Regulation of Hematopoietic Stem Cell Quiescence by the Novel Tumor Suppressor Dmtf1.

Abstract 700

Cell cycle status of hematopoietic stem cells (HSC) is tightly regulated most likely to balance quiescence and proliferation/differentiation of these cells which fulfil the immediate and continuous demands for maintenance of the size of the stem cell pool as well as the production of all the formed elements of the blood, respectively. Tumor suppressor genes, which negatively influence cell cycle regulation and control cell survival have different important roles in HSC function. Recently, Dmtf1 (Cyclin D-binding Myb-like Transcription Factor 1) was recognized as a tumor suppressor gene that is deleted in lung cancer and leukemic cells. Dmtf1 has been shown to regulate cell cycle progression by mainly an Arf-Mdm2-p53 dependent mechanism. Since the role of Dmtf1 in the hematopoietic system has not been explored, we investigated weather Dmtf1 plays a role in regulating HSC quiescence by analyzing Dmtf1 KO mice. Dmtf1 was expressed in many stages and lineages of hematopoietic cells, including Kit+Sca-1+Lineage- (KSL) cells, Kit+/lin- cells, T-cells, B-cells, and granulocytes. As previously reported, KO mice were smaller than WT mice (25.1 vs 28.0 g at 13-14 weeks old male, p<0.01), however, bone marrow (BM) of KO mice contained a higher number of total nucleated cells than that of WT mice (6.8 vs 5.1 × 10e7 in 2 femurs, p<0.01). Population of myeloid and T/B-cell were similar in both genotype. Absolute KSL count in KO BM was more than 2-fold higher than that of WT marrow (6.1 vs 2.2 × 10e4/femur, p<0.03). Although the frequency of ST-HSC (CD34+CD135+ KSL) was higher in KO mice (0.17 vs 0.09%, p<0.05), the frequency of LT-HSC (CD34-CD135- KSL) was similar in both genotypes (0.03 vs 0.03%). Sorted KSL from KO mice contained higher numbers of clonogenic cells (62.3 vs 32.3 CFU/1000KSL, p<0.01) and exhibited a higher proliferative potential in liquid culture. Competitively transplanted low-density BM cells from KO mice sustained a higher level of chimerism in recipient mice than their WT counterparts at 16 weeks post-transplantation (83 vs 48%, p<0.05). To evaluate differences in the repopulation potential of LT-HSC, we transplanted 100 sorted CD34-CD135- KSL (CD45.2) with 5×10e5 competitor BM low-density cells (CD45.1). KO LT-HSC supported markedly higher chimerism than WT cells at 16 weeks (45.6 vs 3.8%, p<0.01). Results of secondary transplantation are pending. Because it has been shown that Dmtf1 is induced by oncogenic or proliferative RAS/ERK signals as an initiation of negative feed back regulation, we investigated the relationship between Dmtf1 expression and cell cycle status of KSL cells in the early phases of proliferation. At steady state (0 hr), sorted KO-KSL showed a higher percentage of cycling cells (S+G2/M) by Hst/Pyronin staining (16.8 vs 11.2%) and the same percentage of G0 cells (51.2 vs 52.6%). Percentage of cycling KO- & WT-KSL cells after 24hr in culture was 37.8% and 24.2%, respectively, and at 48hr, the fraction of KO-KSL in G0 was lower than that among WT cells (1.6 vs 7.8%). Apoptosis was not increased among KO BM cells. Accordingly, expression of Dmtf1 was increased at 24 & 48 hr compared to 0 hr by real-time qPCR analysis. Interestingly, a marked suppression of CDKN1a (p21) expression in KO-KSL cells was observed at both 0 and 48hr. Given that Arf expression is induced by Dmtf1 in epithelial cells, we examined the relationship between Arf and Dmtf1 in KSL cells. Arf was not detected at 0hr in WT and KO KSL cells. While cultured KO-KSL cells failed to express Arf after 48 & 72hr in culture, WT cells expressed Arf after 72hr suggesting that induction of Arf may be partially responsible for the dysregulation of cell cycle progression in cultured KO-KSL cells and Arf does not have important role for regulating the cell cycle of steady state HSCs. Taken together, our data suggest that loss of Dmtf1 make LT-HSC acquire a higher long-term repopulating potential compared to WT cells and that in the hematopoietic system that might be involved in development of leukemic stem cell. Dmtf1 regulates HSC quiescence by the induction of CDKN1a via an Arf independent mechanism.