[Show abstract][Hide abstract] ABSTRACT: Background
Leukemia is a systemic malignancy originated from hematopoietic cells. The extracellular environment has great impacts on the survival, proliferation and dissemination of leukemia cells. The spleen is an important organ for extramedullary hematopoiesis and a common infiltration site in lymphoid malignancies. Splenomegaly, frequently observed in T cell acute lymphoblastic leukemia (T-ALL), is associated with poor prognosis. However, how the spleen microenvironment distinctly affects T-ALL cells as opposed to bone marrow (BM) microenvironment has not been addressed.MethodsA Notch1-induced mouse T-ALL model was applied in this study. Flow cytometry and two-photon fluorescence microscopy were used to analyze early distribution of T-ALL cells. MILLIPLEX® MAP Multiplex Immunoassay was performed to measure cytokine/chemokine levels in different microenvironments. Transwell and co-culture experiments were used to test the effects of splenic microenvironment in vitro. Splenectomy was performed to assess the organ specific impact on the survival of T-ALL-bearing mice.ResultsMore leukemia cells were detected in the spleen than in the BM after injection of T-ALL cells by flow cytometry and two-photon fluorescence microscopy analysis. By screening a panel of cytokines/chemokines, a higher level of MIP-3ß was found in the splenic microenvironment than BM microenvironment. In vitro transwell experiment further confirmed that MIP-3ß recruits T-ALL cells which express a high level of MIP-3ß receptor, CCR7. Furthermore, the splenic microenvironment stimulates T-ALL cells to express a higher level of MIP-3ß, which further recruits T-ALL cells to the spleen. Co-culture experiment found that the splenic microenvironment more potently stimulated the proliferation and migration of T-ALL cells than BM. Moreover, the mice transplanted with T-ALL cells from the spleen had a shorter life span than those transplanted from BM, suggesting increased potency of the T-ALL cells induced by the splenic microenvironment. In addition, splenectomy prolonged the survival of leukemic mice.Conclusions
Our study demonstrates an organ specific effect on leukemia development. Specifically, T-ALL cells can be potentiated by splenic microenvironment and thus spleen may serve as a target organ for the treatment of some types of leukemia.
Journal of hematology & oncology. 11/2014; 7(1):71.
[Show abstract][Hide abstract] ABSTRACT: Immuno-compromised mice, such as the non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice, have been widely used to examine the in vivo self-renewal and differentiation of human hematopoietic stem cells (HSCs). However, the efficiency of human HSC engraftment remains very low. Here, we report that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in their bone marrow (BM) than other commonly used mouse strains (C57BL/6 and BALB/C). Treatment with the antioxidant N-acetyl-L-cysteine (NAC) decreased ROS levels in the BM of NOD/SCID mice. Furthermore, the NAC-treated mice displayed a significant increase in human HSC engraftment and multi-lineage differentiation compared with the controls. In comparison with the control mice, NAC-treated recipients displayed a 10.8-fold increase in engraftment in the injected tibiae. A beneficial effect of NAC for human hematopoietic engraftment was also observed in an additional immune-deficient mouse strain, namely NOD.Cg-Prkdc(scid)I12rg(tm1Wjl)/SzJ (NOD/SCID/γc(-/-) or NSG). Therefore, our current study uncovers a previously unappreciated negative effect of ROS on human stem cell engraftment in immune-deficient mouse models.
[Show abstract][Hide abstract] ABSTRACT: Cytopenia and delayed immune reconstitution with acute graft-versus-host disease (aGvHD) indicate a poor prognosis. However, how donor-derived cell hematopoiesis is impaired in aGvHD is not well understood. We addressed this issue by studying the kinetics of hematopoiesis and the functions of hematopoietic stem and progenitor cells (HSPCs) in an aGvHD model with haplo-MHC-matched murine bone marrow transplantation (BMT). Although hematopoiesis was progressively suppressed during aGvHD, the hematopoietic regenerative potential of donor-derived HSCs remains intact. There was a dramatic reduction in primitive hematopoietic cells and a defect in the ability of these cells to generate common myeloid progenitors (CMPs) and megakaryocyte/erythrocyte progenitors (MEPs). These effects were observed along with a concomitant increase in granulocyte/macrophage progenitors (GMPs), suggesting that differentiation into MEPs is blocked during aGvHD. Interestingly, cyclosporine A (CsA) was able to partially reverse the hematopoietic suppression as well as the differentiation blockage of CMPs. These data provide new insights into the pathogenesis of aGvHD and may improve the clinical management of aGvHD.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 05/2014; · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To analyze the impact of the occurrence and severity of acute and chronic graft versus host disease (GVHD) on the long-term outcome of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for leukemia.
[Show abstract][Hide abstract] ABSTRACT: Acute leukemia characterized by chromosomal rearrangements requires additional molecular disruptions to develop into full-blown malignancy, yet the cooperative mechanisms remain elusive. Using whole-genome sequencing of a pair of monozygotic twins discordant for MLL (also called KMT2A) gene-rearranged leukemia, we identified a transforming MLL-NRIP3 fusion gene and biallelic mutations in SETD2 (encoding a histone H3K36 methyltransferase). Moreover, loss-of-function point mutations in SETD2 were recurrent (6.2%) in 241 patients with acute leukemia and were associated with multiple major chromosomal aberrations. We observed a global loss of H3K36 trimethylation (H3K36me3) in leukemic blasts with mutations in SETD2. In the presence of a genetic lesion, downregulation of SETD2 contributed to both initiation and progression during leukemia development by promoting the self-renewal potential of leukemia stem cells. Therefore, our study provides compelling evidence for SETD2 as a new tumor suppressor. Disruption of the SETD2-H3K36me3 pathway is a distinct epigenetic mechanism for leukemia development.
[Show abstract][Hide abstract] ABSTRACT: Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)-haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4+, but not CD8+ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4+ donor T cells through the Fas/FasL pathway.
PLoS ONE 01/2014; 9(8):e104607. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian target of rapamycin (mTOR) is composed of two distinct biochemical complexes, mTORC1 and mTORC2. mTORC1 is known to control cellular growth in response to nutrients and growth factors by regulating the translational regulators S6K1 and 4E-BP1, whereas mTORC2 mediates cell proliferation and survival by activating AKT through phosphorylation at Ser473. Studies have shown that the deregulation of mTORC2 leads to the development of myeloproliferative disorder and leukemia in the PTEN-deleted mouse model. However, the mechanism by which mTORC2 specifically affects leukemogenesis is still not fully understood. Here, we investigated the role of mTORC2 in NOTCH1-driven T-ALL in a Rictor deficient mouse model. We found that by deleting Rictor, an essential component of mTORC2, leukemia progression was significantly suppressed by arresting a greater proportion of Rictor△/△ leukemic cells at the G0 phase of the cell cycle. Furthermore, the absence of Rictor led to the overexpression of chemotaxis-related genes, such as CCR2, CCR4 and CXCR4, which contributed to the homing and migration of Rictor deficient T-ALL cells to the spleen but not the bone marrow. In addition, we demonstrated that inactivation of mTORC2 caused the overexpression of FoxO3a and its downstream effectors and eased the progression of leukemia in T-ALL mice. Our study thus indicates that FoxO3a could be a potential drug target for the treatment of T-ALL leukemia.
[Show abstract][Hide abstract] ABSTRACT: The Notch1 signaling pathway plays an essential role in cell growth and differentiation. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-cell acute lymphoblastic leukemia (T-ALL) in mice. Here we explored the drug sensitivity of T-ALL cells in two subpopulations of CD8(+)CD4(+) and CD8(+)CD4(-) cells in Notch1-induced T-ALL mice. We found that Notch1 induced T-ALL cells could be decreased by chemotherapeutic drug cyclophosphamide (CTX). CD8(+)CD4(-) T-ALL cells were more sensitive to CTX treatment than CD8(+)CD4(+) T-ALL cells. The percentage of apoptotic cells induced by CTX treatment was higher in CD8(+)CD4(-) T-ALL cells. T-ALL cells were also inhibited by inhibitor of mTORC1 rapamycin. CD8(+)CD4(+) T-ALL cells were more susceptible to rapamycin treatment than CD8(+)CD4(-) T-ALL cells. Rapamycin treatment selectively arrested more CD8(+)CD4(+) T-ALL cells at G0 phase of cell cycle. A combination of the two drugs significantly improved overall survival of T-ALL bearing mice when compared with CTX or rapamycin alone. These results indicated that CD8(+)CD4(+) and CD8(+)CD4(-) leukemia cell populations had distinct drug sensitivity.
[Show abstract][Hide abstract] ABSTRACT: Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 05/2013; 21(3):735-740.
[Show abstract][Hide abstract] ABSTRACT: The prognosis of patients with refractory/relapsed acute myelogenous leukemia (rAML) is poor. Recent studies have shown that more transplant centers are choosing allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for recipients, even with a higher leukemia burden. The purpose of the present study is to evaluate the outcome of rAML patients undergoing allo-PBSCT and to determine whether the disease status can predict the posttransplantation survival. The outcome of 58 patients (median age 34 years, range: 14-52) with rAML who underwent allo-PBSCT in our institution from 01/2000 to 9/2011 were retrospectively studied. Thirty-three patients had complete remission (CR) prior to PBSCT while 25 patients had no remission (NR). Donors are matched-related (31 patients) and unrelated (27 patients). Reduced-intensity conditioning (RIC-FBA) was used for 18 patients of rAML, and myeloablative conditioning was used for others. Sixty-six consecutive non-rAML patients (median age 33 years, range 15-51) who received an allo-PBSCT at the same period were used as a control. Full donor-type engraftment was achieved in all patients. After a median follow-up of 61 months, the 5-year overall survival (OS) of rAML patients was 54.21±7.06%, which was lower than non-rAML patients (71.82±6.4%, P=.0386). However, the 5-year event-free survival (EFS) for rAML and non-rAML patients had no statistical significance (53.54±6.87% vs. 62.07±6.78%, P= .2626). The 5-year OS between rAML patients who had CR and NR prior to PBSCT were 56.06±9.2% and 51.85±10.83%, respectively (P=.6408). These data demonstrate that allo-PBSCT is a promising and safe choice for the treatment of rAML, and the results were partially due to the rapid tapering of immunosuppressants in the early stage after PBSCT and prophylactic DLI. Meanwhile, the patients who were not able to get CR prior to PBSCT could also benefit from allo-PBSCT.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 01/2013; · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) are capable of self-renewal and multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. As such, bone marrow transplantation (BMT) is a major assay commonly used to examine murine HSC activity. BMT traditionally involves injection of HSCs into lethally irradiated recipients via the tail vein, then subsequently analyzing donor engraftment. Here, we describe the methods for assaying HSC reconstitution in direct, competitive, and serial BMT.
[Show abstract][Hide abstract] ABSTRACT: Bone marrow microenvironment (niche) plays essential roles in the fate of hematopoietic stem cells (HSCs). Intracellular and extracellular redox metabolic microenvironment is one of the critical factors for the maintenance of the niche. Cytochrome P450 reductase (CPR) is an obligate electron donor to all microsomal cytochrome P450 enzymes (P450 or CYP), and contributes to the redox metabolic process. However, its role in maintaining HSCs is unknown.
To examine the effects of low CPR expression on HSCs function using a mouse model of globally suppressed Cpr gene expression (Cpr Low, CL mice).
Hematopoietic cell subpopulations in bone marrow (BM) and peripheral blood (PB) from WT and CL mice were examined for their repopulation and differentiation ability upon BM competitive transplantation and enriched HSC (LKS(+)) transplantation. Effects of low CPR expression on hematopoiesis were examined by transplanting normal BM cells into CL recipients. Reactive oxygen species (ROS), cell cycle, and apoptosis in CL mice were analyzed by flow cytometry for DCF-DA fluorescence intensity, Ki67 protein, and Annexin-V, respectively.
The levels of ROS in BM cells, HPCs and HSCs were comparable between CL and WT mice. In comparison to WT mice, the number of LT-HSCs or ST-HSCs was lower in CL mice while CMPs, GMPs and MEPs in CL mice were higher than that in WT control. Competitive transplantation assay revealed enhanced repopulation capacity of HSCs with low CPR expression, but no difference in differentiation potential upon in vitro experiments. Furthermore, lymphoid differentiation of donor cells decreased while their myeloid differentiation increased under CL microenvironment although the overall level of donor hematopoietic repopulation was not significantly altered.
Our studies demonstrate that suppressing CPR expression enhances the repopulation efficiency of HSCs and a low CPR expression microenvironment favors the differentiation of myeloid over lymphoid lineage cells.
PLoS ONE 01/2013; 8(7):e69913. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bortezomib has been widely used in the treatment of various cancers; however, its exact mechanisms of action are not fully understood, particularly in acute T lymphoblast leukemia (T-ALL). Here, we visualize the anti-leukemia effect of bortezomib in both human T-ALL cell line and animal models. In vitro study, a human T-ALL cell line bearing Notch1 mutations, MOLT-4, was treated with bortezomib. At clinically achievable concentrations, bortezomib inhibited cell growth by inducing G1 phase arrest and apoptosis with a dose-dependent manner. A murine tumor xenograft model was achieved by subcutaneous injection of MOLT-4 cells for in vivo study. Administration of bortezomib significantly reduced tumor mass volume when compared with controls. Of note, bortezomib inhibited growth of leukemia cells in a Notch1-induced murine T-ALL model, and the life span of leukemia-bearing mice was markedly increased. Further studies revealed that bortezomib led to inhibited expression of Notch1 target genes. Taken together, our results demonstrate that bortezomib shows significant anti-leukemia effect in T-ALL bearing Notch1 mutations in vitro and in vivo. The present study provides evidence that bortezomib might be a candidate therapeutic reagent in the treatment of T-ALL.
Cancer Chemotherapy and Pharmacology 09/2012; · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cells (HSC) are the source of all blood cells, which can differentiate into various hematopoietic hierarchy cells. Physiological level of reactive oxygen species (ROS) plays an important role in regulating functions of HSC as excessive ROS is harmful to HSC. Oxidative reductases and antioxidants can eliminate cellular ROS to maintain ROS homeostasis and thus avoid excessive ROS-caused damages. There are several types of oxidative reductases in cells such as catalase, manganese superoxide dismutase (MnSOD), glutathione peroxidase 1 (GPX1), thioredoxin reductase 1 (Txrnd1) and Nqo1 [NAD(P)H dehydrogenenase quinone 1]. However, the functional roles of various oxidative reductases in regulating ROS level in hematopoietic cells remain unclear. This study was to investigate the expression patterns of these oxidative reductases in mouse hematopoietic cells that were sorted out via flow cytometry and to find out important oxidative reductases involving in HSC ROS regulation. The expression of various oxidative reductases was detected by semi-quantitative real-time PCR. The results showed that the expression level of catalase in T cell population was 0.14 times that in LT-HSC population (P < 0.05). The expression levels of MnSOD in CLP population and myeloid cells were 0.56 and 0.47 times that in LT-HSC population respectively (P < 0.05). The expression levels of GPX1 in ST-HSC, GMP, Myeloid cells, MEP, T lymphocytes and B lymphocytes were 1.79, 2.96, 2.07, 0.58, 0.10, 0.6 times that in LT-HSC population respectively (P < 0.05). The expression levels of Txrnd1 in ST-HSC, MPP, CMP, GMP, Myeloid cells, T lymphocytes and B lymphocytes were 3.36, 3.18, 4.19, 6.39, 4.27, 0.016, 0.56 time that in LT-HSC population, respectively (P < 0.05). The expression levels of Nqo1 in ST-HSC, MPP, CMP, GMP, CLP and B cell were 0.30, 0.17, 0.25, 0.10, 0.04, 0.01 times that in LT-HSC population, respectively (P < 0.05). It is concluded that the expression levels of oxidative reductases (catalase, MnSOD, GPX1, Txrnd1 and Nqo1) in hematopoietic hierarchy cells are cell-type specific. It suggests that reductases may play divergent roles in various hematopoietic cell populations. More importantly, the expression level of Nqo1 in LT-HSC population significantly increased as compared with other cell populations, thereby suggesting its unique regulatory role in HSC.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 06/2012; 20(3):686-91.
[Show abstract][Hide abstract] ABSTRACT: In this study, we sought to determine whether the expression of inducible co-stimulator ligand (ICOSLG) on mouse hematologic neoplasm cells influences cytotoxicity. Using flow cytometry, we examined the expression of ICOSLG in mouse hematologic neoplasm cell lines for the first time and used FBL3/A20 as targets to study. Effectors and targets were incubated at effector:target (E:T) ratios ranging from 5:1 to 50:1 for 10 h. Enzyme-linked immunosorbent assay was used to assess the expression of cytokines associated with type 2 T helper cells. We found that FBL3, A20 and P388 cells expressed ICOSLG. At E:T ratios ranging from 20:1 to 30:1, the cytotoxic activity of alloreactive T cells was significantly increased when ICOSLG was blocked (p < 0.05). We also found that ICOSLG blockade was associated with decreased expression of interleukin 4 and interleukin 10. Our results indicate that ICOSLG blockade may enhance cytotoxity in allogeneic mixed lymphocyte-hematologic neoplasm cell reactions.