Negative Regulation of Cell Cycle Kinetics of Human Hematopoietic Cells by the Transcription Factor Dmtf1.

Abstract

Hematopoietic stem cells (HSCs) are predominantly quiescent with only a small number entering active phases of cell cycle at any time point. With such tightly regulated cell cycle kinetics, HSCs ensure preservation of the stem cell pool. Many cell cycle-related proteins, mainly tumor suppressor genes, are involved in maintenance of HSCs quiescence. Dmtf1 (Cyclin D-binding Myb-like Protein1) is a transcription factor that negatively regulates cell cycle by inducing Arf expression, and its deletion has been reported in some leukemias (Bodner SM et al, 1999). As there is no information regarding the role of Dmtf1 in hematopoiesis, we examined the impact of Dmtf1 on regulating cell cycle kinetics of hematopoietic progenitor cells. Dmtf1 mRNA was expressed in human granulocytes, lymphocytes, CD34+ cells, and in murine Sca1+lin-CD117+ (KSL) cells. Using retroviral vectors (MIEG3/IRES-GFP), we first investigated cell cycle progression in 293 cells transduced with four different constructs; GFP only control vector (−), wild type Dmtf1 (WT), and two dominant negative mutants expressing a point mutation (K319E) or a deleted myb-like repeat box (dMHR). A total of 36.0% of sorted and cultured GFP+ control (−) cells were in S/G2+M 24hr after initiation of culture. Whereas 30.8% of cells expressing (WT) were in S/G2+M at the same time point, expression of the two dominant negative mutants K319E and dMHR induced 46.6% and 45.5% of the cells into S/G2+M, respectively suggesting that loss of Dmtf1 activity results in rapid cell proliferation. Interestingly, a 2.5-fold increase in the ecxpression of Arf and p21 mesured by qPCR was detected in cells transduced with WT only whereas other transduced cells did not show any change in expression of both Arf and p21. The impact of these constructs was then evaluated in cord blood cells using CFU assays. Cord blood CD34+ cells were transduced with the four vectors mentioned above and GFP+ cells were subsequently sorted and cultured. Both K319E and dMHR induced a 25% increase in the number of clonogenic progenitors relative to (−) while a modest decrease of 10% in colony numbers was detected in the WT group. Cells cycle analysis of these cells is currently under investigation. These results demonstrate that Dmtf1 acts as a negative regulator of cell cycle control in hematopoietic cells and suggests that it may play a role in the maintenance of HSC quiescence.