参考文献

Although it has been shown that unfractionated bone marrow,hematopoietic stem cells,common myeloid progenitors,and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture,monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here,we use a monoclonal antibody to the megakaryocyte-associated surface protein,CD9,to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes,with a plating efficiency textgreater60% at the single-cell level. In vivo,MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures,indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns. View Publication

Kit receptor tyrosine kinase and erythropoietin receptor (Epo-R) cooperate in regulating blood cell development. Mice that lack the expression of Kit or Epo-R die in utero of severe anemia. Stimulation of Kit by its ligand,stem cell factor activates several distinct early signaling pathways,including phospholipase C gamma,phosphatidylinositol 3-kinase,Src kinase,Grb2,and Grb7. The role of these pathways in Kit-induced growth,proliferation,or cooperation with Epo-R is not known. We demonstrate that inactivation of any one of these early signaling pathways in Kit significantly impairs growth and proliferation. However,inactivation of the Src pathway demonstrated the most profound defect. Combined stimulation with Epo also resulted in impaired cooperation between Src-defective Kit mutant and Epo-R and,to a lesser extent,with Kit mutants defective in the activation of phosphatidylinositol 3-kinase or Grb2. The impaired cooperation between the Src-defective Kit mutant and Epo-R was associated with reduced transphosphorylation of Epo-R and expression of c-Myc. Remarkably,restoration of only the Src pathway in a Kit receptor defective in the activation of all early signaling pathways demonstrated a 50% correction in proliferation in response to Kit stimulation and completely restored the cooperation with Epo-R. These data demonstrate an essential role for Src pathway in regulating growth,proliferation,and cooperation with Epo-R downstream from Kit. View Publication

Caveolae,specialized flask-shaped lipid rafts on the cell surface,are composed of cholesterol,sphingolipids,and structural proteins termed caveolins; functionally,these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study,we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1,which is usually absent in blood cells,is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells,as shown by transmission electron microscopy,and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I,growth and survival factors in multiple myeloma,induce caveolin-1 phosphorylation,which is abrogated by pre-treatment with beta-cyclodextrin. Importantly,inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore,cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together,this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma. View Publication

Epstein-Barr virus (EBV) is associated with the development of malignant lymphomas and lymphoproliferative disorders in immunocompromised individuals. The LMP2A protein of EBV is thought to play a central role in this process by allowing the virus to persist in latently infected B lymphocytes. We have demonstrated that LMP2A,when expressed in B cells of transgenic mice,allows normal B-cell developmental checkpoints to be bypassed. To identify cellular genes targeted by LMP2A that are involved in this process,we have utilized DNA microarrays to compare gene transcription in B cells from wild-type versus LMP2A transgenic mice. In B cells from LMP2A transgenic mice,we observed decreased expression of many genes associated with normal B-cell development as well as reduced levels of the transcription factors that regulate their expression. In particular,expression of the transcription factor E2A was down-regulated in bone marrow and splenic B cells. Furthermore,E2A activity was inhibited in these cells as determined by decreased DNA binding and reduced expression of its target genes,including the transcription factors early B-cell factor and Pax-5. Expression of two E2A inhibitors,Id2 and SCL,was up-regulated in splenic B cells expressing LMP2A,suggesting a possible mechanism for E2A inhibition. These results indicate that LMP2A deregulates transcription factor expression and activity in developing B cells,and this likely allows for a bypass of normal signaling events required for proper B-cell development. The ability of LMP2A to interfere with B-cell transcription factor regulation has important implications regarding its role in EBV latency. View Publication

4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases,including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling,we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders,and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition,PP1 (and PP2) inhibited the in vitro kinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity. View Publication

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines,suggesting that WT1 participates in regulating the proliferation of leukemic cells. However,the role of WT1 in normal hematopoiesis is not well understood. To investigate this question,we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1,although contributing efficiently to other organ systems,make only a minimal contribution to the hematopoietic system in chimeras,indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition,fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E),erythroid colony-forming unit (CFU-E),and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However,transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1. View Publication

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures,respectively. In erythrocytes,the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However,the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme,designated IGnT3. The genes for IGnT1 (reported in 1993),IGnT2 (also presented in this study),and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells,IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover,reticulocytes expressed the IGnT3 transcript,but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree,individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006GtextgreaterA; Gly336Arg) and exon 3 (1049GtextgreaterA; Gly350Glu),respectively,of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3,not by IGnT1 or IGnT2. View Publication

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling,quiescent progenitors are present in most samples. In the current study,(3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0),G(1),and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results,with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly,after 72 hours in serum-free culture with or without Steel factor (SF),Flt-3 ligand (FL),and interleukin-3 (IL-3),most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours,1.2% to 37%,and 0% to 7.6% in cultures without and with growth factors [GFs],respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF,FL,IL-3,and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition,3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary,quiescent leukemic cells,including NOD/SL-ICs,are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs. View Publication

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM,respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck),A-431 (vulva),BT474 (breast),CaLu-3 (lung),and N87 (gastric). Normal human foreskin fibroblasts,nontumorigenic epithelial cells (HB4a),and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure,average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were textless 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator,AKT,was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest,the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016,and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines,concentrations of GW2016 were reached where outgrowth did not occur. Furthermore,growth arrest and cell death were observed in parallel experiments,as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally,twice daily,with complete inhibition of tumor growth at the higher dose. Together,these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2. View Publication

Purmorphamine,which is a 2,6,9-trisubstituted purine compound,was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development. View Publication

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription,nuclear import,and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date,cells have been activated via their cognate antigen receptor. To couple activation with gene transfer,we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However,transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here,we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7,these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore,a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells,preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether,these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy. View Publication

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines,but its role in proliferation,differentiation,and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells,we generated green fluorescent protein (GFP)-tagged versions of wild type CIS,a mutant harboring an inactivated SH2 domain (CIS R107K),and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence,both are essential for the growth inhibitory effect of CIS,whereas the CIS SOCS box is dispensable. Accordingly,the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly,apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus,CIS negatively regulates signaling at two levels,apoptosis and proliferation,and thereby sets a threshold for signal transduction. View Publication