The Biology of the Germ line in Echinoderms

PubMed Central

Wessel, Gary M.; Brayboy, Lynae; Fresques, Tara; Gustafson, Eric A.; Oulhen, Nathalie; Ramos, Isabela; Reich, Adrian; Swartz, S. Zachary; Yajima, Mamiko; Zazueta, Vanessa

2014-01-01

SUMMARY The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed. PMID:23900765

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

PubMed Central

Albright, Emily R.

2013-01-01

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34+ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34+ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34+ cells. However, in contrast to what occurs in primary CD34+ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34+ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed. PMID:23824798

In vitro and in vivo studies on antitumor effects of gossypol on human stomach adenocarcinoma (AGS) cell line and MNNG induced experimental gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gunassekaran, G.R., E-mail: gunassekaran@yahoo.co.in; Kalpana Deepa Priya, D.; Gayathri, R.

2011-08-12

Highlights: {yields} Gossypol is a well known polyphenolic compound used for anticancer studies but we are the first to report that gossypol has antitumor effect on MNNG induced gastric cancer in experimental animal models. {yields} Our study shows that gossypol inhibits the proliferation of AGS (human gastric adenocarcinoma) cell line. {yields} In animal models, gossypol extends the survival of cancer bearing animals and also protects the cells from carcinogenic effect. {yields} So we suggest that gossypol would be a potential chemotherapeutic and chemopreventive agent for gastric cancer. -- Abstract: The present study has evaluated the chemopreventive effects of gossypol onmore » N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and on human gastric adenocarcinoma (AGS) cell line. Gossypol, C{sub 30}H{sub 30}O{sub 8}, is a polyphenolic compound that has anti proliferative effect and induces apoptosis in various cancer cells. The aim of this work was to delineate in vivo and in vitro anti-initiating mechanisms of orally administered gossypol in target (stomach) tissues and in human gastric adenocarcinoma (AGS) cell line. In vitro results prove that gossypol has potent cytotoxic effect and inhibit the proliferation of adenocarcinoma (AGS) cell line. In vivo results prove gossypol to be successful in prolonging the survival of MNNG induced cancer bearing animals and in delaying the onset of tumor in animals administrated with gossypol and MNNG simultaneously. Examination of the target (stomach) tissues in sacrificed experimental animals shows that administration of gossypol significantly reduces the level of tumor marker enzyme (carcino embryonic antigen) and pepsin. The level of Nucleic acid contents (DNA and RNA) significantly reduces, and the membrane damage of glycoprotein subsides, in the target tissues of cancer bearing animals, with the administration of gossypol. These data suggest that gossypol may create a beneficial effect in patients with gastric cancer.« less

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma.

PubMed

Yang, Yang; Hui, Lv; Yuqin, Che; Jie, Li; Shuai, Hou; Tiezhu, Zhou; Wei, Wang

2014-08-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 10 4 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction.

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Marshall, Marianne E.; Hinz, Trista K.; Kono, Scott A.; Singleton, Katherine R.; Bichon, Brady; Ware, Kathryn E.; Marek, Lindsay; Frederick, Barbara A.; Raben, David; Heasley, Lynn E.

2011-01-01

Purpose We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and EGFR-dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed while only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and ERK signaling in three HNSCC cell lines while three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR TKIs with no contribution from FGFR pathways. Conclusions FGFR signaling was dominant or co-dominant with EGFR in six HNSCC lines while three lines exhibited little or no role for FGFRs and were highly EGFR-dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. PMID:21673064

The effect of differentiation agents on inflammatory and oxidative responses of the human neuroblastoma cell line SK-N-SH.

PubMed

Niewiarowska-Sendo, Anna; Patrzalek, Katarzyna; Kozik, Andrzej; Guevara-Lora, Ibeth

2015-01-01

Obtaining a suitable experimental cellular model is a major problem for neuroscience studies. Neuroblastoma cell lines have been often applied in studies related to pathological disorders of nervous system. However, in the search for an ideal model, these cells must be differentiated to cancel their tumor character. The subsequent reactions that are caused by differentiation are not always indifferent to the same model. We evaluated the effect of two well known substances, used for SH-N-SK cell line differentiation, retinoic acid (RA) and phorbol-12-myristate-13-acetate (PMA), on the induction of pro-inflammatory and pro-oxidative reactions in these cells. Cells differentiated with PMA were able to produce significantly higher amounts of pro-inflammatory cytokines whereas the release of nitric oxide radicals was similar to that in undifferentiated cells. On the contrary, in RA-differentiated cells no significant changes in cytokine production were observed and the nitric oxide release was decreased. Additionally, the RA-differentiated neuronal model was more sensible to lipopolysaccharide stimulation, producing pro-inflammatory cytokines abundantly. These results suggest that RA-differentiated SH-N-SK cells provide a more suitable experimental model for the study of molecular and cellular mechanisms of the inflammation and oxidative stress in neuronal cells.

Subcloning the RBL-2H3 mucosal mast cell line reduces Ca2+ response heterogeneity at the single-cell level.

PubMed

Kuchtey, J; Fewtrell, C

1996-03-01

Ca2+ imaging experiments have revealed that for a wide variety of cell types, including RBL-2H3 mucosal mast cells, there are considerable cell-to-cell differences of the Ca2+ responses of individual cells. This heterogeneity is evident in both the shape and latency of the responses. Mast cells within a single microscopic field of view, which have experienced identical culture conditions and experimental preparation, display a wide variety of responses upon antigen stimulation. We have subcloned the RBL-2H3 mucosal mast cell line to test the hypothesis that genetic heterogeneity within the population is the cause of the Ca2+ response heterogeneity. We found that cell-to-cell variability was significantly reduced in four of five clonal lines. The response heterogeneity remaining within the clones was not an experimental artifact caused by differences in the amount of fura-2 loaded by individual cells. Factors other than genetic heterogeneity must partly account for Ca2+ response heterogeneity. It is possible that the complex shapes and variability of the Ca2+ responses are reflections of the fact that there are multiple factors underlying the Ca2-response to antigen stimulation. Small differences from cell to cell in one or more of these factors could be a cause of the remaining Ca2+ response heterogeneity.

CellLineNavigator: a workbench for cancer cell line analysis

PubMed Central

Krupp, Markus; Itzel, Timo; Maass, Thorsten; Hildebrandt, Andreas; Galle, Peter R.; Teufel, Andreas

2013-01-01

The CellLineNavigator database, freely available at http://www.medicalgenomics.org/celllinenavigator, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources. PMID:23118487

Mechanism of metformin action in MCF-7 and MDA-MB-231 human breast cancer cells involves oxidative stress generation, DNA damage, and transforming growth factor β1 induction.

PubMed

Marinello, Poliana Camila; da Silva, Thamara Nishida Xavier; Panis, Carolina; Neves, Amanda Fouto; Machado, Kaliana Larissa; Borges, Fernando Henrique; Guarnier, Flávia Alessandra; Bernardes, Sara Santos; de-Freitas-Junior, Júlio Cesar Madureira; Morgado-Díaz, José Andrés; Luiz, Rodrigo Cabral; Cecchini, Rubens; Cecchini, Alessandra Lourenço

2016-04-01

The participation of oxidative stress in the mechanism of metformin action in breast cancer remains unclear. We investigated the effects of clinical (6 and 30 μM) and experimental concentrations of metformin (1000 and 5000 μM) in MCF-7 and in MDA-MB-231 cells, verifying cytotoxicity, oxidative stress, DNA damage, and intracellular pathways related to cell growth and survival after 24 h of drug exposure. Clinical concentrations of metformin decreased metabolic activity of MCF-7 cells in the MTT assay, which showed increased oxidative stress and DNA damage, although cell death and impairment in the proliferative capacity were observed only at higher concentrations. The reduction in metabolic activity and proliferation in MDA-MB-231 cells was present only at experimental concentrations after 24 h of drug exposition. Oxidative stress and DNA damage were induced in this cell line at experimental concentrations. The drug decreased cytoplasmic extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT and increased nuclear p53 and cytoplasmic transforming growth factor β1 (TGF-β1) in both cell lines. These findings suggest that metformin reduces cell survival by increasing reactive oxygen species, which induce DNA damage and apoptosis. A relationship between the increase in TGF-β1 and p53 levels and the decrease in ERK1/2 and AKT was also observed. These findings suggest the mechanism of action of metformin in both breast cancer cell lineages, whereas cell line specific undergoes redox changes in the cells in which proliferation and survival signaling are modified. Taken together, these results highlight the potential clinical utility of metformin as an adjuvant during the treatment of luminal and triple-negative breast cancer.

CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation.

PubMed

El-Ashmawy, Mariam; Delgado, Oliver; Cardentey, Agnelio; Wright, Woodring E; Shay, Jerry W

2014-01-01

Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.

CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation

PubMed Central

El-Ashmawy, Mariam; Delgado, Oliver; Cardentey, Agnelio; Wright, Woodring E.; Shay, Jerry W.

2014-01-01

Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients. PMID:25536195

Withaferin A induces mitochondrial-dependent apoptosis in non-small cell lung cancer cells via generation of reactive oxygen species.

PubMed

Liu, Xi; Chen, Lei; Liang, Tao; Tian, Xiao-Dong; Liu, Yang; Zhang, Tao

2017-01-01

Withaferin A (WA) is a bioactive lactone, isolated from natural sources, mainly found in Withania somnifera, and was known to be highly effective against a variety of tumor cells both in vitro and in vivo. Accumulating experimental evidence suggests the involvement of reactive oxygen species (ROS) in WA-mediated cytotoxicity against cancer cells. Hence, the purpose of this study was to investigate the effect of WA in non-small cell lung cancer (NSCLC) cells and also the role of ROC in WA-mediated cytotoxicity. In the present study we investigated the cytotoxic potential of WA against NSCLC cell line A549 and also highlighted the mechanism of cytotoxicity of this compound. Non-carcinoma WI-38 and PBMC cell lines were used as controls. WA treatment resulted in a dose-dependent cytotoxicity in A549 cells, while the non-carcinoma cells WI-38 and PBMC were unaffected. Further experimental approaches revealed that ROS plays a major role in WAinduced apoptosis in NSCLC cells. WA induces oxidative damage to NSCLC cells with minimum toxicity to normal cells.

Genomic instability of osteosarcoma cell lines in culture: impact on the prediction of metastasis relevant genes.

PubMed

Muff, Roman; Rath, Prisni; Ram Kumar, Ram Mohan; Husmann, Knut; Born, Walter; Baudis, Michael; Fuchs, Bruno

2015-01-01

Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages. The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines. Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer

PubMed Central

Young, Jonathan H.; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D.; White, Michael A.; Marcotte, Edward M.

2016-01-01

Motivation: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Results: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. Availability and implementation: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper. Contact: marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26755624

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer.

PubMed

Young, Jonathan H; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D; White, Michael A; Marcotte, Edward M

2016-05-01

Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Active microwave negative-index metamaterial transmission line with gain.

PubMed

Jiang, Tao; Chang, Kihun; Si, Li-Ming; Ran, Lixin; Xin, Hao

2011-11-11

We studied the active metamaterial transmission line at microwave frequency. The active composite right-handed or left-handed transmission line was designed to incorporate a germanium tunnel diode with a negative differential resistance property as the gain device at the unit cell level. Measurements of the fabricated planar transmission line structures with one-, two-, and three-unit cells showed that the addition of the dc pumped tunnel diodes not only provided gain but also maintained the left handedness of the transmission line metamaterial. Simulation results agree well with experimental observation. This work demonstrated that negative index material can be obtained with a net gain when an external source is incorporated.

Infrared line intensities of chlorine monoxide

NASA Technical Reports Server (NTRS)

Kostiuk, T.; Faris, J. L.; Mumma, M. J.; Deming, D.; Hillman, J. J.

1986-01-01

Absolute infrared line intensities of several ClO lines in the rotational-vibrational (1-0) band were measured using infrared heterodyne spectroscopy near 12 microns. A measurement technique using combined ultraviolet absorption and infrared line measurements near 9.5 microns and 12 microns permitted an accurate determination of the column densities of O3 and ClO in the absorption cell and thus improved ClO line intensities. Results indicate ClO line and band intensities approximately 2.4 times lower than previous experimental results. Effects of possible failure of local thermodynamic equilibrium conditions in the absorption cell and the implication of the results for stratospheric ClO measurements in the infrared are discussed.

Effects of femara and tamoxifen on proliferation of FM3A cells in culture.

PubMed

Topcul, Mehmet; Topcul, Funda; Cetin, Idil

2013-01-01

In this study, antiproliferative effects of the selective estrogen receptor modulator Tamoxifen and the aromatase inhibitor letrozole (Femara) were evaluated and compared using the FM3A cell line, originating from a C3H mouse mammary carcinoma and positive in terms of estrogen receptor (ER) expression. Cell kinetic parameters including labelling index, mitotic index and labelling index were assessed after exposure of the. FM3A cell line to 0.001μg/ml of Tamoxifen and 0.25μg/ml of Femara for 4, 8, 16 and 32 h for all parameters. The results showed that cell growth was inhibited by both agents. There was a significant decrease in labelling index and mitotic index and significant increase in apoptotic index for all experimental groups. The differences between control and all experimental groups were statistically significant (p<0.001) for all applications.

