技术资料

Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions,while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress,such as occurs in the Rb-null fetal liver,that Rb(-/-) macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function. View Publication

Survivin,which is the smallest member of the inhibitor of apoptosis protein (IAP) family,is a chromosomal passenger protein that mediates the spindle assembly checkpoint and cytokinesis,and also functions as an inhibitor of apoptosis. Frequently overexpressed in human cancers and not expressed in most adult tissues,survivin has been proposed as an attractive target for anticancer therapies and,in some cases,has even been touted as a cancer-specific gene. Survivin is,however,expressed in proliferating adult cells,including human hematopoietic stem cells,T-lymphocytes,and erythroid cells throughout their maturation. Therefore,it is unclear how survivin-targeted anticancer therapies would impact steady-state blood development. To address this question,we used a conditional gene-targeting strategy and abolished survivin expression from the hematopoietic compartment of mice. We show that inducible deletion of survivin leads to ablation of the bone marrow,with widespread loss of hematopoietic progenitors and rapid mortality. Surprisingly,heterozygous deletion of survivin causes defects in erythropoiesis in a subset of the animals,with a dramatic reduction in enucleated erythrocytes and the presence of immature megaloblastic erythroblasts. Our studies demonstrate that survivin is essential for steady-state hematopoiesis and survival of the adult,and further,that a high level of survivin expression is critical for proper erythroid differentiation. View Publication

Myelodysplastic syndromes (MDSs) are a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias. Lenalidomide has dramatic therapeutic effects in patients with low-risk MDS and a chromosome 5q31 deletion,resulting in complete cytogenetic remission in textgreater60% of patients. The molecular basis of this remarkable drug response is unknown. To gain insight into the molecular targets of lenalidomide we investigated its in vitro effects on growth,maturation,and global gene expression in isolated erythroblast cultures from MDS patients with del(5)(q31). Lenalidomide inhibited growth of differentiating del(5q) erythroblasts but did not affect cytogenetically normal cells. Moreover,lenalidomide significantly influenced the pattern of gene expression in del(5q) intermediate erythroblasts,with the VSIG4,PPIC,TPBG,activin A,and SPARC genes up-regulated by textgreater2-fold in all samples and many genes involved in erythropoiesis,including HBA2,GYPA,and KLF1,down-regulated in most samples. Activin A,one of the most significant differentially expressed genes between lenalidomide-treated cells from MDS patients and healthy controls,has pleiotropic functions,including apoptosis of hematopoietic cells. Up-regulation and increased protein expression of the tumor suppressor gene SPARC is of particular interest because it is antiproliferative,antiadhesive,and antiangiogenic and is located at 5q31-q32,within the commonly deleted region in MDS 5q- syndrome. We conclude that lenalidomide inhibits growth of del(5q) erythroid progenitors and that the up-regulation of SPARC and activin A may underlie the potent effects of lenalidomide in MDS with del(5)(q31). SPARC may play a role in the pathogenesis of the 5q- syndrome. View Publication

This review discusses the challenges of chemotherapy for human African trypanosomiasis (HAT). The few drugs registered for use against the disease are unsatisfactory for a number of reasons. HAT has two stages. In stage 1 the parasites proliferate in the haemolymphatic system. In stage 2 they invade the central nervous system and brain provoking progressive neurological dysfunction leading to symptoms that include the disrupted sleep wake patterns that give HAT its more common name of sleeping sickness. Targeting drugs to the central nervous system offers many challenges. However,it is the cost of drug development for diseases like HAT,that afflict exclusively people of the world's poorest populations,that has been the principal barrier to new drug development and has led to them becoming neglected. Here we review drugs currently registered for HAT,and also discuss the few compounds progressing through clinical trials. Finally we report on new initiatives that might allow progress to be made in developing new and satisfactory drugs for this terrible disease. View Publication

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells,Lin(-)c-Kit+ cells,and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid,B-lymphoid,and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3,MPO,Neutrophil elastase,Cathepsin G,and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1,Notch1,LEF-1,Mcl-1,and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development. View Publication

UNLABELLED: Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors. INTRODUCTION: Imatinib mesylate,an orally active inhibitor of the c-abl,c-kit,and platelet-derived growth factor receptor (PDGFR) tyrosine kinases,is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia,suggesting that imatinib might have adverse effects on the skeleton. However,biochemical markers of bone formation increase in patients with CML starting imatinib therapy,whereas bone resorption is unchanged,despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients. MATERIALS AND METHODS: Osteoblast differentiation was assessed using a mineralization assay,proliferation by [(3)H]thymidine incorporation,and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples,osteoblastic cells,and murine bone marrow cultures. Osteoprotegerin was measured by ELISA. RESULTS: The molecular targets of imatinib are expressed in bone cells. In vitro,imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures,imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells. CONCLUSIONS: Taken together with recent in vivo data,these results suggest a role for the molecular targets of imatinib in bone cell function,that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation,and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors. View Publication

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA,followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample,whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles,we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP,CTCGAGCTGHHHHHAACTAC,where H is a mixture of base consisting of 50% A,25% T,and 25% C; and second 5' DOP,CTCGAGCTGDDDDDGTTTAG,where D is a mixture of base consisting of 50% T,25% G,and 25% A. Our results showed that bisulfite-modified DNAs from a cell line,cord blood cells,or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template. View Publication

Human embryonic stem cells,because of their unique combination of long-term self-renewal properties and pluripotency,are providing new avenues of investigation of stem cell biology and human development and show promise in providing a new source of human cells for transplantation therapies and pharmaceutical testing. Current methods of propagating these cells using combinations of mouse fibroblast feeder cultures and bovine serum components are inexpensive and,in general,useful. However,the systematic investigation of the regulation of self-renewal and the production of safer sources of cells for transplantation depends on the elimination of animal products and the use of defined culture conditions. Both goals are served by the development of serum-free culture methods for human embryonic stem cells. View Publication

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2,making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent,selective,and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205,Calu-6,and SW-620 xenografts,whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3,a marker of apoptosis,was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation,but the response varies between different xenografts. Moreover,enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. View Publication

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms,active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell,decreased proliferation,and increased apoptosis. Cells became binucleated,suggesting a failure of cytokinesis,and micronuclei were formed,indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis,filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation,and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization. View Publication

We have studied the mechanism of A-769662,a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators,it directly activates native rat AMPK by mimicking both effects of AMP,i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated alpha subunit kinase domain,with or without the associated autoinhibitory domain,or on interaction of glycogen with the beta subunit glycogen-binding domain. Although it mimics actions of AMP,it has no effect on binding of AMP to the isolated Bateman domains of the gamma subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC),effects that are completely abolished in AMPK-alpha1(-/-)alpha2(-/-) cells but not in TAK1(-/-) mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1,a major upstream kinase for AMPK in this tissue. However,in HeLa cells,which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-beta,phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells,the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication