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

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging,intraoperative visualization,and therapeutic applications. These APC analogs derived from chemically-synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB),APC permeability across intact BBB remains unknown. Three of our APC analogs,CLR1404 (PET radiotracer),CLR1501 (green fluorescence),and CLR1502 (near infrared fluorescence),were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEERtextgreater1500 Ω-cm(2)) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the iPSC-BMEC (35±5.7 (CLR1404),54±3.2 (CLR1501),and 26±4.9 (CLR1502) x10(-5) cm/min) compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29). Only our fluorescent APC analogs (CLR1501,CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover,addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP,and may affect fluorescence-guided applications. Overall,the characterization of APC analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents. View Publication

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication

BACKGROUND: Recurrence is a common problem in bladder cancer; this has been attributed to cancer stem cells. In this study,we characterized potential cancer stem cell populations isolated from three cell lines that demonstrate different responses to cisplatin. MATERIALS AND METHODS: The ALDEFLUOR® assay was used to isolate cells from TCCSUP,T24,and 5637 cell lines,and these cells were evaluated for their ability to form colonies,differentiate,migrate and invade. RESULTS: The cell lines demonstrate a spectrum of aldehyde dehydrogenase high (ALDH(High)) populations that correlate with resistance to cisplatin. In the two resistant cell lines,T24 and 5637,the ALDH(High) cells demonstrate increased colony formation,migration,invasion,and ability to differentiate. The resistant T24 and 5637 cell lines may serve as models to investigate alternative therapies for bladder cancer. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. 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

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool,as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies. However,implementation of this assay into clinical routine has been cumbersome,as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers,and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step. This review addresses the assays that have been described so far in the literature,including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication