigate the expression of MKL1. Comparing to PB CD34+ control group (with 0% CD41+ cell and polyploid cell), MKL1 expression of 1.5% BSA group (with 67.2% CD41+ cells and 1.3% polyploid cells), 3% BSA group (with 74.4% CD41+ cells and 39.3% polyploid cells) and FACS sorted group (with 98.0% CD41+ cells and 21.5% polyploid cells) was 3.0, 6.8 and 5.0 folds higher (p<0.01), respectively. Moreover, the expression of MKL1 in FACS sorted group was higher than 1.5% BSA group (p<0.01), and in 3% BSA group was higher than FACS sorted group (p<0.05). These results indicated that the expression of MKL1 was higher in CD41+ cells than in PB CD34+ cells and higher in more matured polyploid cells than mononuclear megakaryocytes, which suggested MKL1 was upregulated within the process of megakaryocyte differentiation and maturation.3. Effect of ectopic MKL1 expression on megakaryopoiesis of PB CD34+ cellsLentivirus was constructed to express MKL1 in PB CD34+ cells and megakaryocyte differentiation and polyploidization were studied. After cultured in optimized two-phase condition, pCCL-MKL1 group with enhanced MKL1 expression obtained 61.49% of CD41+ cells, which was significantly higher than 36.26% of pCCL control group (p<0.05). Comparing the DNA distribution, ectopic MKL1 expression decreased the percentage of cells with 2N ploidy from 35.46% to 23.86% (p=0.013), 4N ploidy from 30.52% to 21.52% (p=0.001), and increased the 16N ploidy from 9.15% to 19.44% (p=0.014), 32N ploidy from 2.39% to 7.77% (p=0.010). The total percentage of cells with polyploidy (8N and above) was increased from 31.10% to 50.81% (p=0.001). Thus, the ectopic MKL1 promoted the differentiation of PB CD34+ cells and increased the percentage of polyploid cells, which suggested that ectopic MKL1 expression promoted the maturation of megakaryocytes.4. Effect of MKL1 deficiency on mouse megakaryopoiesisMKL1 gene knockout mice were produced to investigate the effect of MKL1 deficiency on mouse megakaryopoiesis in vivo. MKL1 deficiency didn't affect the number of red blood cells and white blood cells in peripheral blood, however the platelet number of MKL1 knockout mice was 5.2×105/μL, significantly lower than 8.2×105/μL of wild-type mice (p<0.001), suggesting a thrombocytopenia in MKL1 deficiency mice. There was no significant difference between MKL1 knockout mice and wild-type mice in bone marrow cell number, LSK cell percentage, pre-MegE cell percentage and the ability to form CFU-MK. The percentages of CD41+ cells (p<0.0001) and CD41+c-kit+ cells (p<0.01) in the bone marrow of MKL1 knockout mice were higher than wild-type. Comparing the DNA distribution of CD41+ cells in bone marrow, in MKL1 knockout mice, the cells with 2N ploidy was 32.15%, higher than 14.97% in wild-type (p<0.001). Meanwhile, the percentages of cells with 8N and 16N ploidy were 18.20% and 19.67% in MKL1 knockout mice, which were lower than 25.90% (p<0.01) and 30.65% (p<0.05) in wild-type mice. The initial differentiation from hematopoietic stem/progenitor cells to megakaryocyte progenitor cells was not affected by the MKL1 deficiency, however the maturation of megakaryocyte progenitor cells or early stage megakaryocytes was blocked and resulted in the accumulation of immature megakryoblasts in the bone marrow. The deficiency of megakaryocyte maturation may be responsible for the decreased platelet volume in peripheral blood.In summary, our results suggested that MKL1 acts an important role in megakaryocyte maturation. For this reason, RBM15-MKL1 fusion protein may affect the function of endogenous MKL1, inhibit the polyploidization of megakaryocyte differentiation and results in t (1;22) (p13;q13) AMKL. |
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