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

PubMed Central

Parton, Angela; Bayne, Christopher J.; Barnes, David W.

2010-01-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories “envelope” and “oxidoreductase activity” but the SAE transcripts did not. GO analysis of SAE transcripts identified the category “anatomical structure formation” that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. PMID:20471924

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

PubMed

Parton, Angela; Bayne, Christopher J; Barnes, David W

2010-09-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. Copyright 2010 Elsevier Inc. All rights reserved.

Toward computer simulation of high-LET in vitro survival curves.

PubMed

Heuskin, A-C; Michiels, C; Lucas, S

2013-09-21

We developed a Monte Carlo based computer program called MCSC (Monte Carlo Survival Curve) able to predict the survival fraction of cells irradiated in vitro with a broad beam of high linear energy transfer particles. Three types of cell responses are studied: the usual high dose response, the bystander effect and the low-dose hypersensitivity (HRS). The program models the broad beam irradiation and double strand break distribution following Poisson statistics. The progression of cells through the cell cycle is taken into account while the repair takes place. Input parameters are experimentally determined for A549 lung carcinoma cells irradiated with 10 and 20 keV µm(-1) protons, 115 keV µm(-1) alpha particles and for EAhy926 endothelial cells exposed to 115 keV µm(-1) alpha particles. Results of simulations are presented and compared with experimental survival curves obtained for A549 and EAhy296 cells. Results are in good agreement with experimental data for both cell lines and all irradiation protocols. The benefits of MCSC are several: the gain of time that would have been spent performing time-consuming clonogenic assays, the capacity to estimate survival fraction of cell lines not forming colonies and possibly the evaluation of radiosensitivity parameters of given individuals.

CRISPR/Cas9 Genetic Modification of CYP3A5 *3 in HuH-7 Human Hepatocyte Cell Line Leads to Cell Lines with Increased Midazolam and Tacrolimus Metabolism.

PubMed

Dorr, Casey R; Remmel, Rory P; Muthusamy, Amutha; Fisher, James; Moriarity, Branden S; Yasuda, Kazuto; Wu, Baolin; Guan, Weihua; Schuetz, Erin G; Oetting, William S; Jacobson, Pamala A; Israni, Ajay K

2017-08-01

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 engineering of the CYP3A5 *3 locus (rs776746) in human liver cell line HuH-7 ( CYP3A5 *3/*3 ) has led to three CYP3A5 *1 cell lines by deletion of the exon 3B splice junction or point mutation. Cell lines CYP3A5 *1/*3 sd (single deletion), CYP3A5 *1/*1 dd (double deletion), or CYP3A5 *1/*3 pm (point mutation) expressed the CYP3A5 *1 mRNA and had elevated CYP3A5 mRNA ( P < 0.0005 for all engineered cell lines) and protein expression compared with HuH-7. In metabolism assays, HuH-7 had less tacrolimus (all P < 0.05) or midazolam (MDZ) (all P < 0.005) disappearance than all engineered cell lines. HuH-7 had less 1-OH MDZ (all P < 0.0005) or 4-OH (all P < 0.005) production in metabolism assays than all bioengineered cell lines. We confirmed CYP3A5 metabolic activity with the CYP3A4 selective inhibitor CYP3CIDE. This is the first report of genomic CYP3A5 bioengineering in human cell lines with drug metabolism analysis. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

PubMed Central

Atwell, Kathryn; Qin, Zhao; Gavaghan, David; Kugler, Hillel; Hubbard, E. Jane Albert; Osborne, James M.

2015-01-01

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems. PMID:26428008

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma

PubMed Central

YANG, YANG; HUI, LV; YUQIN, CHE; JIE, LI; SHUAI, HOU; TIEZHU, ZHOU; WEI, WANG

2014-01-01

Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 104 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; P<0.001). In addition, the two cell lines had a higher expression level of p53 protein in the experimental group as compared with the control group (t=40.810; P<0.001). Protein expression levels of Bcl-xL decreased significantly in the experimental group as compared with the control group (t=19.640; P=0.000). Therefore, saw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction. PMID:25009620

Classification of human carcinoma cells using multispectral imagery

NASA Astrophysics Data System (ADS)

Ćinar, Umut; Y. Ćetin, Yasemin; Ćetin-Atalay, Rengul; Ćetin, Enis

2016-03-01

In this paper, we present a technique for automatically classifying human carcinoma cell images using textural features. An image dataset containing microscopy biopsy images from different patients for 14 distinct cancer cell line type is studied. The images are captured using a RGB camera attached to an inverted microscopy device. Texture based Gabor features are extracted from multispectral input images. SVM classifier is used to generate a descriptive model for the purpose of cell line classification. The experimental results depict satisfactory performance, and the proposed method is versatile for various microscopy magnification options.

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

PubMed

Zhou, Yizhou; Shaw, David; Lam, Cynthia; Tsukuda, Joni; Yim, Mandy; Tang, Danming; Louie, Salina; Laird, Michael W; Snedecor, Brad; Misaghi, Shahram

2017-09-23

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

The efficacy of 9-cis retinoic acid in experimental models of cancer.

PubMed

Gottardis, M M; Lamph, W W; Shalinsky, D R; Wellstein, A; Heyman, R A

1996-01-01

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

NASA Astrophysics Data System (ADS)

Pavlovich, Elena; Volkova, Nataliia; Yakymchuk, Elena; Perepelitsyna, Olena; Sydorenko, Michail; Goltsev, Anatoliy

2017-08-01

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6-12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1-3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6-12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: II—Mathematical prediction and experimental validation of optimal cryopreservation protocols☆

PubMed Central

Kashuba, Corinna M.; Benson, James D.; Critser, John K.

2014-01-01

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1 M propylene glycol, a cooling rate of 1 °C/minute, and plunging into liquid nitrogen at −41 °C, combined with subsequent warming in a 22 °C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5 °C/minute in the presence of 1.0 M dimethyl sulfoxide or 1.0 M propylene glycol, combined with plunge temperature of −80 °C was optimal. PMID:24560712

Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

PubMed

Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

2016-07-15

Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. Copyright © 2016 Elsevier B.V. All rights reserved.

Chemical species of sulfur in prostate cancer cells studied by XANES spectroscopy

NASA Astrophysics Data System (ADS)

Czapla, Joanna; Kwiatek, Wojciech M.; Lekki, Janusz; Dulińska-Litewka, Joanna; Steininger, Ralph; Göttlicher, Jörg

2013-12-01

The role of sulfur in prostate cancer progression may be significant for understanding the process of carcinogenesis. This work, based on X-ray Absorption Near Edge Structure (XANES) spectroscopy, is focused on determination of sulfur chemical species occurring in prostate cancer cell lines. The experimental material consisted of four commercially available cell lines: three from metastasized prostate cancer (PC3, LNCaP, and DU145) and one, used as a control, from the non-tumourigenic peripheral zone of the prostate (PZ-HPV-7). The experiment was performed at the SUL-X beamline of the synchrotron radiation source ANKA, Karlsruhe (Germany). The K-edge XANES spectra of sulfur were analyzed by deconvolution in order to establish sulfur species that occur in prostate cancer cells and to find out whether there are any differences in their content between various cell lines. Experimental spectra were fitted in two ways: with two Gaussian peaks and one arctangent step function, and additionally by a Linear Combination Fit with spectra of reference compounds in order to obtain quantitative chemical information. All fitting procedures were performed with the Athena code (Ravel and Newville, 2005) and the results of deconvolution were used to determine the fraction of each sulfur form. The results of data analysis showed that cell lines from different metastasis had different ratio of reduced to oxidized sulfur species. The LCF analysis demonstrated that the highest content of GSH, one of the most important sulfur-bearing compounds in cells, was observed in DU145 cells. These findings may confirm the hypothesis of changes in redox balance in case of cancer initiation and progression.

Studies of Pressure-Broadening of Alkali Atom Resonance Lines for Modeling Atmospheres of Extrasolar Giant Planets and Brown Dwarfs

NASA Technical Reports Server (NTRS)

Kirby, Kate; Babb, J.; Yoshino, K.

2004-01-01

In L-dwarfs and T-dwarfs the resonance lines of sodium and potassium are so profoundly pressure-broadened that their wings extend several hundred nanometers from line center. With accurate knowledge of the line profiles as a function of temperature and pressure: such lines can prove to be valuable diagnostics of the atmospheres of such objects. We have initiated a joint program of theoretical and experimental research to study the line-broadening of alkali atom resonance lines due to collisions with species such as helium and molecular hydrogen. Although potassium and sodium are the alkali species of most interest in the atmospheres of cool brown dwarfs and extrasolar giant planets, some of our theoretical focus this year has involved the calculation of pressure-broadening of lithium resonance lines by He, as a test of a newly developed suite of computer codes. In addition, theoretical calculations have been carried out to determine the leading long range van der Waals coefficients for the interactions of ground and excited alkali metal atoms with helium atoms, to within a probable error of 2%. Such data is important in determining the behavior of the resonance line profiles in the far wings. Important progress has been made on the experimental aspects of the program since the arrival of a postdoctoral fellow in September. A new absorption cell has been designed, which incorporates a number of technical improvements over the previous cell, including a larger cell diameter to enhance the signal, and fittings which allow for easier cleaning, thereby significantly reducing the instrument down-time.

T cells in the lesion of experimental autoimmune encephalomyelitis. Enrichment for reactivities to myelin basic protein and to heat shock proteins.

PubMed Central

Mor, F; Cohen, I R

1992-01-01

To characterize the cellular immune response in an autoimmune lesion, we investigated the accumulation of specific T cells in the central nervous system in actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats, using a limiting dilution analysis (LDA) assay for T cells that proliferate in response to antigens. Lymphocytes isolated from the spinal cord infiltrate were compared with cells from the popliteal lymph nodes with respect to frequency of cells responding to basic protein (BP), mycobacterium tuberculosis (MT), the 65-kD heat shock protein (hsp65), allogeneic brown norway spleen cells, and concanavalin A. Additionally, we compared the BP frequency in acute EAE of cells from the spinal cord, peripheral blood, spleen and lymph nodes, and the spinal cord and lymph node after recovery from EAE. We found that acute EAE was associated with marked enrichment of BP-reactive T cells in the spinal cord relative to their frequency in the lymphoid organs and peripheral blood. The infiltrate was also enriched for T cells responding to hsp65; alloreactive T cells, in contrast, were not enriched. The frequency of BP reactive T cells in the spinal cord was highest at the peak of paralysis; however, BP-reactive T cells could still be detected at moderate frequencies after clinical recovery. We established BP- and Mycobacteria-reactive T cell lines from the spinal infiltrates that were CD4+ and TcR alpha beta +. Most of the BP lines were found to react to the major encephalitogenic epitope of guinea pig BP for rats (amino acids 71-90); these lines were found to mediate EAE in naive recipients. T cell lines recognizing other epitopes of BP were not encephalitogenic. All of the lines responsive to Mycobacteria recognized hsp65 or hsp70. These results indicating that the immune infiltrate in active EAE is enriched with cells responding to the autoantigen and to hsp65 were confirmed in EAE adoptively transferred by anti-BP T cell clone. Images PMID:1281835

Complexity of expression of the intermediate filaments of six new human ovarian carcinoma cell lines: new expression of cytokeratin 20.

PubMed

Yanagibashi, T; Gorai, I; Nakazawa, T; Miyagi, E; Hirahara, F; Kitamura, H; Minaguchi, H

1997-01-01

Six permanent human ovarian carcinoma cell lines (OVISE, OVTOKO, OVMANA and OVSAYO from clear cell adenocarcinoma, and OVSAHO and OVKATE from serous papillary adenocarcinoma) were established from solid tumours. The cell lines have been in culture for 5-8 years, the passage number varying from 62 to 246. Immunohistochemical analysis has shown that five of the six cell lines express at least six cytokeratin (CK) polypeptides. OVISE and OVSAYO expressed CKs 6, 7, 8, 18, 19 and 15 and/or 16. OVTOKO was positive for CKs 7, 8, 18, 19 and 15 and/or 16. OVSAHO expressed CKs 6, 7, 8, 14, 18, 19 and 15 and/or 16. OVMANA expressed CKs 6, 7, 8, 18, 19, 20 and 15 and/or 16. OVKATE expressed CKs 6, 7, 8, 13, 17, 18, 19, 20 and 15 and/or 16. The expression of CK7, additional expression of vimentin, and clinical and histopathological findings enabled us to confirm that six cell lines had been established from primary ovarian cancers. Two of the six cell lines were positive for CK20, although CK20 was not expressed in the original tumours. The heterotransplanted tumours produced by CK20-positive cells also expressed CK20. This is the first report of ovarian carcinoma cell lines that express CK20 irrespective of their histological type. CK20 has been found in all colon carcinoma cell lines, but only in the mucinous type of ovarian tumours. These new ovarian carcinoma cell lines will therefore provide a relevant experimental system for elucidating the regulatory control mechanisms of intermediate filament expression.

