技术资料

Polo-like kinases (PLKs) play critical roles throughout mitosis. Here,we report that wortmannin,which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases,is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly,we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together,our results indicate that,at the concentrations of wortmannin commonly used to inhibit PI 3-kinases,PLK1 is also significantly inhibited. View Publication

TEL-TRKC is a fusion gene generated by chromosomal translocation and encodes an activated tyrosine kinase. Uniquely,it is found in both solid tumors and leukemia. However,a single exon difference (in TEL) in TEL-TRKC fusions is associated with the two sets of cancer phenotypes. We expressed the two TEL-TRKC variants in vivo by using the 3' regulatory element of SCL that is selectively active in a subset of mesodermal cell lineages,including endothelial and hematopoietic stem cells and progenitors. The leukemia form of TEL-TRKC (-exon 5 of TEL) enhanced hematopoietic stem cell renewal and initiated leukemia. In contrast,the TEL-TRKC solid tumor variant (+ TEL exon 5) elicited an embryonic lethal phenotype with impairment of both angiogenesis and hematopoiesis indicative of an effect at the level of the hemangioblasts. The ability of TEL-TRKC to repress expression of Flk1,a critical regulator of early endothelial and hematopoietic cells,depended on TEL exon 5. These data indicate that related oncogenic fusion proteins similarly expressed in a hierarchy of early stem cells can have selective,cell type-specific developmental impacts. View Publication

OBJECTIVE To investigate the long-term effect of cryopreservation on hepatocyte function,as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution,HypoThermosol (HTS),as the carrier solution. SUMMARY BACKGROUND DATA Advances in the field of bioartificial liver support have led to an increasing demand for successful,efficient means of cryopreservation of hepatocytes. METHODS Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group),both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen,cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion,urea synthesis,deethylation of ethoxyresorufin (CYT P450 activity),and responsiveness to stimulation with interleukin-6 (IL-6) were performed. RESULTS Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo,in comparison with control (90%). Albumin secretion,urea synthesis and CYT P450 activity yielded 33%,55%,and 59% in Media-cryo and 71%,80%,and 88% in HTS-cryo,respectively,compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. CONCLUSIONS This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability,long-term hepatospecific function,and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices. View Publication

A series of substituted 2-(aminopyridyl)- and 2-(aminopyrimidinyl)thiazole-5-carboxamides was identified as potent Src/Abl kinase inhibitors with excellent antiproliferative activity against hematological and solid tumor cell lines. Compound 13 was orally active in a K562 xenograft model of chronic myelogenous leukemia (CML),demonstrating complete tumor regressions and low toxicity at multiple dose levels. On the basis of its robust in vivo activity and favorable pharmacokinetic profile,13 was selected for additional characterization for oncology indications. View Publication

Whether cytokines can modulate the fate of primitive hematopoietic progenitor cells (HPCs) through successive in vitro cell divisions has not been established. Single human marrow CD34+CD38-/lo cells in the G0 phase of cell cycle were cultured under 7 different cytokine combinations,monitored for proliferation on days 3,5,and 7,then assayed for long-term culture-initiating cell (LTC-IC) function on day 7. LTC-IC function was then retrospectively correlated with prior number of in vitro cell divisions to determine whether maintenance of LTC-IC function after in vitro cell division is dependent on cytokine exposure. In the presence of proliferation progression signals,initial cell division was independent of cytokine stimulation,suggesting that entry of primitive HPCs into the cell cycle is a stochastic property. However,kinetics of proliferation beyond day 3 and maintenance of LTC-IC function were sensitive to cytokine stimulation,such that LTC-IC underwent an initial long cell cycle,followed by more synchronized shorter cycles varying in length depending on the cytokine combination. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation studies revealed analogous results to those obtained with LTC-ICs. These data suggest that although exit from quiescence and commitment to proliferation might be stochastic,kinetics of proliferation,and possibly fate of primitive HPCs,might be modulated by extrinsic factors. View Publication

In the present study,we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor-ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts,similar to neuroblastoma cell lines,involves NKp46 and NKp30 (natural cytotoxicity receptors). However,freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover,a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably,susceptibility to lysis directly correlated with the surface expression,on neuroblasts,of poliovirus receptor [PVR (CD155)],a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed,PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover,monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus,assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing. View Publication

Monocyte cytokines (ie,monokines) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma),which is critical for monocyte clearance of infectious pathogens and tumor surveillance. Human CD56bright NK cells produce far more IFN-gamma in response to monokines than do CD56dim NK cells. The kinases and phosphatases involved in regulating IFN-gamma production by monokine-activated NK cells are not clearly identified. SHIP1 is a 5' inositol phosphatase that dephosphorylates the phosphatidylinositol-3 kinase (PI-3K) product PI3,4,5P3. Here,we show that constitutive expression of SHIP1 is distinctly lower in CD56bright NK cells compared with CD56dim NK cells,suggesting it could be an important negative regulator of IFN-gamma production in monokine-activated NK cells. Indeed,overexpression of SHIP1 in CD56bright NK cells followed by monokine activation substantially lowered IFN-gamma production. This effect was not seen when NK cells were infected with a SHIP1 mutant containing an inactive catalytic domain. Finally,NK cells in SHIP1-/- mice produced more IFN-gamma in response to monokines in vivo than did NK cells from wild-type mice. Collectively,these results demonstrate that SHIP1 negatively regulates monokine-induced NK cell IFN-gamma production in vitro and in vivo and provide the first molecular explanation for an important functional distinction observed between CD56bright and CD56dim human NK subsets. View Publication

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA,IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells,as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA,while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells,which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab,while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together,these results suggest that at an early stage of recurrent viral infection,NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells. View Publication

Because osteoblasts and marrow adipocytes are derived from a common mesenchymal progenitor,increased adipogenesis may occur at the expense of osteoblasts,leading to bone loss. Our previous in vitro studies indicated that activation of the proadipogenic transcription factor peroxisome proliferator-activated receptor isoform gamma 2 with rosiglitazone suppressed osteoblast differentiation. Here,we show that 5-month-old Swiss-Webster mice receiving rosiglitazone for 28 d exhibited bone loss associated with an increase in marrow adipocytes,a decrease in the ratio of osteoblasts to osteoclasts,a reduction in bone formation rate,and a reduction in wall width--an index of the amount of bone formed by each team of osteoblasts. Rosiglitazone had no effect on the number of early osteoblast or osteoclast progenitors,or on osteoblast life span,but decreased the expression of the key osteoblastogenic transcription factors Runx2 and Osterix in cultures of marrow-derived mesenchymal progenitors. These effects were associated with diversion of bipotential progenitors from the osteoblast to the adipocyte lineage,and suppression of the differentiation of monopotential osteoblast progenitors. However,rosiglitazone had no effect on osteoblastic cells at later stages of differentiation. Hence,rosiglitazone attenuates osteoblast differentiation and thereby reduces bone formation rate in vivo,leading to bone loss. These findings provide a mechanistic explanation for the recent evidence that peroxisome proliferator-activated receptor isoform gamma activation is a negative regulator of bone mass and suggest that the increased production of oxidized fatty acids with age may indeed be an important mechanism for age-related osteoporosis in humans. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

Human mesenchymal stem cells (hMSCs) are capable of differentiating into several cell types including adipocytes,osteoblasts,and chondrocytes,under appropriate culture conditions. We found that SP600125,an inhibitor of Jun N-terminal kinase (JNK),promoted adipogenesis whereas it repressed osteogenesis from hMSCs. SP600125 increased the expression of adipogenic transcription factors,CCAAT/enhancer-binding proteins alpha and beta as well as peroxisome proliferator-activated receptor gamma2,which suggested that the chemical acted on the early steps of transcriptional regulatory cascade in adipogenesis. A gene reporter assay showed that SP600125 and a dominant negative JNK promoted a transcriptional activity dependent on the cAMP-response element (CRE). Thus,JNK represses adipogenesis from hMSCs probably by,at least in part,inhibiting the transactivating function of CRE-binding protein. Another action of JNK,phosphorylation at Ser(307) of insulin receptor substrate-1,was also predicted to contribute to the repression of adipogenesis. View Publication

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication