Background: In patients with myelodysplastic syndrome (MDS), bone marrow cells have an increased\npredisposition to apoptosis, yet MDS cells outcompete normal bone marrow (BM)-- suggesting that factors\nregulating growth potential may be important in MDS. We previously identified v-Erb A related-2 (EAR-2, NR2F6) as\na gene involved in control of growth ability.\nMethods: Bone marrow obtained from C57BL/6 mice was transfected with a retrovirus containing EAR-2-IRES-GFP.\nEx vivo transduced cells were flow sorted. In some experiments cells were cultured in vitro, in other experiments\ncells were injected into lethally irradiated recipients, along with non-transduced bone marrow cells. Short-hairpin\nRNA silencing EAR-2 was also introduced into bone marrow cells cultured ex vivo.\nResults: Here, we show that EAR-2 inhibits maturation of normal BM in vitro and in vivo and that EAR-2 transplant\nchimeras demonstrate key features of MDS. Competitive repopulation of lethally irradiated murine hosts with EAR-\n2-transduced BM cells resulted in increased engraftment and increased colony formation in serial replating\nexperiments. Recipients of EAR-2-transduced grafts had hypercellular BM, erythroid dysplasia, abnormal localization\nof immature precursors and increased blasts; secondary transplantation resulted in acute leukemia. Animals were\ncytopenic, having reduced numbers of erythrocytes, monocytes and granulocytes. Suspension culture confirmed\nthat EAR-2 inhibits granulocytic and monocytic differentiation, while knockdown induced granulocytic\ndifferentiation. We observed a reduction in the number of BFU-E and CFU-GM colonies and the size of erythroid\nand myeloid colonies. Serial replating of transduced hematopoietic colonies revealed extended replating potential\nin EAR-2-overexpressing BM, while knockdown reduced re-plating ability. EAR-2 functions by recruitment of histone\ndeacetylases, and inhibition of differentiation in 32D cells is dependent on the DNA binding domain.\nConclusions: This data suggest that NR2F6 inhibits maturation of normal BM in vitro and in vivo and that the\nNR2F6 transplant chimera system demonstrates key features of MDS, and could provide a mouse model for MDS.
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