Establishment of a Brazilian line of human embryonic stem cells in defined medium: implications for cell therapy in an ethnically diverse population.

PubMed

Fraga, Ana M; Sukoyan, Marina; Rajan, Prithi; Braga, Daniela Paes de Almeida Ferreira; Iaconelli, Assumpto; Franco, José Gonçalves; Borges, Edson; Pereira, Lygia V

2011-01-01

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-1, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-1 maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-1 and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Combined use of the ASK and SHK-1 cell lines to enhance the detection of infectious salmon anemia virus

USGS Publications Warehouse

Rolland, J.B.; Bouchard, D.; Coll, J.; Winton, J.R.

2005-01-01

Infectious salmon anemia (ISA) is a severe disease primarily affecting commercially farmed Atlantic salmon (Salmo salar) in seawater. The disease has been reported in portions of Canada, the United Kingdom, the Faroe Islands, and the United States. Infectious salmon anemia virus (ISAV), the causative agent of ISA, has also been isolated from several asymptomatic marine and salmonid fish species. Diagnostic assays for the detection of ISAV include virus isolation in cell culture, a reverse transcriptase-PCR, an enzyme-linked immunosorbent assay, and an indirect fluorescent antibody test. Virus isolation is considered the gold standard, and 5 salmonid cell lines are known to support growth of ISAV. In this study, the relative performance of the salmon head kidney 1 (SHK-1), Atlantic salmon kidney (ASK), and CHSE-214 cell lines in detecting ISAV was evaluated using samples from both experimentally and naturally infected Atlantic salmon. Interlaboratory comparisons were conducted using a quality control-quality assurance ring test. Both the ASK and SHK-1 cell lines performed well in detecting ISAV, although the SHK-1 line was more variable in its sensitivity to infection and somewhat slower in the appearance of cytopathic effect. Relative to the SHK-1 and ASK lines, the CHSE-214 cell line performed poorly. Although the ASK line appeared to represent a good alternative to the more commonly used SHK-1 line, use of a single cell line for diagnostic assays may increase the potential for false-negative results. Thus, the SHK-1 and ASK cell lines can be used in combination to provide enhanced ability to detect ISAV.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

PubMed

Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

2013-11-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

The neuroendocrine phenotype, genomic profile and therapeutic sensitivity of GEPNET cell lines

PubMed Central

Hofving, Tobias; Arvidsson, Yvonne; Almobarak, Bilal; Inge, Linda; Pfragner, Roswitha; Persson, Marta; Stenman, Göran; Kristiansson, Erik; Johanson, Viktor; Nilsson, Ola

2018-01-01

Experimental models of neuroendocrine tumour disease are scarce, and no comprehensive characterisation of existing gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines has been reported. In this study, we aimed to define the molecular characteristics and therapeutic sensitivity of these cell lines. We therefore performed immunophenotyping, copy number profiling, whole-exome sequencing and a large-scale inhibitor screening of seven GEPNET cell lines. Four cell lines, GOT1, P-STS, BON-1 and QGP-1, displayed a neuroendocrine phenotype while three others, KRJ-I, L-STS and H-STS, did not. Instead, these three cell lines were identified as lymphoblastoid. Characterisation of remaining authentic GEPNET cell lines by copy number profiling showed that GOT1, among other chromosomal alterations, harboured losses on chromosome 18 encompassing the SMAD4 gene, while P-STS had a loss on 11q. BON-1 had a homozygous loss of CDKN2A and CDKN2B, and QGP-1 harboured amplifications of MDM2 and HMGA2. Whole-exome sequencing revealed both disease-characteristic mutations (e.g. ATRX mutation in QGP-1) and, for patient tumours, rare genetic events (e.g. TP53 mutation in P-STS, BON-1 and QGP-1). A large-scale inhibitor screening showed that cell lines from pancreatic NETs to a greater extent, when compared to small intestinal NETs, were sensitive to inhibitors of MEK. Similarly, neuroendocrine NET cells originating from the small intestine were considerably more sensitive to a group of HDAC inhibitors. Taken together, our results provide a comprehensive characterisation of GEPNET cell lines, demonstrate their relevance as neuroendocrine tumour models and explore their therapeutic sensitivity to a broad range of inhibitors. PMID:29444910

Chemopreventive effect of Cynodon dactylon (L.) Pers. extract against DMH-induced colon carcinogenesis in experimental animals.

PubMed

Albert-Baskar, Arul; Ignacimuthu, Savarimuthu

2010-07-01

The present study was aimed at evaluating the chemopreventive property of Cynodon dactylon. The antioxidant, antiproliferative and apoptotic potentials of the plant were investigated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, nitric oxide radical scavenging activity (NO(-)) and MTT assay on four cancer cell lines (COLO 320 DM, MCH-7, AGS, A549) and a normal cell line (VERO). In vivo chemopreventive property of the plant extract was studied in DMH-induced colon carcinogenesis. The methanolic extract of C. dactylon was found to be antiproliferative and antioxidative at lower concentrations and induced apoptotic cell death in COLO 320 DM cells. Treatment with methanolic extract of C. dactylon increased the levels of antioxidant enzymes and reduced the number of dysplastic crypts in DMH-induced colon of albino rats. The present investigation revealed the anticancer potential of methanolic extract of C. dactylon in COLO 320 DM cells and experimentally induced colon carcinogenesis in rats.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Use of Microsphere Technology for Targeted Delivery of Rifampin to Mycobacterium tuberculosis-Infected Macrophages

PubMed Central

Barrow, Esther L. W.; Winchester, Gary A.; Staas, Jay K.; Quenelle, Debra C.; Barrow, William W.

1998-01-01

Microsphere technology was used to develop formulations of rifampin for targeted delivery to host macrophages. These formulations were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive rifampin during a 7-day experimental period. Treatment of Mycobacterium tuberculosis H37Rv-infected monocyte cell lines with rifampin-loaded microspheres resulted in a significant decrease in numbers of CFU at 7 days following initial infection, even though only 8% of the microsphere-loaded rifampin was released. The levels of rifampin released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of rifampin given as a free drug. These results demonstrate that rifampin-loaded microspheres can be formulated for effective sustained and targeted delivery to host macrophages. PMID:9756777

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development and characterization of two cell lines from gills of Atlantic salmon

USGS Publications Warehouse

Gjessing, Mona C.; Aamelfot, Maria; Batts, William N.; Benestad, Sylvie L.; Dale, Ole B.; Thoen, Even; Weli, Simon C.; Winton, James R.

2018-01-01

Gill disease in Atlantic salmon, Salmo salar L., causes big losses in the salmon farming industry. Until now, tools to cultivate microorganisms causing gill disease and models to study the gill responses have been lacking. Here we describe the establishment and characterization of two cell lines from the gills of Atlantic salmon. Atlantic salmon gill cell ASG-10 consisted of cells staining for cytokeratin and e-cadherin and with desmosomes as seen by transmission electron microscopy suggesting the cells to be of epithelial origin. These structures were not seen in ASG-13. The cell lines have been maintained for almost 30 passages and both cell lines are fully susceptible to infection by infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), infectious pancreatic necrosis virus (IPNV), Atlantic salmon reovirus TS (TSRV) and Pacific salmon paramyxovirus (PSPV). While infectious salmon anemia virus (ISAV) did not cause visible CPE, immunofluorescent staining revealed a sub-fraction of cells in both the ASG-10 and ASG-13 lines may be permissive to infection. ASG-10 is able to proliferate and migrate to close scratches in the monolayer within seven days in vitro contrary to ASG-13, which does not appear to do have the same proliferative and migratory ability. These cell lines will be useful in studies of gill diseases in Atlantic salmon and may represent an important contribution for alternatives to experimental animals and studies of epithelial–mesenchymal cell biology.

Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research.

PubMed

Drexler, H G; Matsuo, A Y; MacLeod, R A

2000-11-01

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species

PubMed Central

Penketh, Philip G.; Baumann, Raymond P.; Ishiguro, Kimiko; Shyam, Krishnamurthy; Seow, Helen A.; Sartorelli, Alan C.

2010-01-01

Cloretazine [1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]-hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The number of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produce a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line. PMID:18479747

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

Tibbe, Arjan G J; de Grooth, Bart G; Greve, Jan; Dolan, Gerald J; Rao, Chandra; Terstappen, Leon W M M

2002-03-01

Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension. Copyright 2002 Wiley-Liss, Inc.

Dynamic mathematical modeling of IL13-induced signaling in Hodgkin and primary mediastinal B-cell lymphoma allows prediction of therapeutic targets.

PubMed

Raia, Valentina; Schilling, Marcel; Böhm, Martin; Hahn, Bettina; Kowarsch, Andreas; Raue, Andreas; Sticht, Carsten; Bohl, Sebastian; Saile, Maria; Möller, Peter; Gretz, Norbert; Timmer, Jens; Theis, Fabian; Lehmann, Wolf-Dieter; Lichter, Peter; Klingmüller, Ursula

2011-02-01

Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.

Modelling cell population growth with applications to cancer therapy in human tumour cell lines.

PubMed

Basse, Britta; Baguley, Bruce C; Marshall, Elaine S; Wake, Graeme C; Wall, David J N

2004-01-01

In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines. Copyright 2004 Elsevier Ltd.

Palladium(II) complexes with N-heteroaromatic bidentate hydrazone ligands: the effect of the chelate ring size and lipophilicity on in vitro cytotoxic activity.

PubMed

Filipović, Nenad; Grubišić, Sonja; Jovanović, Maja; Dulović, Marija; Marković, Ivanka; Klisurić, Olivera; Marinković, Aleksandar; Mitić, Dragana; Anđelković, Katarina; Todorović, Tamara

2014-09-01

Novel Pd(II) complex with N-heteroaromatic Schiff base ligand, derived from 8-quinolinecarboxaldehyde (q8a) and ethyl hydrazinoacetate (haOEt), was synthesized and characterized by analytical and spectroscopy methods. The structure of novel complex, as well as structures of its quinoline and pyridine analogues, was optimized by density functional theory calculations, and theoretical data show good agreement with experimental results. A cytotoxic action of the complexes was evaluated on cultures of human promyelocytic leukemia (HL-60), human glioma (U251), rat glioma (C6), and mouse fibrosarcoma (L929) cell lines. Among investigated compounds, only complexes with quinoline-based ligands reduce the cell numbers in a dose-dependent manner in investigated cell lines. The observed cytotoxic effect of two isomeric quinoline-based complexes is predominantly mediated through the induction of apoptotic cell death in HL-60 cell line. The cytotoxicity of most efficient novel Pd(II) complex is comparable to the activity of cisplatin, in all cell lines investigated. © 2014 John Wiley & Sons A/S.

[Study on garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M].

PubMed

Wu, Fayin; Zhou, Hefeng; Fan, Zhiying; Zhu, Yawen; Li, Yongye; Yao, Yukun; Ran, Dan

2014-02-01

To observe the effect of garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M. Human salivary in adenoid cystic carcinoma cell line AC-M was cultured, divided into the experimental group (5-FU group, garlic oil group, garlic oil + 5-FU group) and the control group, to observe the growth activity of tumor cells by MTT methods; to analyse the changes of cell cycle and apoptosis rate by flow cytometry. MTT experiments showed that 5-FU, garlic oil, garlic oil and 5-FU on ACC-M cells have inhibition in different concentration, with the increase of concentration and action time of the rise; Cell cycle analysis showed significant changes in flow cytometry. With the increase of concentration and the acting time, the G0/G1, phase of the cell ratio increased, S had no significant change, but G2/M phase cells decreased. Apoptosis rate display showed garlic oil combined with 5-FU induced apoptosis of ACC-M cells was significantly stronger than single group. Garlic oil can effectively induce the apoptosis of adenoid cystic carcinoma cell line ACC-M. The effect of garlic oil combined with 5-FU on ACC-M cells was stronger than the garlic oil, 5-FU used alone.

The combined effects of high-energy shock waves and ionising radiation on a human bladder cancer cell line.

PubMed

Fickweiler, S; Steinbach, P; Wörle, K; Hofstädter, F

1996-01-01

The effects of high-energy shock waves (HESW) generated by an experimental Siemens lithotripter in combination with 137Cs gamma-rays were examined in vitro. Proliferation after treatment of immobilised pellets of either single cells or multicellular spheroids of the bladder cancer cell line RT4 was determined using colony-forming assays and cell cycle analysis. Surviving and cell cycle fractions were calculated for each shock wave and radiation application mode separately, and for sequential combination in different successions for the purpose of characterizing the interaction of both treatment modalities. Combination of HESW and ionising radiation turned out to act additively or slightly supra-additively on both biologic models.

Cancer-cells on a chip for label-free optic detection of secreted molecules

NASA Astrophysics Data System (ADS)

Berthuy, Ophélie I.; Blum, Loïc. J.; Marquette, Christophe A.

2015-05-01

To unravel cell complexity, living-cell chips have been developed that allow delivery of experimental stimuli but also measurement of the resulting cellular responses. We have been developing a new concept for multiplexed detection of biomolecules secreted by different cancer cells. In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. For that purpose, antibodies and different cell lines were spotted using a piezoelectric spotter. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. There is no doubt that our chip will, in a near future, be applied to more multiplexed and complex biological secretion systems for which kinetic data are at the moment not reachable using standard cellular biology tools.

Development and Characterisation of a Human Chronic Skin Wound Cell Line-Towards an Alternative for Animal Experimentation.

PubMed

Caley, Matthew; Wall, Ivan B; Peake, Matthew; Kipling, David; Giles, Peter; Thomas, David W; Stephens, Phil

2018-03-27

Background : Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives : To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results : Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions : These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.

Genetically fluorescent melanoma bone and organ metastasis models.

PubMed

Yang, M; Jiang, P; An, Z; Baranov, E; Li, L; Hasegawa, S; Al-Tuwaijri, M; Chishima, T; Shimada, H; Moossa, A R; Hoffman, R M

1999-11-01

We report here the establishment and metastatic properties of bright, highly stable, green fluorescent protein (GFP) expression transductants of the B16 mouse malignant melanoma cell line and the LOX human melanoma line. The highly fluorescent malignant melanoma cell lines allowed the visualization of skeletal and multiorgan metastases after i.v. injection of B16 cells in C57BL/6 mice and intradermal injection of LOX cells in nude mice. The melanoma cell lines were transduced with the pLEIN expression retroviral vector containing the GFP and neomycin resistance genes. Stable B16F0 and LOX clones expressing high levels of GFP were selected stepwise in vitro in levels of G418 of up to 800 microg/ml. Extensive bone and bone marrow metastases of B16F0 were visualized by GFP expression when the animals were sacrificed 3 weeks after cell implantation. Metastases for both cell lines were visualized in many organs, including the brain, lung, pleural membrane, liver, kidney, adrenal gland, lymph nodes, skeleton, muscle, and skin by GFP fluorescence. This is the first observation of experimental skeletal metastases of melanoma, which was made possible by GFP expression. These models should facilitate future studies of the mechanism and therapy of bone and multiorgan metastasis of melanoma.

Molecular predictors of therapeutic response to specific anti-cancer agents

DOEpatents

Spellman, Paul T.; Gray, Joe W.; Sadanandam, Anguraj; Heiser, Laura M.; Gibb, William J.; Kuo, Wen-lin; Wang, Nicholas J.

2016-11-29

Herein is described the use of a collection of 50 breast cancer cell lines to match responses to 77 conventional and experimental therapeutic agents with transcriptional, proteomic and genomic subtypes found in primary tumors. Almost all compounds produced strong differential responses across the cell lines produced responses that were associated with transcriptional and proteomic subtypes and produced responses that were associated with recurrent genome copy number abnormalities. These associations can now be incorporated into clinical trials that test subtype markers and clinical responses simultaneously.

Detection of Virus-Specific CD8+ T Cells With Cross-Reactivity Against Alloantigens: Potency and Flaws of Present Experimental Methods

PubMed Central

van den Heuvel, Heleen; Heutinck, Kirstin M.; van der Meer-Prins, Ellen P.M.W.; Yong, Si La; Claas, Frans H.J.; ten Berge, Ineke J.M.

2015-01-01

Background Virus-specific T cells have the intrinsic capacity to cross-react against allogeneic HLA antigens, a phenomenon known as heterologous immunity. In transplantation, these cells may contribute to the alloimmune response and negatively impact graft outcome. This study describes the various techniques that can be used to detect heterologous immune responses of virus-specific CD8+ T cells against allogeneic HLA antigens. The strengths and weaknesses of the different approaches are discussed and illustrated by experimental data. Methods Mixed-lymphocyte reactions (MLRs) were performed to detect allo-HLA cross-reactivity of virus-specific CD8+ T cells in total peripheral blood mononuclear cells. T-cell lines and clones were generated to confirm allo-HLA cross-reactivity by IFNγ production and cytotoxicity. In addition, the conventional MLR protocol was adjusted by introducing a 3-day resting phase and subsequent short restimulation with alloantigen or viral peptide, whereupon the expression of IFNγ, IL-2, CD107a, and CD137 was determined. Results The accuracy of conventional MLR is challenged by potential bystander activation. T-cell lines and clones can circumvent this issue, yet their generation is laborious and time-consuming. Using the adjusted MLR and restimulation protocol, we found that only truly cross-reactive T cells responded to re-encounter of alloantigen and viral peptide, whereas bystander-activated cells did not. Conclusions The introduction of a restimulation phase improved the accuracy of the MLR as a screening tool for the detection of allo-HLA cross-reactivity by virus-specific CD8+ T cells at bulk level. For detailed characterization of cross-reactive cells, T-cell lines and clones remain the golden standard. PMID:27500209

Identification of possible genetic alterations in the breast cancer cell line MCF-7 using high-density SNP genotyping microarray

PubMed Central

Wang, Hui-Yun; Greenawalt, Danielle; Cui, Xiangfeng; Tereshchenko, Irina V; Luo, Minjie; Yang, Qifeng; Azaro, Marco A; Hu, Guohong; Chu, Yi; Li, James Y; Shen, Li; Lin, Yong; Zhang, Lianjun

2009-01-01

Context: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. Aims: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. Materials and Methods: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. Results: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. Conclusions: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues. PMID:19439911

Study of small-cell lung cancer cell-based sensor and its applications in chemotherapy effects rapid evaluation for anticancer drugs.

PubMed

Guohua, Hui; Hongyang, Lu; Zhiming, Jiang; Danhua, Zhu; Haifang, Wan

2017-11-15

Small cell lung cancer (SCLC) is a smoking-related cancer disease. Despite improvement in clinical survival, SCLC outcome remains extremely poor. Cisplatin (DDP) is the first-line chemotherapy drug for SCLC, but the choice of second-line chemotherapy drugs is not clear. In this paper, a SCLC cell-based sensor was proposed, and its applications in chemotherapy effects rapid evaluation for anticancer drugs were investigated. SCLC cell lines lung adenocarcinoma cell (LTEP-P) and DDP-resistant lung adenocarcinoma cell (LTEP-P/DDP-1.0) are cultured on carbon screen-printed electrode (CSPE) to fabricate integrated cell-based sensor. Several chemotherapy anticancer drugs, including cisplatin, ifosmamide, gemcitabine, paclitaxel, docetaxel, vinorelbine, etoposide, camptothecin, and topotecan, are selected as experimental chemicals. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests are conducted to evaluate chemotherapy drug effects on LTEP-P and LTEP-P/DDP-1.0 cell lines. Electrical cell-substrate impedance sensing (ECIS) responses to anti-tumor chemicals are measured and processed by double-layered cascaded stochastic resonance (DCSR). Cisplatin solutions in different concentrations measurement results demonstrate that LTEP-P cell-based sensor presents quantitative analysis abilities for cisplatin and topotecan. Cisplatin and its mixtures can also be discriminated. Results demonstrate that LTEP-P cell-based sensor sensitively evaluates chemotherapy drugs' apoptosis function to SCLC cells. LTEP-P/DDP-1.0 cell-based sensor responses demonstrate that gemcitabine, vinorelbine, and camptothecin are ideal second-line drugs for clinical post-cisplatin therapy than other drugs according to MTT test results. This work provides a novel way for SCLC second-line clinical chemotherapy drug screening. Copyright © 2017 Elsevier B.V. All rights reserved.

An integrated bioinformatics analysis to dissect kinase dependency in triple negative breast cancer.

PubMed

Ryall, Karen A; Kim, Jihye; Klauck, Peter J; Shin, Jimin; Yoo, Minjae; Ionkina, Anastasia; Pitts, Todd M; Tentler, John J; Diamond, Jennifer R; Eckhardt, S Gail; Heasley, Lynn E; Kang, Jaewoo; Tan, Aik Choon

2015-01-01

Triple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis. We integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. We validated our predictions using published and new experimental data. In summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

Brush border membrane vesicle and Caco-2 cell line: Two experimental models for evaluation of absorption enhancing effects of saponins, bile salts, and some synthetic surfactants

PubMed Central

Moghimipour, Eskandar; Tabassi, Sayyed Abolghassem Sajadi; Ramezani, Mohammad; Handali, Somayeh; Löbenberg, Raimar

2016-01-01

The aim of this study was to investigate the influence of absorption enhancers in the uptake of hydrophilic compounds. The permeation of the two hydrophilic drug models gentamicin and 5 (6)-carboxyfluorescein (CF) across the brush border membrane vesicles and Caco-2 cell lines were evaluated using total saponins of Acanthophyllum squarrosum, Quillaja saponaria, sodium lauryl sulfate, sodium glycocholate, sodium taurodeoxycholate, and Tween 20 as absorption enhancers. Transepithelial electrical resistance (TEER) measurement was utilized to assess the paracellular permeability of cell lines. Confocal laser scanning microscopy (CLSM) was performed to obtain images of the distribution of CF in Caco-2 cells. These compounds were able to loosen tight junctions, thus increasing paracellular permeability. CLSM confirmed the effect of these absorption enhancers on CF transport across Caco-2 lines and increased the Caco-2 permeability via transcellular route. It was also confirmed that the decrease in TEER was transient and reversible after removal of permeation enhancers. PMID:27429925

Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

NASA Astrophysics Data System (ADS)

Tormoen, Garth W.; Recht, Olivia; Gruber, András; Levine, Ross L.; McCarty, Owen J. T.

2013-10-01

Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

NFATC3-PLA2G15 Fusion Transcript Identified by RNA Sequencing Promotes Tumor Invasion and Proliferation in Colorectal Cancer Cell Lines.

PubMed

Jang, Jee-Eun; Kim, Hwang-Phill; Han, Sae-Won; Jang, Hoon; Lee, Si-Hyun; Song, Sang-Hyun; Bang, Duhee; Kim, Tae-You

2018-06-14

This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer lines. We performed paired-end RNA sequencing of 28 colorectal cancer (CRC) cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. 1,380 FT candidates were detected through bioinformatics filtering. We selected 6 candidate FTs, including 4 inter-chromosomal and 2 intra-chromosomal FTs and each FT was found in at least 1 of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in 2. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Inferring Molecular Processes Heterogeneity from Transcriptional Data.

PubMed

Gogolewski, Krzysztof; Wronowska, Weronika; Lech, Agnieszka; Lesyng, Bogdan; Gambin, Anna

2017-01-01

RNA microarrays and RNA-seq are nowadays standard technologies to study the transcriptional activity of cells. Most studies focus on tracking transcriptional changes caused by specific experimental conditions. Information referring to genes up- and downregulation is evaluated analyzing the behaviour of relatively large population of cells by averaging its properties. However, even assuming perfect sample homogeneity, different subpopulations of cells can exhibit diverse transcriptomic profiles, as they may follow different regulatory/signaling pathways. The purpose of this study is to provide a novel methodological scheme to account for possible internal, functional heterogeneity in homogeneous cell lines, including cancer ones. We propose a novel computational method to infer the proportion between subpopulations of cells that manifest various functional behaviour in a given sample. Our method was validated using two datasets from RNA microarray experiments. Both experiments aimed to examine cell viability in specific experimental conditions. The presented methodology can be easily extended to RNA-seq data as well as other molecular processes. Moreover, it complements standard tools to indicate most important networks from transcriptomic data and in particular could be useful in the analysis of cancer cell lines affected by biologically active compounds or drugs.

Inferring Molecular Processes Heterogeneity from Transcriptional Data

PubMed Central

Wronowska, Weronika; Lesyng, Bogdan; Gambin, Anna

2017-01-01

RNA microarrays and RNA-seq are nowadays standard technologies to study the transcriptional activity of cells. Most studies focus on tracking transcriptional changes caused by specific experimental conditions. Information referring to genes up- and downregulation is evaluated analyzing the behaviour of relatively large population of cells by averaging its properties. However, even assuming perfect sample homogeneity, different subpopulations of cells can exhibit diverse transcriptomic profiles, as they may follow different regulatory/signaling pathways. The purpose of this study is to provide a novel methodological scheme to account for possible internal, functional heterogeneity in homogeneous cell lines, including cancer ones. We propose a novel computational method to infer the proportion between subpopulations of cells that manifest various functional behaviour in a given sample. Our method was validated using two datasets from RNA microarray experiments. Both experiments aimed to examine cell viability in specific experimental conditions. The presented methodology can be easily extended to RNA-seq data as well as other molecular processes. Moreover, it complements standard tools to indicate most important networks from transcriptomic data and in particular could be useful in the analysis of cancer cell lines affected by biologically active compounds or drugs. PMID:29362714

Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

PubMed

Nagamoto, Seiji; Iijima, Norio; Ishii, Hirotaka; Takumi, Ken; Higo, Shimpei; Aikawa, Satoko; Anzai, Megumi; Matsuo, Izumi; Nakagawa, Shinji; Takashima, Naoyuki; Shigeyoshi, Yasufumi; Sakamoto, Atsuhiro; Ozawa, Hitoshi

2016-05-04

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

Mechanisms of Acquired Drug Resistance to the HDAC6 Selective Inhibitor Ricolinostat Reveals Rational Drug-Drug Combination with Ibrutinib.

PubMed

Amengual, Jennifer E; Prabhu, Sathyen A; Lombardo, Maximilian; Zullo, Kelly; Johannet, Paul M; Gonzalez, Yulissa; Scotto, Luigi; Serrano, Xavier Jirau; Wei, Ying; Duong, Jimmy; Nandakumar, Renu; Cremers, Serge; Verma, Akanksha; Elemento, Olivier; O'Connor, Owen A

2017-06-15

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model. Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC 50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: The development of this ACY-1215-resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084-96. ©2016 AACR . ©2016 American Association for Cancer Research.

Nanoimprinted ultrafine line and space nanogratings for liquid crystal alignment.

PubMed

Liu, Yan Jun; Loh, Wei Wei; Leong, Eunice Sok Ping; Kustandi, Tanu Suryadi; Sun, Xiao Wei; Teng, Jing Hua

2012-11-23

Ultrafine 50 nm line and space nanogratings were fabricated using nanoimprint lithography, and were further used as an alignment layer for liquid crystals. The surface morphologies of the nanogratings were characterized and their surface energies were estimated through the measurement of the contact angles for two different liquids. Experimental results show that the surface energies of the nanogratings are anisotropic: the surface free energy towards the direction parallel to the grating lines is higher than that in the direction perpendicular to the grating lines. Electro-optical characteristics were tested from a twisted nematic liquid crystal cell, which was assembled using two identical nanogratings. Experimental results show that such a kind of nanograting is promising as an alternative to the conventional rubbing process for liquid crystal alignment.

RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

PubMed Central

Forest, David; Nishikawa, Ryuhei; Kobayashi, Hiroshi; Parton, Angela; Bayne, Christopher J.; Barnes, David W.

2007-01-01

We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3′ UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3′ mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology. PMID:17227856

Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.

PubMed

Plaza, N; Simón, D; Sierra, J; Moreno-Flores, M T

2016-01-26

Olfactory ensheathing glia (OEG) cells are known to foster axonal regeneration of central nervous system (CNS) neurons. Several lines of reversibly immortalized human OEG (ihOEG) have been previously established that enabled to develop models for their validation in vitro and in vivo. In this work, a constitutively GFP-expressing ihOEG cell line was obtained, and named Ts14-GFP. Ts14-GFP neuroregenerative ability was similar to that found for the parental line Ts14 and it can be assayed using in vivo transplantation experimental paradigms, after spinal cord or optic nerve damage. Additionally, we have engineered a low-regenerative ihOEG line, hTL2, using lentiviral transduction of the large T antigen from SV40 virus, denominated from now on Ts12. Ts12 can be used as a low regeneration control in these experiments. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

PubMed

Ma, Jie; Jiang, Nan; LaPatra, Scott E; Jin, Ling; Xu, Jin; Fan, Yuding; Zhou, Yong; Zeng, Lingbing

2015-06-12

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

PubMed Central

NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

2016-01-01

BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in vitro cell line model that could be used to study the biological basis of disparity in PCa between African–Americans and Caucasians. PMID:26764245

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials.

PubMed

Lin, Ann; Giuliano, Christopher J; Sayles, Nicole M; Sheltzer, Jason M

2017-03-24

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied.

Biomechanics of Tetrahymena escaping from a dead end

PubMed Central

Kikuchi, Kenji

2018-01-01

Understanding the behaviours of swimming microorganisms in various environments is important for understanding cell distribution and growth in nature and industry. However, cell behaviour in complex geometries is largely unknown. In this study, we used Tetrahymena thermophila as a model microorganism and experimentally investigated cell behaviour between two flat plates with a small angle. In this configuration, the geometry provided a ‘dead end' line where the two flat plates made contact. The results showed that cells tended to escape from the dead end line more by hydrodynamics than by a biological reaction. In the case of hydrodynamic escape, the cell trajectories were symmetric as they swam to and from the dead end line. Near the dead end line, T. thermophila cells were compressed between the two flat plates while cilia kept beating with reduced frequency; those cells again showed symmetric trajectories, although the swimming velocity decreased. These behaviours were well reproduced by our computational model based on biomechanics. The mechanism of hydrodynamic escape can be understood in terms of the torque balance induced by lubrication flow. We therefore conclude that a cell's escape from the dead end was assisted by hydrodynamics. These findings pave the way for understanding cell behaviour and distribution in complex geometries. PMID:29491169

Neuroblastoma Cell Lines Are Refractory to Genotoxic Drug-Mediated Induction of Ligands for NK Cell-Activating Receptors

PubMed Central

Veneziani, Irene; Brandetti, Elisa; Ognibene, Marzia; Pezzolo, Annalisa; Pistoia, Vito

2018-01-01

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted. PMID:29805983

In vivo physiological recording from the lateral line of juvenile zebrafish

PubMed Central

Olt, Jennifer; Allen, Claire E.

2016-01-01

Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. PMID:27161862

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone.

PubMed

Sakumoto, Marimu; Takahashi, Mami; Oyama, Rieko; Takai, Yoko; Kito, Fusako; Shiozawa, Kumiko; Qiao, Zhiwei; Yoshida, Akihiko; Endo, Makoto; Kawai, Akira; Kondo, Tadashi

2017-10-01

Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fluorescent cellular assay for screening agents inhibiting Pseudomonas aeruginosa adherence.

PubMed

Nosková, Libuše; Kubíčková, Božena; Vašková, Lucie; Bláhová, Barbora; Wimmerová, Michaela; Stiborová, Marie; Hodek, Petr

2015-01-16

Antibodies against Pseudomonas aeruginosa (PA) lectin, PAIIL, which is a virulence factor mediating the bacteria binding to epithelium cells, were prepared in chickens and purified from egg yolks. To examine these antibodies as a prophylactic agent preventing the adhesion of PA we developed a well plate assay based on fluorescently labeled bacteria and immortalized epithelium cell lines derived from normal and cystic fibrosis (CF) human lungs. The antibodies significantly inhibited bacteria adhesion (up to 50%) in both cell lines. In agreement with in vivo data, our plate assay showed higher susceptibility of CF cells towards the PA adhesion as compared to normal epithelium. This finding proved the reliability of the developed experimental system.

WE-H-BRA-06: Experimental Investigation of RBE for Lung Cancer Cell Lines as a Function of Dose and LET in Proton, Helium and Carbon Beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, D; Titt, U; Bronk, L

2016-06-15

Purpose: Investigate and quantify the effect of dose and LET on the RBE of protons, helium and carbon ions. Methods: High throughput, high accuracy experimental setups were custom designed to investigate the Relative Biological Effectiveness (RBE) dependence on the dose and Linear Energy Transfer (LET) values for proton, helium and carbon ion beams. The experiment was conducted at the HIT facility in collaboration with the DKFZ in Heidelberg/Germany. Clonogenic assays of two human lung cancer cell lines, H460 and H1437, were investigated in this study. γH2AX foci staining on the H460 cell line was also undertaken to facilitate the studymore » of differential DNA double-strand break induction and repair between low-design available at the HIT facility. Specific points along the Bragg curve corresponding to well-defined doses and LET values were chosen by appropriate selection of the pre-absorber thicknesses. With a setup design for horizontal beam lines we were able to minimize ion scattering in the cell plate, resulting in narrower energy spectra and hence LET distributions in the Bragg peak and in the distal falloff regions, compared to the earlier experiments. Results: Approximately 16,000 samples of cancer cells were irradiated during 23 hours of beam time. The preliminary results of the survival curves for both cell lines show a distinct dependence on LET for a given dose with decreased survival fractions at increasing LET values, encountered at the Bragg peak and in the distal falloff. Conclusion: Our preliminary findings are indicative of the importance of novel variable-RBE models for proton therapy and provide insight into the RBE of heavy ions for possible future heavy ion therapy facilities in the US. Funding support: SINF 2015/16.« less

Effect of heat dissipation of superparamagnetic nanoparticles in alternating magnetic field on three human cancer cell lines in magnetic fluid hyperthermia.

PubMed

Attar, Mohammad Mahdi; Haghpanahi, Mohammad

2016-01-01

The purpose of this study was to propose a method for constructing the software setup required for investigating thermal effect of superparamagnetic nanoparticles on three human cell lines. This article aimed to examine the required nanoparticle dose, frequency, field intensity and the exposure time. In the present study, first some general details were given about design and construction of the setup required for generating a safe magnetic field in order to examine the thermal effect of superparamagnetic nanoparticles on three human cancer cell lines, cultured under laboratory conditions. Next, a series of experimental tests were conducted to study the effect of magnetic field, on the cells. Finally, by applying three types of iron-based nanoparticles with mean diameters of 8, 15 and 20 nm, for 30 min, the temperature rise and specific absorption rate (SAR) were calculated. By conducting experimental tests, the maximum temperature rise at the resonance frequency of the coil was reported to be 80 kHz, and it was observed that all the cells died when temperature of the cells reached 42°C/30 min. Based on the experiments, it was observed that magnetic field with intensity of 8 kA/m within the frequency range of 80-180 kHz did not have any effect on the cells. Based on the results, it can be concluded that the nanoparticle dose of 80 µg/ml with diameter of 8 nm at the resonance frequency of coil for 30 min was sufficient to destroy all the cancerous cells in the flask.

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

Wagner, Stefanie; Münzer, Simon; Behrens, Peter; Scheper, Thomas; Bahnemann, Detlef; Kasper, Cornelia

2009-05-01

Different TiO2 and SiO2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect.

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

PubMed Central

Shipley, Mackenzie M.; Mangold, Colleen A.; Szpara, Moriah L.

2016-01-01

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease. PMID:26967710

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

PubMed

Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

2016-02-17

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells.

PubMed

Mollet, Mike; Godoy-Silva, Ruben; Berdugo, Claudia; Chalmers, Jeffrey J

2008-06-01

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

PubMed

Ko, Hyun-Ja; Kinkel, Sarah A; Hubert, François-Xavier; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Hirubalan, Premila; Toh, Ban-Hock; Scott, Hamish S; Alderuccio, Frank

2010-12-01

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficacy and safety of cell-associated vaccines against Marek's disease virus grown in QT35 cells or JBJ-1 cells.

PubMed

Geerligs, Harm; Spijkers, Ine; Rodenberg, Jeff

2013-06-01

The Marek's disease virus (MDV) vaccine strain CVI 988 usually is grown in primary chicken embryo fibroblasts (CEFs). We found that the strains could be grown also in the QT35 and JBJ-1 cell lines to titers in the same range as in the CEFs. Both cell lines are fibroblast-like cell lines, which can be grown in flat-bottomed tissue-culture flasks, roller bottles, and on microcarriers. For growth in QT35 cells it was necessary to adapt the virus to the cell line; for growth in JBJ-1 cells this was not necessary. We investigated the efficacy of experimental CVI 988 vaccines grown in QT35 cells and JBJ-1 cells. The efficacy studies were performed in accordance with European Pharmacopoeia (EP) monograph for live MDV disease vaccines. Groups of 1-day-old specific-pathogen-free chicks were vaccinated. Nonvaccinated control groups were included in the studies. Five to 7 days after vaccination all chickens were challenged with the very virulent MDV strain RB1B. After challenge the chickens were observed for a period of 70 days for signs of MD. The protection induced by CVI 988 grown in QT35 cells as well as JBJ-1 cells complied with the requirements of the EP that prescribe that the protection index should be at least 80%. The safety of the vaccines grown in QT35 cells and JBJ-1 cells was tested in a field study in commercial layer chickens. The vaccine virus was not safe after passaging in QT35 cells. This can be explained by the presence of fragments of the genome of MDV strains in the QT35 cell line. No signs of MD were noticed in the study in which CVI988 grown in JBJ-1 cells was tested. It is concluded that the JBJ-1 cell line is a suitable substrate for the current vaccines against MD.

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro.

PubMed

Garcia, Patricia; Leal, Pamela; Ili, Carmen; Brebi, Priscilla; Alvarez, Hector; Roa, Juan C

2013-06-01

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G-415 cells, and the effects on cell viability, anchorage-independent growth and migration was assessed by comparing them to scrambled vector-transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage-independent growth (P < 0.05). An increased p27 expression was observed in G-415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line.

PubMed

Zhang, Yufei; Shi, Jing; Liu, Shuying

2016-01-01

The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-term in vitro studies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunit β (CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes.

Synthesis, crystal structure and spectroscopy of bioactive Cd(II) polymeric complex of the non-steroidal anti-inflammatory drug diclofenac sodium: Antiproliferative and biological activity

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; McArdle, Patrick

2015-02-01

The interaction of Cd(II) with the non-steroidal anti-inflammatory drug diclofenac sodium (Dic) leads to the formation of the complex [Cd2(L)41.5(MeOH)2(H2O)]n(L = Dic), 1, which has been isolated and structurally characterized by X-ray crystallography. Diclofenac sodium and its metal complex 1 have also been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma), and a mouse fibroblast L-929 cell line. The results of cytotoxic activity in vitro expressed as IC50 values indicated the diclofenac sodium and cadmium chloride are non active or less active than the metal complex of diclofenac (1). Complex 1 was also found to be a more potent cytotoxic agent against T-24 and MCF-7 cancer cell lines than the prevalent benchmark metallodrug, cisplatin, under the same experimental conditions. The superoxide dismutase activity was measured by Fridovich test which showed that complex 1 shows a low value in comparison with Cu complexes. The binding properties of this complex to biomolecules, bovine or human serum albumin, are presented and evaluated. Antibacterial and growth inhibitory activity is also higher than that of the parent ligand compound.

Identification of a putative germ plasm in the amphipod Parhyale hawaiensis

PubMed Central

2013-01-01

Background Specification of the germ line is an essential event during the embryonic development of sexually reproducing animals, as germ line cells are uniquely capable of giving rise to the next generation. Animal germ cells arise through either inheritance of a specialized, maternally supplied cytoplasm called 'germ plasm’ or though inductive signaling by somatic cells. Our understanding of germ cell determination is based largely on a small number of model organisms. To better understand the evolution of germ cell specification, we are investigating this process in the amphipod crustacean Parhyale hawaiensis. Experimental evidence from previous studies demonstrated that Parhyale germ cells are specified through inheritance of a maternally supplied cytoplasmic determinant; however, this determinant has not been identified. Results Here we show that the one-cell stage Parhyale embryo has a distinct cytoplasmic region that can be identified by morphology as well as the localization of germ line-associated RNAs. Removal of this cytoplasmic region results in a loss of embryonic germ cells, supporting the hypothesis that it is required for specification of the germ line. Surprisingly, we found that removal of this distinct cytoplasm also results in aberrant somatic cell behaviors, as embryos fail to gastrulate. Conclusions Parhyale hawaiensis embryos have a specialized cytoplasm that is required for specification of the germ line. Our data provide the first functional evidence of a putative germ plasm in a crustacean and provide the basis for comparative functional analysis of germ plasm formation within non-insect arthropods. PMID:24314239

Questioning the utility of pooling samples in microarray experiments with cell lines.

PubMed

Lusa, L; Cappelletti, V; Gariboldi, M; Ferrario, C; De Cecco, L; Reid, J F; Toffanin, S; Gallus, G; McShane, L M; Daidone, M G; Pierotti, M A

2006-01-01

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

PubMed

Matuskova, Miroslava; Kozovska, Zuzana; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Cierna, Zuzana; Bohovic, Roman; Kucerova, Lucia

2015-04-09

Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

Electrical Field Guided Electrospray Deposition for Production of Gradient Particle Patterns.

PubMed

Yan, Wei-Cheng; Xie, Jingwei; Wang, Chi-Hwa

2018-06-06

Our previous work demonstrated the uniform particle pattern formation on the substrates using electrical field guided electrospray deposition. In this work, we reported for the first time the fabrication of gradient particle patterns on glass slides using an additional point, line, or bar electrode based on our previous electrospray deposition configuration. We also demonstrated that the polydimethylsiloxane (PDMS) coating could result in the formation of uniform particle patterns instead of gradient particle patterns on glass slides using the same experimental setup. Meanwhile, we investigated the effect of experimental configurations on the gradient particle pattern formation by computational simulation. The simulation results are in line with experimental observations. The formation of gradient particle patterns was ascribed to the gradient of electric field and the corresponding focusing effect. Cell patterns can be formed on the particle patterns deposited on PDMS-coated glass slides. The formed particle patterns hold great promise for high-throughput screening of biomaterial-cell interactions and sensing.

STL-based Analysis of TRAIL-induced Apoptosis Challenges the Notion of Type I/Type II Cell Line Classification

PubMed Central

Bertaux, François; Maler, Oded; Batt, Gregory

2013-01-01

Extrinsic apoptosis is a programmed cell death triggered by external ligands, such as the TNF-related apoptosis inducing ligand (TRAIL). Depending on the cell line, the specific molecular mechanisms leading to cell death may significantly differ. Precise characterization of these differences is crucial for understanding and exploiting extrinsic apoptosis. Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2. These differences are attributed to the activation of one of two pathways, leading to classification of cell lines into two groups: type I and type II. In this work we challenge this type I/type II cell line classification. We encode the three aforementioned distinguishing behaviors in a formal language, called signal temporal logic (STL), and use it to extensively test the validity of a previously-proposed model of TRAIL-induced apoptosis with respect to experimental observations made on different cell lines. After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors. In particular, this finding sheds light on the role of a feedback loop between caspases, and reconciliates two apparently-conflicting views regarding the importance of either upstream or downstream processes for cell-type determination. More generally, our work suggests that these three distinguishing behaviors should be merely considered as type I/II features rather than cell-type defining criteria. On the methodological side, this work illustrates the biological relevance of STL-diagrams, STL population data, and STL-guided parameter search implemented in the tool Breach. Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways. PMID:23675292

SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study

PubMed Central

Laga, Alvaro C.; Zhan, Qian; Weishaupt, Carsten; Ma, Jie; Frank, Markus H.; Murphy, George F.

2012-01-01

SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the periperhal nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma. PMID:21410764

[Reparative regeneration of rat skin under influence of hollow cathode lamp (HCL) with manganese and copper line spectrum emission].

PubMed

Mel'nikova, V I; Izvol'skaia, M S; Voronova, S N; Sharipova, M M; Rukin, E M; Zakharova, L A

2010-01-01

Influence of local light exposure by hollow cathode lamp with typical manganese and copper (HCL-Mn, Cu) line emission spectrum on posttraumatic regeneration rate of rat skin has been investigated. We performed the comparative analysis of the morphology and the differentiation ability of rat skin on the 15th and 24th days after full-thickness skin wound had been inflicted on rat dorsums. On the 15th day after injury, the experimental group (daily 30 s exposure for two weeks) showed scab loss, re-epithelialization, and hair regrowth, in contrast to the control rats, where scabs were still observed on the 24th day. Histological analysis revealed that in contrast to the control group the treatment with HCL-Mn, Cu resulted in the increased number of hair follicles and sebaceous glands, the decreased number of blood vessels and horizontal orientation of collagen fibers. The immunohistochemistry for OX-62 revealed that the number of dermal dendritic cells in the experimental groups was maximal on the 15th day, and then decreased to the 24th day after injury. The number of dermal dendritic cells was significantly lower in the control group. The immunohistochemistry for pan-keratins in the control animals revealed a high number of cells expressing different types of keratins, distributed in the main part of the epidermis on the 15th day after surgery, whereas in the experimental group the number of such cells was significantly lower and the cells were concentrated more close to the external part of the epidermis. The number of cells stained for keratin 19 was higher in the experimental group on the 15th day after surgery, whereas this number decreased in this group on the 24th day after surgery as compared to the control group. Thus, typical manganese and copper line spectrum emission emitted by hollow cathode lamp stimulates innate immunity, accelerates restoration of derma, skin epithelium and other skin derivates, and stimulates wound healing in general.

Comparative evaluation of maintenance of cell viability of an experimental transport media “coconut water” with Hank's balanced salt solution and milk, for transportation of an avulsed tooth: An in vitro cell culture study

PubMed Central

Thomas, Toby; Gopikrishna, Velayutham; Kandaswamy, Deivanayagam

2008-01-01

The purpose of this study was to evaluate the efficiency of a new storage medium, coconut water, in comparison with other traditional storage media like Hank's balanced salt solution (HBBS) and milk, in maintaining the viability of an established cell line BHK-21/C13 (baby hamster kidney fibroblasts) using the direct suspension cell culture technique. The storage media tested in the study were divided into three major groups and two control groups - Group A: HBBS, Group B: milk, and Group C: coconut water. The positive and negative controls corresponded to 0-minute and 24-hour dry times respectively. The three groups were then divided into five subgroups, each denoting the storage time periods 15 min, 30 min, 45 min, 60 min and 120 min respectively. The cell line BHK-21/C13 was subcultured and the number of cells was standardized by making a cell suspension using Minimal Essential Medium in five culture plates. One ml of each experimental group (HBBS, milk and coconut water) was added to eight wells of each culture plate. The culture plates containing the cells and the experimental groups were incubated for the respective time periods. The cells were then counted with a Neubauer counting chamber, under light microscope. The results were statistically analyzed using One-way ANOVA and Multiple Range Test using the Tukey-HSD procedure to identify the significant groups at p ≤ 0.05. Within the parameters of this study, it appears that coconut water may be a better alternative to HBSS or milk, in terms of maintaining cell viability. Coconut water can be used as a superior transport medium for avulsed teeth. PMID:20142880

Quantum-Dot Cellular Automata

NASA Astrophysics Data System (ADS)

Snider, Gregory

2000-03-01

Quantum-dot Cellular Automata (QCA) [1] is a promising architecture which employs quantum dots for digital computation. It is a revolutionary approach that holds the promise of high device density and low power dissipation. A basic QCA cell consists of four quantum dots coupled capacitively and by tunnel barriers. The cell is biased to contain two excess electrons within the four dots, which are forced to opposite "corners" of the four-dot cell by mutual Coulomb repulsion. These two possible polarization states of the cell will represent logic "0" and "1". Properly arranged, arrays of these basic cells can implement Boolean logic functions. Experimental results from functional QCA devices built of nanoscale metal dots defined by tunnel barriers will be presented. The experimental devices to be presented consist of Al islands, which we will call quantum dots, interconnected by tunnel junctions and lithographically defined capacitors. Aluminum/ aluminum-oxide/aluminum tunnel junctions were fabricated using a standard e-beam lithography and shadow evaporation technique. The experiments were performed in a dilution refrigerator at a temperature of 70 mK. The operation of a cell is evaluated by direct measurements of the charge state of dots within a cell as the input voltage is changed. The experimental demonstration of a functioning cell will be presented. A line of three cells demonstrates that there are no metastable switching states in a line of cells. A QCA majority gate will also be presented, which is a programmable AND/OR gate and represents the basic building block of QCA systems. The results of recent experiments will be presented. 1. C.S. Lent, P.D. Tougaw, W. Porod, and G.H. Bernstein, Nanotechnology, 4, 49 (1993).

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

PubMed

van Lent, P L; Licht, R; Dijkman, H; Holthuysen, A E; Berden, J H; van den Berg, W B

2001-11-01

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

Antimetastatic Efficacy of the Combination of Caffeine and Valproic Acid on an Orthotopic Human Osteosarcoma Cell Line Model in Nude Mice.

PubMed

Igarashi, Kentaro; Kawaguchi, Kei; Kiyuna, Tasuku; Murakami, Takashi; Yamamoto, Norio; Hayashi, Katsuhiro; Kimura, Hiroaki; Miwa, Shinji; Tsuchiya, Hiroyuki; Hoffman, Robert M

2017-03-01

We have previously reported that caffeine can enhance chemotherapy efficacy of bone and soft tissue sarcoma via cell-cycle perturbation. Valproic acid has histone deacetylase (HDAC) inhibitory activity. We have also reported the anti-tumor efficacy of combination treatment with caffeine and valproic acid against osteosarcoma primary tumors in a cell-line orthotopic mouse model. In this study, we performed combination treatment of caffeine and valproic acid on osteosarcoma cell lines in vitro and in spontaneous and experimental lung metastasis mouse models of osteosarcoma. Survival of 143B-RFP human osteosarcoma cells after exposure to caffeine and valproic acid for 72 hours was determined using the WST-8 assay. IC 50 values and combination indices were calculated. Mouse models of primary osteosarcoma and spontaneous lung metastasis were obtained by orthotopic intra-tibial injection of 143B-RFP cells. Valproic acid, caffeine, and combination of both drugs were administered from day 7, five times a week, for four weeks. Six weeks after orthotopic injection, lung samples were excised and observed with a fluorescence imaging system. A mouse model of experimental lung metastasis was obtained by tail vein injection of 143B-RFP cells. The mice were treated with these agents from day 0, five times a week for four weeks. Both caffeine and valproic acid caused concentration-dependent cell kill in vitro. Synergistic efficacy of the combination treatment was observed. In the spontaneous lung-metastasis model, the number of lung metastasis was 9.0±2.6 in the untreated group (G1); 10.8±2.9 in the caffeine group (G2); 10.0±3.1 in the valproic-acid group (G3); and 3.0±1.1 in the combination group (G4); (p=6.78E-5 control vs. combination; p=0.006 valproic acid vs. combination; p=0.003 caffeine vs. combination). In the experimental lung-metastasis model, the combination group significantly reduced lung metastases and improved overall survival (p=0.0005). Efficacy of the combination of caffeine and valproic acid was observed in vitro and in spontaneous and experimental lung-metastasis mouse models of osteosarcoma. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Purpose and regulation of stem cells: a systems-biology view from the Caenorhabditis elegans germ line.

PubMed

Cinquin, Olivier

2009-01-01

Stem cells are expected to play a key role in the development and maintenance of organisms, and hold great therapeutic promises. However, a number of questions must be answered to achieve an understanding of stem cells and put them to use. Here I review some of these questions, and how they relate to the model system provided by the Caenorhabditis elegans germ line, which is exceptional in its thorough genetic characterization and experimental accessibility under in vivo conditions. A fundamental question is how to define a stem cell; different definitions can be adopted that capture different features of interest. In the C. elegans germ line, stem cells can be defined by cell lineage or by cell commitment ('commitment' must itself be carefully defined). These definitions are associated with two other important questions about stem cells: their functions (which must be addressed following a systems approach, based on an evolutionary perspective) and their regulation. I review possible functions and their evolutionary groundings, including genome maintenance and powerful regulation of cell proliferation and differentiation, and possible regulatory mechanisms, including asymmetrical division and control of transit amplification by a developmental timer. I draw parallels between Drosophila and C. elegans germline stem cells; such parallels raise intriguing questions about Drosophila stem cells. I conclude by showing that the C. elegans germ line bears similarities with a number of other stem cell systems, which underscores its relevance to the understanding of stem cells.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line.

PubMed

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133(+), CD133(-) and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133(+) cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Although CD133(+) derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

PubMed Central

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

An non-uniformity voltage model for proton exchange membrane fuel cell

NASA Astrophysics Data System (ADS)

Li, Kelei; Li, Yankun; Liu, Jiawei; Guo, Ai

2017-01-01

The fuel cell used in transportation has environmental protection, high efficiency and no line traction power system which can greatly reduce line construction investment. That makes it a huge potential. The voltage uniformity is one of the most important factors affecting the operation life of proton exchange membrane fuel cell (PEMFC). On the basis of principle and classical model of the PEMFC, single cell voltage is calculated and the location coefficients are introduced so as to establish a non-uniformity voltage model. These coefficients are estimated with the experimental datum at stack current 50 A. The model is validated respectively with datum at 60 A and 100 A. The results show that the model reflects the basic characteristics of voltage non-uniformity and provides the beneficial reference for fuel cell control and single cell voltage detection.

Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: a potential improvement of intravesical chemotherapy in bladder cancer.

PubMed

Vásquez, Juan L; Gehl, Julie; Hermann, Gregers G

2012-12-01

Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporation improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3F cells, the RRs of survival were: 28%, 29%, and 33%, by concentrations 0μM, 500μM and 1000μM respectively. In conclusion, electroporation and mitomycin together are about 30% more effective than mitomycin alone. The results help to elucidate the additive effect of mitomycin and electric pulses and support the use of this combination in the treatment of bladder cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

D2O self-broadening study in 2.5 μ

NASA Astrophysics Data System (ADS)

Lavrentieva, N.; Lugovskoi, A.; Sinitsa, L.; Sherbakov, A.; Svetlichny, O.

2014-11-01

The absorption spectra of the D2O monomer in 3600…4200 cm-1 were recorded using Fourier Transform spectrometer FS-125M at room temperature and pressure of 15 and 33 mbar with spectral resolution of 0.03 cm-1 using 2.5 cm long absorption cell. Strong unblended D2O lines lying on the wing of the H2O stretching band were used to determine the line broadening parameters. They were determined from the line profile by Program VxpProfile. The differences between fitted line profiles and experimental ones do not exceed 2%. Registered D2O lines belong to (011) - (000) and (110) - (000) bands of the second triad. Self-broadening coefficients vary from 0.27 cm-1/atm to 0.445 cm-1/atm and they exceed 3 times the D2O-N2 line broadening coefficients in the v3. Calculations of self-broadening coefficients of the D2O lines were performed using semiempirical method based on the impact theory of broadening and included the correction factors. The calculated results well agree with experimental data.

Systematic modelling and design evaluation of unperturbed tumour dynamics in xenografts.

PubMed

Parra Guillen, Zinnia P Patricia; Mangas Sanjuan, Victor; Garcia-Cremades, Maria; Troconiz, Inaki F; Mo, Gary; Pitou, Celine; Iversen, Philip W; Wallin, Johan E

2018-04-24

Xenograft mice are largely used to evaluate the efficacy of oncological drugs during preclinical phases of drug discovery and development. Mathematical models provide a useful tool to quantitatively characterise tumour growth dynamics and also optimise upcoming experiments. To the best of our knowledge, this is the first report where unperturbed growth of a large set of tumour cell lines (n=28) has been systematically analysed using the model proposed by Simeoni in the context of non-linear mixed effect (NLME). Exponential growth was identified as the governing mechanism in the majority of the cell lines, with constant rate values ranging from 0.0204 to 0.203 day -1 No common patterns could be observed across tumour types, highlighting the importance of combining information from different cell lines when evaluating drug activity. Overall, typical model parameters were precisely estimated using designs where tumour size measurements were taken every two days. Moreover, reducing the number of measurement to twice per week, or even once per week for cell lines with low growth rates, showed little impact on parameter precision. However, in order to accurately characterise parameter variability (i.e. relative standard errors below 50%), a sample size of at least 50 mice is needed. This work illustrates the feasibility to systematically apply NLME models to characterise tumour growth in drug discovery and development, and constitutes a valuable source of data to optimise experimental designs by providing an a priori sampling window and minimising the number of samples required. The American Society for Pharmacology and Experimental Therapeutics.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines

PubMed Central

Weigelt, Britta; Warne, Patricia H; Lambros, Maryou B; Reis-Filho, Jorge S; Downward, Julian

2013-01-01

Purpose Endometrioid endometrial cancers (EECs) frequently harbor coexisting mutations in PI3K pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Experimental Design Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and MAPK signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, MEK and RAF inhibitors. RNA interference (RNAi) was employed to assess effects of KRAS silencing in EEC cells. Results EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor Temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2/6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66, a decrease in cell viability was observed. Conclusions Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA-mutant and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required. PMID:23674493

In Vitro Growth Inhibitory Activities of Natural Products from Irciniid Sponges against Cancer Cells: A Comparative Study

PubMed Central

BenRedjem Romdhane, Yosr; Elbour, Monia; Carbone, Marianna; Ciavatta, Maria Letizia; Gavagnin, Margherita; Mathieu, Véronique; Lefranc, Florence; Ktari, Leila; Ben Mustapha, Karim; Boudabous, Abdellatif; Kiss, Robert

2016-01-01

Marine sponges of the Irciniidae family contain both bioactive furanosesterterpene tetronic acids (FTAs) and prenylated hydroquinones (PHQs). Both classes of compounds are known for their anti-inflammatory, antioxidant, and antimicrobial properties and known to display growth inhibitory effects against various human tumor cell lines. However, the different experimental conditions of the reported in vitro bioassays, carried out on different cancer cell lines within separate studies, prevent realistic actual discrimination between the two classes of compounds from being carried out in terms of growth inhibitory effects. In the present work, a chemical investigation of irciniid sponges from Tunisian coasts led to the purification of three known FTAs and three known PHQs. The in vitro growth inhibitory properties of the six purified compounds have been evaluated in the same experiment in a panel of five human and one murine cancer cell lines displaying various levels of sensitivity to proapoptotic stimuli. Surprisingly, FTAs and PHQs elicited distinct profiles of growth inhibitory-responses, differing by one to two orders of magnitude in favor of the PHQs in all cell lines. The obtained comparative results are discussed in the light of a better selection of drug candidates from natural sources. PMID:27597966

CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials

PubMed Central

Lin, Ann; Giuliano, Christopher J; Sayles, Nicole M; Sheltzer, Jason M

2017-01-01

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been reported to be a genetic dependency in several cancer types. MELK RNAi and small-molecule inhibitors of MELK block the proliferation of various cancer cell lines, and MELK knockdown has been described as particularly effective against the highly-aggressive basal/triple-negative subtype of breast cancer. Based on these preclinical results, the MELK inhibitor OTS167 is currently being tested as a novel chemotherapy agent in several clinical trials. Here, we report that mutagenizing MELK with CRISPR/Cas9 has no effect on the fitness of basal breast cancer cell lines or cell lines from six other cancer types. Cells that harbor null mutations in MELK exhibit wild-type doubling times, cytokinesis, and anchorage-independent growth. Furthermore, MELK-knockout lines remain sensitive to OTS167, suggesting that this drug blocks cell division through an off-target mechanism. In total, our results undermine the rationale for a series of current clinical trials and provide an experimental approach for the use of CRISPR/Cas9 in preclinical target validation that can be broadly applied. DOI: http://dx.doi.org/10.7554/eLife.24179.001 PMID:28337968

Effects of celecoxib on cell apoptosis and Fas, FasL and Bcl-2 expression in a BGC-823 human gastric cancer cell line.

PubMed

Li, Qian; Peng, Jie; Liu, Ting; Zhang, Guiying

2017-09-01

Fas, which is an apoptotic-related protein, has an important role in cell apoptosis. Fas ligand (FasL) binds to Fas and activates apoptosis signal transduction. We previously demonstrated that the efficiency of celecoxib inhibited the proliferation and apoptosis of HT-29 colon cancer cell line. The BGC823 cell line was used as an experimental model to evaluate the potential role of celecoxib on gastric cancer cell apoptosis. Inhibitory effects of celecoxib on cell viability were determined by MTT assay. Cell apoptosis was evaluated by flow cytometric analysis and laser confocal microscopy. The results of the present study demonstrated that celecoxib inhibited the viability of BGC823 cells in a concentration- and time-dependent manner. Furthermore, the effect of BGC823 cells apoptosis was increased in a concentration-dependent manner. Western blotting was used to determine the protein expression levels of Fas, FasL, and B-cell lymphoma-2 (Bcl-2). During the celecoxib-induced apoptosis of BGC823 cells, celecoxib upregulated Fas expression and downregulated FasL and Bcl-2 expression in a concentration-dependent manner. These results suggest that celecoxib inhibited the growth and induced apoptosis of BGC823 gastric cancer cells by regulating the protein expression of Fas, FasL and Bcl-2.

Increased efficiency of mammalian somatic cell hybrid production under microgravity conditions during ballistic rocket flight

NASA Technical Reports Server (NTRS)

Schnettler, R.; Gessner, P.; Zimmermann, U.; Neil, G. A.; Urnovitz, H. B.

1989-01-01

The electrofusion of hybridoma cell lines under short-duration microgravity during a flight of the TEXUS 18 Black Brand ballistic sounding rocket at Kiruna, Sweden is reported. The fusion partners, growth medium, cell fusion medium, cell fusion, cell viability in the fusion medium, and postfusion cell culture are described, and the rocket, cell fusion chamber, apparatus, and module are examined. The experimental timeline, the effects of fusion medium and incubation time on cell viability and hybrid yields, and the effect of microgravity on hybrid yields are considered.

Three-photon-excited luminescence from unsymmetrical cyanostilbene aggregates: morphology tuning and targeted bioimaging.

PubMed

Mandal, Amal Kumar; Sreejith, Sivaramapanicker; He, Tingchao; Maji, Swarup Kumar; Wang, Xiao-Jun; Ong, Shi Li; Joseph, James; Sun, Handong; Zhao, Yanli

2015-05-26

We report an experimental observation of aggregation-induced enhanced luminescence upon three-photon excitation in aggregates formed from a class of unsymmetrical cyanostilbene derivatives. Changing side chains (-CH3, -C6H13, -C7H15O3, and folic acid) attached to the cyanostilbene core leads to instantaneous formation of aggregates with sizes ranging from micrometer to nanometer scale in aqueous conditions. The crystal structure of a derivative with a methyl side chain reveals the planarization in the unsymmetrical cyanostilbene core, causing luminescence from corresponding aggregates upon three-photon excitation. Furthermore, folic acid attached cyanostilbene forms well-dispersed spherical nanoaggregates that show a high three-photon cross-section of 6.0 × 10(-80) cm(6) s(2) photon(-2) and high luminescence quantum yield in water. In order to demonstrate the targeted bioimaging capability of the nanoaggregates, three cell lines (HEK293 healthy cell line, MCF7 cancerous cell line, and HeLa cancerous cell line) were employed for the investigations on the basis of their different folate receptor expression level. Two kinds of nanoaggregates with and without the folic acid targeting ligand were chosen for three-photon bioimaging studies. The cell viability of three types of cells incubated with high concentration of nanoaggregates still remained above 70% after 24 h. It was observed that the nanoaggregates without the folic acid unit could not undergo the endocytosis by both healthy and cancerous cell lines. No obvious endocytosis of folic acid attached nanoaggregates was observed from the HEK293 and MCF7 cell lines having a low expression of the folate receptor. Interestingly, a significant amount of endocytosis and internalization of folic acid attached nanoaggregates was observed from HeLa cells with a high expression of the folate receptor under three-photon excitation, indicating targeted bioimaging of folic acid attached nanoaggregates to the cancer cell line. This study presents a paradigm of using organic nanoaggregates for targeted three-photon bioimaging.

