Hesl Defends Normal HSC Against T Cell Leukemic Environment

ObjectiveLeukemia is the hematopoiesis malignancy, in which normal blood cells in bone marrow (BM) and other hematopoietic organizations are reduced whereas leukemic cells dominate the hematopoietic compartments and infiltrate many other tissues/organs. Clinical manifestations of leukemia are anemia, bleeding, infection and fever. Many intrinsic and extrinsic factors play roles in the development of leukemia. But the impacts and mechanisms of leukemic environment on normal hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are poorly understood. The aim of our research is to study the changes and mechanisms of normal hematopoiesis in acute T lymphocytic leukemia (T-ALL).MethodsBone marrow nucleated cells (BMNCs;107/host) from B6.SJL mice were transplanted into lethally irradiated C57BL/6J recipients with106intracellular domain of Notch1(ICN1) plasmid (MSCV-ICN1-IRES-GFP)-transduced Lin-Sca-1+cells from C57BL/6J mice. In control group, recipients were transplanted with10BMNCs from C57BL/6J mice and107BMNCs from B6.SJL mice. After transplantation, the frequency and cell cycle of normal HSC/HPC were analyzed. On day10after transplantation, HSCs were sorted for gene microarray (Affymatrix mouse4302.0) with FACS Aria Ⅱ sorter (BD Biosciences) and then the results were valided by Realtime RT-PCR analyses. The retrovirus vector with Hesl overexpression (MSCV-Hesl-IRES-GFP) was constructed. To study the in vitro effects of Hesl on mouse HPCs, Lin-cells were transduced with Hesl or blank retrovector. Successfully transduced (GFP+) cells were enriched by FACS sorting and plated in standard methylcellulose-containing CFC assays. To test the function of Hesl on normal HSPCs in T-ALL leukemic mice, Hesl-transduced mice models were established. Lin-cells from B6.SJL mice were transduced with either MSCV-Hesl-IRES-GFP or blank plasmid. After transduction, Hesl-GFP+or blank-GFP+cells (CD45.1) were sorted and then injected into lethally irradiated C57BL/6J (CD45.2) by tail vein at a quantity of5×104cells per mouse with106cells of Notchl-induced leukemia cells (CD45.2).107CD45.1+Lin+cells were cotransplanted as protection cells. After transplantation, the proportion and absolute number of CD45.1+GFP+Lin-cells in BM were analyzed. For the T-ALL mice model, on day10after transplantation, the expression of SCF in peripheral blood serum and BM were assayed by ELISA and western blot.ResultsEnforced expression of ICN1was able to induce T-ALL with100%penetration. During the leukemogenesis, leukemic cells were accumulated while normal hematopoietic cells declined, and HSC and HPC were also suppressed. HPC showed accelerated proliferation at the earlier period, while most HSC went to quiescence. Gene expression analysis on normal HSCs in leukemic hosts showed that the expression of many genes related to cell cycle, proliferation, homing and adhesion was changed. Of them,8genes were valided by realtime PCR. CXCR4, Mmp2, c-Kit were down regulated while Itgb3, IL18r1, Fbxw11, p21, Hesl were up-regulated, which were consistent with the results from our microarray analysis. Moreover, Hesl expression in the HSCs-enriched population was higher than that in the HPC-enriched population from both leukemia and control mice. More importantly, its expression in HSC-enriched population from leukemia mice was two-fold higher than that from control mice whereas no significant difference could be found in HPCs-enriched population between leukemia and control mice. In vitro, the colony number in blank retrovector transduced Lin-cells was52±1.732. In constrast, Hesl transduced Lin-cells was5.8±1.128. As for the in vivo experiment, the frequencies and absolute numbers of Lin-cells and Lin-c-Kit+cells in the Hesl mice were higher than those in control mice. In T-ALL mice model, SCF was overexpressed in PB and BM samples from leukemic mice analyzed by ELISA and western blot. But the mRNA level of SCF in peripheral blood, spleen and bone marrow, where leukemia cells accounted in majority, was lower than that of control. Further, normal hematopoietic cells and leukemic cells of BM were sorted to test the mRNA level of SCF. SCF RNA expression level in leukemic cells was also lower than that in control mice, about23%of SCF expression in control mice. In contrast, SCF expression in normal hematopoietic cells was about4.52fold higher than that in control.Conclusions1. Gene chip results showed that under the leukemia environment, the expression of the genes related with cell proliferation, adhesion and homing such as CXCR4Mmp2, Itgb3were altered in normal HSCs.2. Our results suggested that the different responses of Hesl on HSCs and HPCs resulted in the different fates of these cells under the leukemic condition.3. Our data indicated that overexpression of Hesl in normal HSPCs in T-ALL leukemia mice could partialy maintain the number and function of normal HSPCs. Hense, Hesl overexpression is one of the mechanisms that normal HSC is reversibly suppressed in T-ALL leukemia environment.4. In the T-ALL leukemia environment, SCF expression in PB and BM was increased, but SCF mRNA level in normal hematopoietic cells was higher than that in leukemia cells, which suggested that SCF was secreted by non-leukeumic cells as a response to the leukemia development.