参考文献

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work,we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin,osteopontin,collagen I,alpha smooth muscle actin,thrombospondin-1,EDa fibronectin,calponin,Stro-1 antigens,myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein,cytokeratins 8 and 18,albumin,E-cadherin,hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT),often observed during development,including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation,induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment. View Publication

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors,namely insulin receptor substrate-2 (IRS2),Src homology 2 domain-containing inositol 5'-phosphatase (SHIP),Grb2-associated binder-1 (Gab1),and the Epo receptor (EpoR). Using different in vitro systems,we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors,Akt,FKHRL1,and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR),through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR,or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors,but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors,the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally,our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2),which,in turn,down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation. View Publication

RET/papillary thyroid carcinoma (PTC) oncogenes,generated by recombination of the tyrosine kinase-encoding domain of RET with different heterologous genes,are prevalent in papillary carcinomas of the thyroid. Point mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) familial cancer syndrome and are found in sporadic medullary thyroid carcinomas. Here,we show that ZD6474,a low molecular weight tyrosine kinase inhibitor,blocks the enzymatic activity of RET-derived oncoproteins at a one-half maximal inhibitory concentration of 100 nM. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells-treated ZD6474 lost proliferative autonomy and showed morphological reversion. ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Finally,it blocked anchorage-independent growth of RET/PTC3-transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH-RET/PTC3 cells into nude mice. Thus,targeting RET oncogenes with ZD6474 might offer a potential treatment strategy for carcinomas sustaining oncogenic activation of RET. View Publication

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses,we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-gamma in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12,interaction with costimulatory molecules,and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides. View Publication

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