搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. View Publication

Selumetinib (AZD6244; ARRY-142886) is a tight-binding,uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non-small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC(50) textless1 micromol/L (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines,with a correlation between sensitivity and raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies,including a small number of genes in which differential expression was common to both histologies. In total,these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations,respectively. View Publication

Treatment with arsenic trioxide (As(2)O(3)) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As(2)O(3) (iAs(III)) is methylated in the liver to mono- and dimethylated metabolites,including methylarsonic acid,methylarsonous acid,dimethylarsinic acid,and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins,genotoxins,and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAs(III). Therefore,we compared the potency of iAs(III) and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation,growth inhibition,and apoptosis. Methylarsine oxide (MAs(III)O) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAs(III) in NB4 cells,an APL cell line. This was also observed in K562 human leukemia,lymphoma cell lines,and in primary culture of chronic lymphocytic leukemia cells,but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAs(III)O,in contrast to iAs(III),did not induce PML-retinoic acid receptor alpha degradation,or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment,hepatoma-derived HepG2 cells,but not NB4 cells,methylate radiolabeled iAs(III). Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAs(III) may contribute to As(2)O(3)-induced apoptosis but not differentiation of APL cells. MAs(III)O as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma. View Publication

In response to inflammatory stimuli,monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha),interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors,such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex,which plays a vital role in several cellular functions,including the regulation of transcription factors like NF-kappaB. In this study,we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132,a proteasome inhibitor,on the release of TNF-alpha,IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1,IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation,resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors. View Publication

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

PURPOSE: We have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines,the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524,2011). In this report,the ability of PP2 and PP1,the more specific Src inhibitor SU6656,and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1,Colo 357) and primary NSCLC (Tu459) cell lines were investigated. METHODS: The effects of PP2,PP1,SU6656,and SB431542 on TGF-β1-dependent cell scattering/EMT,cell migration/invasion,and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR,respectively. RESULTS: In all three cell lines tested,PP1,PP2,and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT,migration,and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast,SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1,and to a greater extent PP2,also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant. CONCLUSIONS: These data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC. View Publication

A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs),including professional antigen-presenting cells such as dendritic cells (DCs),play a central role in T-cell biology. Here,we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell-derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes,but cell death was significantly increased after monocyte depletion. Furthermore,monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin,and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES),where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively,the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders. View Publication

A subset of cells,tentatively called cancer stem cells (CSCs),in breast cancer have been associated with tumor initiation,drug resistance,and tumor persistence or aggressiveness. They are characterized by CD44 positivity,CD24 negativity,and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed,flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue,with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years,we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1,CD44,and cytokeratin to measure the coexpression of these proteins,putative CSCs appear in variable sized clusters and in 27 cases (of 490),which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size,histological grade,nodal status,ER-,PR,- and HER2-status. In this cohort,ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication

Up to now,no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes,which hampers its application in gene therapy and immunotherapy areas. Here,we report that LVs incorporating measles virus (MV) glycoproteins,H and F,on their surface allowed transduction of 50% of quiescent B cells,which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover,the naive and memory phenotypes of transduced resting B cells were maintained. Importantly,H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells,B-cell chronic lymphocytic leukemia cells,blocked in G(0)/G(1) early phase of the cell cycle. Thus,H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells. View Publication

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases,neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore,midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally,a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression,or by BH3-mimetics such as obatoclax,may be an attractive therapy concept in SM. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication