1.The Role Of Rac1 GTPase In Malignance Transformation Of Hematopoietic Stem/Progenitor Cells 2.Upregulated A20 Promotes Proliferation, Regulates Cell Cycle Progression And Induces Chemotherapy Resistance Of Acute Lymphoblastic Leukemia Cells

Objective:Acute myeloid leukemia is a hematological malignancy resulting from the transformation of normal hematopoietic stem/progenitor cells (HSPC). Except for the intrinsic factors, it is acceptable that some extrinsic events from microenvironment could be the important co-factors in the development of leukemia. The change of interaction between HSPC and microenvironment is involved in leukemogenesis. Racl is an important molecule regulating HSPCs homing and interaction with hematopoietic microenvironment Activation of Rac1 is observed in a serial of leukemia cells. In this study, we investigated the effects of Rac1 GTPase in the transformation of HSPC and determined whether the activation of Racl GTPase could promote the interaction of HSPCs with microenvironment and further contribute to leukemia development and maintenance.Methods:Dominant-positive mutant Racl-V12 and AE9a gene were cloned into retroviral plasmids pMSCV-IRES-GFP respectively. The Racl-V12 and AE9a gene coexpression plasmid pMSCV-AE9a-2A-Racl-V12-IRES-GFP (AER) were also constructed. The c-Kit+ HSPC from mouse BM were purified with anti-CD117 microbeads and then infected with retroviral supernatant. Western blot and pull down were employed to determine the expression level of transduced genes. Cell migration was determined by transwell assay. A quantitative measurement of cell adhesion to fibronectin was used to determine cell adhesion ability. MethoCult M3434 was used for cell colony formation assay. The ratio of GO phase was detected by flow cytometry after Ki67 and 7-AAD staining. The expression of microenvironment associated molecules was measured by qRT-PCR. After transduction HSPCs with AER and transplantation into C57BL/6J mice, an active Racl associated acute myeloid leukemia (AML) model was established. As control, an AML mouse model induced by AE9a was also established. For lineage marker analysis, leukemic cells were incubated with monoclonal antibodies and then applied for analysis by FACS. Wright’s staining and HE staining were used to assess the pathological types of leukemic mice. Homing ability was determined by the ratio of GFP+ cells in BM cells collected from the mouse 18 hours after injection of leukemic cells. After transplantation, the lodging of leukemic cells in BM in the subsequent time point was monitored by FACS and survival time of mice was determined. In Rac1 inhibitor treatment assay, spleen cells from AER mice were cultured with PBS or EHT1864 for 2h and then transplanted into the sublethally irradiated (4.5Gy) recipient mice. Homing ability, the lodging of leukemic cells in BM and the survival time were then determined. Osteoblasts (OBCs) were isolated by collagenase digestion of murine calvarial and multipotent stromal cells (MSCs) were obtained by adherent culture from mouse BM cells. After 4 days coculture, cell cycle, cell apoptosis and colony formation ability of leukemic cells were determined with same methods as described above. For in vivo drug treatment assays, mice were separated into four groups according to the following treatments:(1) PBS (days 5-9), (2) Ara-C (500 mg/kg.d, days 8,9), (3) EHT1864 (40 mg/kg.d, days 5-9), (4) Ara-C (500 mg/kg.d, days 8,9) and EHT1864 (40 mg/kg.d, days 5-9). Then the rate of GFP+cells and WBC counts of peripheral blood were tested dynamically and the survival time was monitored.Results:Three kinds of retroviral plasmids were constructed. The expression of fusion protein AE9a and active Rac1 GTPase were confirmed to be present in retroviral transduced mouse BM cells. After active Racl GTPase was expressed in HSPC, cell migration, adhesion and clonogenic potential was enhanced. Active Rac1 GTPase also promotes HSPC in quiescent state. Higher expression level of several regulators mediated interaction of HSPC with BM niche was found in Rac1-V12 expressing cells and the difference between control and Racl-V12 expressing cells was significant. An AML model induced by active Racl together with AE9a was developed in mice. Meanwhile, an AE9a AML model was established as control. The integration of AE9a and Racl-V12 gene in the genome of leukemic cells were verified. High expression