技术资料

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%,despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival,such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies,including AML,and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover,constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation,although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor,C188-9,inhibited G-CSF-induced Stat3 phosphorylation,induced apoptosis in AML cell lines and primary samples,and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore,Stat3 inhibition may be a valuable strategy for targeted therapies for AML. View Publication

Ionising radiation (IR) is a known carcinogen and poses a significant risk to the haematopoietic system for the development of leukaemia in part by induction of genomic instability. Induction of chronic oxidative stress has been assumed to play an important role in mediating the effect of IR on the haematopoietic system. However,there was no direct evidence to support this hypothesis prior to our studies. In our recent studies,we showed that exposure of mice to total body irradiation (TBI) induces persistent oxidative stress selectively in haematopoietic stem cells (HSCs) at least in part via up-regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4. Now,we found that post-TBI treatment with diphenylene iodonium (DPI),a pan NOX inhibitor,not only significantly reduces TBI-induced increases in reactive oxygen species (ROS) production,oxidative DNA damage and DNA double-strand breaks in HSCs but also dramatically decreases the number of cells with unstable chromosomal aberrations in the clonal progeny of irradiated HSCs. The effects of DPI are comparable to Mn (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin,a superoxide dismutase mimetic and a potent antioxidant. These findings demonstrate that increased production of ROS by NOX in HSCs mediates the induction of haematopoietic genomic instability by IR and that NOX may represent a novel molecular target to inhibit TBI-induced genomic instability. View Publication

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) 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

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

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

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

Patients with heart failure are predisposed to infections and anemia,possibly due to reduced hematopoiesis. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in heart failure,and it inhibits normal hematopoiesis,partly due to apoptosis through the effector molecule Fas. We examined bone marrow progenitor cells of mice with heart failure induced by acute myocardial infarction. The fraction of progenitor cells in mice with heart failure was only approximately 40% of control. Measured with in vitro clonal assays,the proliferative capacity of the progenitor cells in mice with heart failure was reduced to approximately 50% of control. Flow cytometry with specific markers revealed a threefold increase in apoptosis among progenitor cells from mice with heart failure. In these mice,TNF-alpha/Fas expression was increased in bone marrow natural killer (NK) and T cells,and these lymphocytes showed increased cytolytic activity in vitro against progenitor cells. We conclude that the TNF-alpha/Fas pathway in lymphocytes is activated in the bone marrow during heart failure,which may play a pathogenic role in the observed decrease in hematopoiesis. View Publication

In the present study,we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women,13 non-pregnant women who had given birth to male offsprings,12 women who had never been pregnant,and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies� View Publication

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly,we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre,we designed a lentiviral vector that integrates into the host genome,expresses Cre in the target cell,and is subsequently deleted from the genome in a Cre-dependent manner. Thus,the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression,transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo. View Publication