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

Abnormal differentiation of myeloid cells is one of the hallmarks of cancer. However,the molecular mechanisms of this process remain elusive. In this study,we investigated the effect of tumor-derived factors on Janus kinase (Jak)/STAT signaling in myeloid cells during their differentiation into dendritic cells. Tumor cell conditioned medium induced activation of Jak2 and STAT3,which was associated with an accumulation of immature myeloid cells. Jak2/STAT3 activity was localized primarily in these myeloid cells,which prevented the differentiation of immature myeloid cells into mature dendritic cells. This differentiation was restored after removal of tumor-derived factors. Inhibition of STAT3 abrogated the negative effects of these factors on myeloid cell differentiation,and overexpression of STAT3 reproduced the effects of tumor-derived factors. Thus,this is a first demonstration that tumor-derived factors may affect myeloid cell differentiation in cancer via constitutive activation of Jak2/STAT3. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Mammalian cells generally require both mitogens and anchorage signals in order to proliferate. An important characteristic of many tumour cells is that they have lost this anchorage-dependent cell-cycle checkpoint,allowing them to proliferate without signals provided by their normal microenvironment. In the absence of anchorage signals from the extracellular matrix,many cell types arrest cell-cycle progression in G1 phase as a result of Rb-dependent checkpoints. However,despite inactivation of p53 and Rb proteins,SV40LT-expressing cells retain anchorage dependency,suggesting the presence of an uncharacterised cell-cycle checkpoint,which can be overridden by coexpression of oncogenic Ras. We report here that,although cyclin-CDK complexes persisted in suspension,proliferation was inhibited in LT-expressing cells by the CDK inhibitor p27(Kip1) (p27). Interestingly,this did not induce a stable arrest,but aberrant cell-cycle progression associated with stalled DNA replication,rereplication and chromosomal instability,which was sufficient to increase the frequency of oncogenic transformation. These results firstly indicate loss of anchorage in Rb- and p53-deficient cells as a novel mechanism for promotion of genomic instability; secondly suggest that anchorage checkpoints that protect normal cells from inappropriate proliferation act deleteriously in Rb- and p53-deficient cells to promote tumourigenesis; and thirdly indicate caution in the use of CDK inhibitors for cancer treatment. View Publication

Cancer-initiating cells (CIC) comprise a rare subpopulation of cells in tumors that are proposed to be responsible for tumor growth. Starting from CICs identified in head and neck squamous cell carcinomas (HNSCC),termed head and neck cancer-initiating cells (HN-CIC),we determined as a candidate stemness-maintaining molecule for HN-CICs the proinflammatory mediator S100A4,which is also known to be an inducer of epithelial-mesenchymal transition. S100A4 knockdown in HN-CICs reduced their self-renewal capability and their stemness and tumorigenic properties,both in vitro and in vivo. Conversely,S100A4 overexpression in HNSCC cells enhanced their stem cell properties. Mechanistic investigations indicated that attenuation of endogenous S100A4 levels in HNSCC cells caused downregulation of Notch2 and PI3K (phosphoinositide 3-kinase)/pAKT along with upregulation of PTEN,consistent with biological findings. Immunohistochemical analysis of HNSCC clinical specimens showed that S100A4 expression was positively correlated with clinical grading,stemness markers,and poorer patient survival. Together,our findings reveal a crucial role for S100A4 signaling pathways in maintaining the stemness properties and tumorigenicity of HN-CICs. Furthermore,our findings suggest that targeting S100A4 signaling may offer a new targeted strategy for HNSCC treatment by eliminating HN-CICs. 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

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control,we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells,respectively,PC-3,HeLa,CWR22Rv1,and DU-145 cells,and induced accumulation of polyploidal cells with textgreater or =4N DNA content. However,RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover,PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1),a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly,also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6,while simultaneously suppressing the expression of cyclin B and CDK1. View Publication

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS,we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3),including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also,GSK3 phosphorylated PAX3-FKHR in vitro,suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT),underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest,but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs,but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement,while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally,adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL. View Publication

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines,causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified textgreater 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM,whereas no cytotoxicity against EGF-R-negative leukemia cells was observed,even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival,19 days) and all control mice had tumors that rapidly progressed to reach an average size of textgreater 500 mm3 by 58 days,40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size textgreater 50 mm3. Thus,targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme. View Publication