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

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24�?� cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24�?� phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24�?� phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24�?� and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24�?� or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. 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

BACKGROUND: Combining MEK inhibitors with other signalling pathway inhibitors or conventional cytotoxic drugs represents a promising new strategy against cancer. RDEA119/BAY 869766 is a highly potent and selective MEK1/2 inhibitor undergoing phase I human clinical trials. The effects of RDEA119/BAY 869766 as a single agent and in combination with rapamycin were studied in 3 early passage primary pancreatic cancer xenografts,OCIP19,21,and 23,grown orthotopically. METHODS: Anti-cancer effects were determined in separate groups following chronic drug exposure. Effects on cell cycle and downstream signalling were examined by flow cytometry and western blot,respectively. Plasma RDEA119 concentrations were measured to monitor the drug accumulation in vivo. RESULTS: RDEA119/BAY 869766 alone or in combination with rapamycin showed significant growth inhibition in all the 3 models,with a significant decrease in the percentage of cells in S-phase,accompanied by a large decrease in bromodeoxyuridine labelling and cell cycle arrest predominantly in G1. The S6 ribosomal protein was inhibited to a greater extent with combination treatment in all the three models. Blood plasma pharmacokinetic analyses indicated that RDEA119 levels achieved in vivo are similar to those that produce target inhibition and cell cycle arrest in vitro. CONCLUSIONS: Agents targeting the ERK and mTOR pathway have anticancer activity in primary xenografts,and these results support testing this combination in pancreatic cancer patients. View Publication

BACKGROUND: Resistance to imatinib monotherapy frequently emerges in advanced stages of chronic myelogenous leukemia (CML),supporting the rationale for combination drug therapy. In the present study,the activities of the farnesyltransferase inhibitors (FTIs) L744,832 and LB42918,as single agents and in combination with imatinib,were investigated in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. MATERIALS AND METHODS: Growth inhibition of the cell lines and primary patient cells was assessed by MTT assays and colony-forming cell assays,respectively. Drug interactions were analyzed according to the median-effect method of Chou and Talalay. The determination of apoptotic cell death was performed by annexin V/propidium iodide staining. RESULTS: Combinations of both FTIs with imatinib displayed synergism or sensitization (potentiation) in all the cell lines tested. In primary chronic phase CML cells,additive and synergistic effects were discernible for the combination of imatinib plus L744,832 and imatinib plus LB42918,respectively. Annexin V/propidium iodide staining showed enhancement of imatinib-induced apoptosis with either drug combination,both in imatinib-sensitive and -resistant cells. CONCLUSION: The results indicated the potential of L744,832 and LB42918 as combination agents for CML patients on imatinib treatment. View Publication

PURPOSE: Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells,which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. MATERIALS AND METHODS: The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. RESULTS: ALDH1(high) cells were detected in 6.8% of 22Rv1 cells,which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p textless 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. CONCLUSIONS: Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay. View Publication

SALL4,a human homolog to Drosophila spalt,is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR),we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML,n = 81),and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML,most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation,as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells. View Publication

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication

ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication

Adipose-derived stem cells (ASCs) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix (ECM) composition and stiffness,migration,and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A,ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225,and MCF10A overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly,ASCs promoted the self-renewal of all cell types except SUM225. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow basal/stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells. Among multiple factors secreted by ASCs,IFN$$ and HGF displayed unique actions on epithelial cell hierarchy. IFN$$ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres,whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF,whereas adipocytes expressed higher levels of IFN$$. Since luminal progenitor cells are believed to be prone for transformation,IFN$$ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells. IMPLICATIONS This study suggests that the ratio of adipose-derived stem cells to adipocytes influences cancer cell hierarchy,which may impact incidence and progression. View Publication

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover,CaP metastasizes to the bone marrow,which harbors a rich source of lipids stored within adipocytes. Here,we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique,we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition,larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction,the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes,indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication