技术资料

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4,which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast,at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia. View Publication

Many acute and chronic anaemias,including haemolysis,sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia,are not treatable with erythropoietin (Epo),because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently,we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor,burst-forming unit erythroid (BFU-E),and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors,with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis,PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally,both in control and corticosteroid-treated BFU-E cells,PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists,additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias. View Publication

Generating engraftable hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is an ideal approach for obtaining induced HSCs for cell therapy. However,the path from PSCs to robustly induced HSCs (iHSCs) in vitro remains elusive. We hypothesize that the modification of hematopoietic niche cells by transcription factors facilitates the derivation of induced HSCs from PSCs. The Lhx2 transcription factor is expressed in fetal liver stromal cells but not in fetal blood cells. Knocking out Lhx2 leads to a fetal hematopoietic defect in a cell non-autonomous role. In this study,we demonstrate that the ectopic expression of Lhx2 in OP9 cells (OP9-Lhx2) accelerates the hematopoietic differentiation of PSCs. OP9-Lhx2 significantly increased the yields of hematopoietic progenitor cells via co-culture with PSCs in vitro. Interestingly,the co-injection of OP9-Lhx2 and PSCs into immune deficient mice also increased the proportion of hematopoietic progenitors via the formation of teratomas. The transplantation of phenotypic HSCs from OP9-Lhx2 teratomas but not from the OP9 control supported a transient repopulating capability. The upregulation of Apln gene by Lhx2 is correlated to the hematopoietic commitment property of OP9-Lhx2. Furthermore,the enforced expression of Apln in OP9 cells significantly increased the hematopoietic differentiation of PSCs. These results indicate that OP9-Lhx2 is a good cell line for regeneration of hematopoietic progenitors both in vitro and in vivo. View Publication

Aldehyde dehydrogenase (ALDH) activity is a widely used marker for human hematopoietic stem cells (HSCs),yet its relevance and role in murine HSCs remain unclear. We found that murine marrow cells with a high level of ALDH activity as measured by Aldefluor staining (ALDH(br) cells) do not contain known HSCs or progenitors. In contrast,highly enriched murine HSCs defined by the CD48(-)EPCR(+) and other phenotypes contain two subpopulations,one that stains dimly with Aldefluor (ALDH(dim)) and one that stains at intermediate levels (ALDH(int)). The CD48(-)EPCR(+)ALDH(dim) cells are virtually all in G(0) and yield high levels of engraftment via both intravenous and intrabone routes. In contrast the CD48(-)EPCR(+)ALDH(int) cells are virtually all in G(1),have little intravenous engraftment potential,and yet can engraft long-term after intrabone transplantation. These data demonstrate that Aldefluor staining of unfractionated murine marrow does not identify known HSCs or progenitors. However,varying levels of Aldefluor staining when combined with CD48 and EPCR detection can identify novel populations in murine marrow including a highly enriched population of resting HSCs and a previously unknown HSC population in G(1) with an intravenous engraftment defect. View Publication

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination. View Publication

SHP2,a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene,plays a critical role in developmental hematopoiesis in the mouse,and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However,the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition,the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF,and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation,survival,and differentiation of human progenitor cells. View Publication

BACKGROUND: Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro. METHODS: Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study,rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8),kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL). RESULTS: SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03),regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies. CONCLUSION: mTOR inhibition induces microcytosis and polyglobulia,but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells. View Publication

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment,low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82),little/no EpoR protein was detected and it was not functional. In contrast,EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20),and functional responses to rHuEpo were reported with MCF-7 cells. In another study,a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However,the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82,no EpoR protein was detectable in normal human and matching cancer tissues from breast,lung,colon,ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells,but no rHuEpo-induced phosphorylation of AKT,STAT3,pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

Chemotherapy is the most common way to treat malignancies,but myelosuppression,one of its common side-effects,is a formidable problem. The present study described the protective role of dammarane sapogenins (DS),an active fraction from oriental ginseng,on myelosuppression induced by cyclophosphamide (CP) in mice. DS was orally administered at different dosages (37.5,75,and 150 mg/kg) for 10 d after CP administration (200 mg/kg intraperitoneally). The results showed that DS increased the number of white blood cells (WBC) on day 3 and day 7 (P textless 0.05),such that WBC levels were increased by 105.7 ± 29.5% at 75 mg/kg of DS on day 3 (P textless 0.05,compared with the CP group). Similar results were observed in red blood cells and platelets in DS-treated groups. The colony-forming assay demonstrated that the depressed numbers of CFU-GM (colony-forming unit-granulocyte and macrophage),CFU-E (colony-forming unit-erythroid),BFU-E (burst-forming unit-erythroid),CFU-Meg (colony-forming unit-megakaryocyte) and CFU-GEMM (colony-forming unit-granulocyte,-erythrocyte,-monocyte and -megakaryocyte) induced by CP were significantly reversed after DS treatment. Moreover,the ameliorative effect of DS on myelosuppression was also observed in the femur by hematoxylin/eosin staining. In DS-treated groups,ConA-induced splenocyte proliferation was enhanced significantly at all the doses (37.5,75,150 mg/kg) on day 3 at the rate of 50.3 ± 8.0%,77.6 ± 8.5% and 44.5 ± 8.4%,respectively,while lipopolysaccharide-induced proliferation was increased mainly on day 7 (P textless 0.01),with an increased rate of 39.8 ± 5.6%,34.9 ± 6.6% and 38.3 ± 7.3%,respectively. The thymus index was also markedly increased by 70.4% and 36.6% at 75 mg/kg on days 3 and 7,respectively,as compared with the CP group. In summary,DS has a protective function against CP-induced myelosuppression. Its mechanism might be related to stimulating hematopoiesis recovery,as well as enhancing the immunological function. View Publication