of fusion protein AE9a and active Racl were confirmed in the spleen cells of sick mouse. Histological examination showed extensive infiltration of primitive myeloid blasts in BM, spleen and liver. Wright-Giemsa staining revealed that immature cells were increased in PB, BM and spleen. The phenotype of leukemic cells was negative for the myeloid and lymphoid differentiation markers. Compared with AE9a alone, Rac1-V12 cooperated with AE9a drove an AML with a short survival time. Homing capacity and lodging in niche of AER leukemic cell was significantly higher than that of AE9a cells. AER cells exhibited higher expression levels of regulators mediated interaction of leukemic cells with BM niche and the difference between AE9a and AER expressing cells was significant. In Racl inhibitor treatment assay, after treated with EHT1864 for 2h, AER leukemic cells drove an AML with a longer survival time and the homing capacity and lodging in niche was decreased. Expression levels of regulators mediated interaction of leukemic cells with BM niche was decreased after EHT1864 treatment. Coculture of leukemic cells with OBCs promoted leukemic cells in quiescence, protected them from apoptosis and supported colony formation. MSCs can promote leukemic cells in quiescence. In vivo treatment model, compared with untreated mice, there was no obvious difference in GFP+ cells and white blood cells in mice treated with EHT1864 alone. But compared with Ara-C alone treated mice, the mice treated with EHT1864 combined Ara-C have more GFP+ cells and white blood cells in peripheral blood. The survival time was markedly longer in EHT1864 combined Ara-C treated group than that in chemotherapy treated alone and untreated group.Conclusions:Active Rac1 GTPase enhances the migration, adhesion and clonogenic ability of HSPC and promotes HSPC in quiescent state. The change of biological behavior promotes HSPC interaction with niche. Active Rac1 promotes leukemia development through enhancing homing capacity and interaction between leukemic cells and microenvironment. This interaction further promotes leukemic cells in quiescent state and enhances ability to resistance to apoptosis. Treated with the active Racl GTPase inhibitor EHT1864 combined chemotherapy drug could be effective for prolong the survival time of leukemic mice.Objective: A20, also known as tumor necrosis factor-a(TNFa) induced protein 3(TNFAIP3), was ide打tified as 红 key iregulator of cell survival. A20 overexpreasion was found in Breast cancer, re打al clear cell carcinoma and colorectal cancer,eb,but its expKssion in acute lymphoblastic leukemia(ALL) is undefined. I打 this experiment,we analyzed the expressio打 features of A20 and its biological fiinction in ALL.Methods: Western blot was used determine the expression level of A20 in primary ALL patients and ALL cell Knes. Lentiviral shRNA clones targeting A20 were constructed and ALL cell lines Jurkat, Nalm6 and Reh cells were infected with lentivirus.Cell proliferation and IC50 化 daunombicin were measured by MTT assay. CeU-cycle analysis was determined by PI staining, Annexin V and PI staining was.used to assess apoptosis. Cell proliferation, cell-cycle and apoptosis associated signal molecules were measured by WB.Results:(1) High expression levels of A20 were detected in bone marrow(BM) cells from primary ALL patients and ALL cell lines when compared with healthy donors.(2)Lentiviral shRNA clones targeting A20 were constructed and a significant reduction in A20 protein levels was observed in Jurkat cells, Nalm-6 cells and Reh cells after treated with shRNA.(3) Silence of A20 inhibited the cell proliferatio打 and reduced phosphorylation of ERK in three cell lines.(4) Silence of A20 decreased the percentage of cells in the S phase and increased the percentage of cells in the GO/Gl phase and increased the level of p21 and p53 in ALL cells.(5) Apoptosis was significantly increased and BCL-2 was decreased in Jurkat cells and Reh cells with A20 silencing.(6)A2D sdencing increased sensitivity of Jurkat ceUs,Nalm-6 cells and Reh cells chemotherapy drug daunorubicin.Conclusions: Upregulated A20 was found in ALL patients and cdl lines. Reducing expression of A20 can induce cell cycle arrest in GO/Gl stage, inhibit cell proliferation,promote cell apoptosis and increase sensitivity 化 chemotherapy drug. This finding reveals that A20 may be used as a new therapy target in ALL treatment...