Development without germ cells: the role of the germ line in zebrafish sex differentiation.

PubMed

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-03-15

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin-antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.

Development without germ cells: The role of the germ line in zebrafish sex differentiation

PubMed Central

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-01-01

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin–antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males. PMID:15728735

Combined effect of microRNA, nutraceuticals and drug on pancreatic cancer cell lines.

PubMed

Pandita, Archana; Manvati, Siddharth; Singh, Shashank K; Vaishnavi, Samantha; Bamezai, Rameshwar N K

2015-05-25

We proposed to investigate the combination effect of microRNA, nutraceuticals and drug (MND), in two pancreatic cancer cell lines to assess the therapeutic potential. MIA PaCa-2 and PANC-1 cells transfected with miR-101 or miR-24-2 were treated with Betulinic acid or Thymoquinone and gemcitabine independently and in combination and assessed for the extent of synergism in both experimental and control conditions, considering significance at the p value of <0.05. miR-101 or miR-24-2 over-expressing cells when treated with lower than IC50 doses of the dietary compounds and drug showed a reduced (37-50%) viability in two cell lines with differential synergistic effect and the outcome for Pro-caspase3, Poly (ADP-ribose) polymerase (PARP) cleavage and PKM2 expression. Two independent microRNA backgrounds showed promise in therapeutic intervention of gemcitabine sensitive, MIA PaCa-2 and resistant, PANC-1 pancreatic cancer cells, in combination with dietary agents and drug. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Craise, L. M.; Durante, M.

1997-01-01

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

PubMed Central

Liu, Chieh-Lun; Watson, Aaron M.; Place, Allen R.; Jagus, Rosemary

2017-01-01

Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity. PMID:28587087

Optical modeling of fiber organic photovoltaic structures using a transmission line method.

PubMed

Moshonas, N; Stathopoulos, N A; O'Connor, B T; Bedeloglu, A Celik; Savaidis, S P; Vasiliadis, S

2017-12-01

An optical model has been developed and evaluated for the calculation of the external quantum efficiency of cylindrical fiber photovoltaic structures. The model is based on the transmission line theory and has been applied on single and bulk heterojunction fiber-photovoltaic cells. Using this model, optimum design characteristics have been proposed for both configurations, and comparison with experimental results has been assessed.

DeSigN: connecting gene expression with therapeutics for drug repurposing and development.

PubMed

Lee, Bernard Kok Bang; Tiong, Kai Hung; Chang, Jit Kang; Liew, Chee Sun; Abdul Rahman, Zainal Ariff; Tan, Aik Choon; Khang, Tsung Fei; Cheong, Sok Ching

2017-01-25

The drug discovery and development pipeline is a long and arduous process that inevitably hampers rapid drug development. Therefore, strategies to improve the efficiency of drug development are urgently needed to enable effective drugs to enter the clinic. Precision medicine has demonstrated that genetic features of cancer cells can be used for predicting drug response, and emerging evidence suggest that gene-drug connections could be predicted more accurately by exploring the cumulative effects of many genes simultaneously. We developed DeSigN, a web-based tool for predicting drug efficacy against cancer cell lines using gene expression patterns. The algorithm correlates phenotype-specific gene signatures derived from differentially expressed genes with pre-defined gene expression profiles associated with drug response data (IC 50 ) from 140 drugs. DeSigN successfully predicted the right drug sensitivity outcome in four published GEO studies. Additionally, it predicted bosutinib, a Src/Abl kinase inhibitor, as a sensitive inhibitor for oral squamous cell carcinoma (OSCC) cell lines. In vitro validation of bosutinib in OSCC cell lines demonstrated that indeed, these cell lines were sensitive to bosutinib with IC 50 of 0.8-1.2 μM. As further confirmation, we demonstrated experimentally that bosutinib has anti-proliferative activity in OSCC cell lines, demonstrating that DeSigN was able to robustly predict drug that could be beneficial for tumour control. DeSigN is a robust method that is useful for the identification of candidate drugs using an input gene signature obtained from gene expression analysis. This user-friendly platform could be used to identify drugs with unanticipated efficacy against cancer cell lines of interest, and therefore could be used for the repurposing of drugs, thus improving the efficiency of drug development.

Multi-objective experimental design for (13)C-based metabolic flux analysis.

PubMed

Bouvin, Jeroen; Cajot, Simon; D'Huys, Pieter-Jan; Ampofo-Asiama, Jerry; Anné, Jozef; Van Impe, Jan; Geeraerd, Annemie; Bernaerts, Kristel

2015-10-01

(13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi-objective design should stimulate its application within the field of (13)C-based metabolic flux analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Intracellular Ca2+ concentration in the N1E-115 neuronal cell line and its use for peripheric nerve regeneration.

PubMed

Rodrigues, J M; Luís, A L; Lobato, J V; Pinto, M V; Faustino, A; Hussain, N Sooraj; Lopes, M A; Veloso, A P; Freitas, M; Geuna, S; Santos, J D; Maurício, A C

2005-01-01

Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which is capable of growing, differentiating and producing locally nerve growth factors, that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a nerve gap of 10 mm in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for cellular various processes, such as growth and differentiation, be toxic to cells and be involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca2+]i in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment, revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca2+]i was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

PubMed

Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

2015-04-01

Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models.

PubMed

Smits, Jos P H; Niehues, Hanna; Rikken, Gijs; van Vlijmen-Willems, Ivonne M J J; van de Zande, Guillaume W H J F; Zeeuwen, Patrick L J M; Schalkwijk, Joost; van den Bogaard, Ellen H

2017-09-19

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Mechanical properties and cytotoxicity of experimental soft lining materials based on urethane acrylate oligomers.

PubMed

Kanie, Takahito; Tomita, Koichi; Tokuda, Masayuki; Arikawa, Hiroyuki; Fujii, Koichi; Ban, Seiji

2009-07-01

The purpose of this investigation was to determine whether experimental light-curing soft lining materials (ESLMs) based on commercially available urethane acrylate oligomers (UA-160TM, UV-3200B, UV-3500BA, and UV-3700B) are suitable for clinical use by measuring their viscosity, compressive modulus, Shore A hardness, tensile strength, adhesive strength, and cytotoxicity. The viscosities of the four ESLMs at 25 degrees C were 10.5 Pa.s, UV-3500BA; 144.0 Pa.s, UA-160TM; 328.8 Pa.s, UV-3700B; and 1079.7 Pa.s, UV-3200B. Polymerized UV-3700B was very soft, whereas the softness of the other ESLMs was similar to that of conventional soft lining materials. No significant difference in adhesive strength was observed between UV-3500BA and UV-3700B at 1 day and those at 12 months. Cytotoxicity was measured by a MTT-based assay using HeLa S3 and Ca9-22 cells. UV-3200B and UV-3700B oligomers and all four polymerized ESLMs showed cell viability over 95.2% (p < 0.05).

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

PubMed

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-12-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Expression and Functional Significance of HtrA1 Loss in Endometrial Cancer

PubMed Central

Mullany, Sally A.; Moslemi-Kebria, Mehdi; Rattan, Ramandeep; Khurana, Ashwani; Clayton, Amy; Ota, Takayo; Mariani, Andrea; Podratz, Karl C.; Chien, Jeremy; Shridhar, Viji

2010-01-01

Purpose The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Experimental design Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA-1 knocked down clones. Results Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all 7 papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a 3-4 fold increase in the invasive potential. Exogenous expression of HtrA1 in Ark 1 and Ark 2 cells resulted in 3-4 fold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark 2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark 2 cells. Immunohistochemical (IHC) analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (p=0.016). Conclusions Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. PMID:21098697

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

PubMed

Perez-Cunningham, Jessica; Boyer, Scott W; Landon, Mark; Forsberg, E Camilla

2016-08-01

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Experimental observation of the generation of cutoff solitons in a discrete L C nonlinear electrical line

NASA Astrophysics Data System (ADS)

Koon, K. Tse Ve; Marquié, P.; Dinda, P. Tchofo

2014-11-01

We address the problem of supratransmission of waves in a discrete nonlinear system, driven at one end by a periodic excitation at a frequency lying above the phonon band edge. In an experimental electrical transmission line made of 200 inductance-capacitance LC cells, we establish the existence of a voltage threshold for a supratransmission enabling the generation and propagation of cut-off solitons within the line. The decisive role of modulational instability in the onset and development of the process of generation of cut-off solitons is clearly highlighted. The phenomenon of dissipation is identified as being particularly harmful for the soliton generation, but we show that its impact can be managed by a proper choice of the amplitude of the voltage excitation of the system.

The Hidden Side of Disodium Cromolyn: from Mast Cell Stabilizer to an Angiogenic Factor and Antitumor Agent.

PubMed

Cimpean, Anca Maria; Raica, Marius

2016-12-01

Scattered data suggested that disodium cromolyn, well known as a mast cell stabilizer shows some effects on tumor cells and tumor-associated newly formed vascular networks. Most of these studies used tumor cell lines assessed by in vitro studies. Nor disodium cromolyn effects on melanoma cell lines were studied yet, neither its influence on recruited tumor blood vessels or angiogenic growth factors expression. We designed here a study regarding disodium cromolyn effects on A375 melanoma tumor cells implanted on chick embryo chorioallantoic membrane (CAM) and on blood vessels recruited by the experimental melanoma in the absence of mast cells, knowing that within CAM, the existence of mast cells are not certified yet. We also assessed the role of disodium cromolyn on the expression of several angiogenic growth factors. Disodium cromoglycate differentially acts on tumor cells and blood vessels. Extensive necrotic areas of experimental melanoma together with an increased number of peritumor blood vessels were observed in treated specimens as compared with untreated tumors. Disodium cromolyn inhibited VEGF and PDGF-BB expression, and had no effects on EG VEGF expression between treated and non treated specimens in a mast cells free microenvironment. Our results sustain the direct antitumor effects of sodium cromolyn and suggest the involvement of several growth factors in the recruitment of tumor vessels by A375 melanoma tumor cells. The expression of growth factors is differentially influenced by sodium cromolyn treatment.

In vivo physiological recording from the lateral line of juvenile zebrafish.

PubMed

Olt, Jennifer; Allen, Claire E; Marcotti, Walter

2016-10-01

Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More generally, this method would allow functional studies involving live imaging and electrophysiology from juvenile and adult zebrafish. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Karina J.; School of Medicine and Pharmacology, University of Western Australia, Nedlands, WA 6008; Tsykin, Anna

2012-10-19

Highlights: Black-Right-Pointing-Pointer Matrigel alters cancer cell line miRNA expression relative to culture on plastic. Black-Right-Pointing-Pointer Many identified Matrigel-regulated miRNAs are implicated in cancer. Black-Right-Pointing-Pointer miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. Black-Right-Pointing-Pointer miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence ofmore » Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a valuable approach to the in vitro study of miRNAs.« less

Experimental Study of Nasopharyngeal Carcinoma Radionuclide Imaging and Therapy Using Transferred Human Sodium/Iodide Symporter Gene

PubMed Central

Zhong, Xing; Shi, Changzheng; Gong, Jian; Guo, Bin; Li, Mingzhu; Xu, Hao

2015-01-01

Purpose The aim of this study was to design a method of radionuclide for imaging and therapy of nasopharyngeal carcinoma (NPC) using the transferred human sodium/iodide symporter (hNIS) gene. Methods A stable NPC cell line expressing hNIS was established (CNE-2-hNIS). After 131I treatment, we detected proliferation and apoptosis of NPC cells, both in vitro and vivo. In vivo, the radioactivity of different organs of nude mice was counted and 99mTc imaging using SPECT was performed. The apparent diffusion coefficient (ADC) value changes of tumor xenografts were observed by diffusion-weighted magnetic resonance imaging (DW-MRI) within 6–24 days of 131I treatment. The correlation of ADC changes with apoptosis and proliferation was investigated. Post-treatment expression levels of P53, Bax, Bcl-2, Caspase-3, and Survivin proteins were detected by western blotting. Results 131I uptake was higher in CNE-2-hNIS than in CNE-2 cells. The proliferation and apoptosis rate decreased and increased respectively both in vitro and vivo in the experimental group after 131I treatment. The experimental group tumors accumulated 99mTc in vivo, leading to a good visualization by SPECT. DW-MRI showed that ADC values increased in the experimental group 6 days after treatment, while ADC values were positively and negatively correlated with the apoptotic and Ki-67 proliferation indices, respectively. After treatment, CNE-2-hNIS cells up-regulated the expression of P53 and Survivin proteins and activated Caspase-3, and down-regulated the expression of Bcl-2 proteins. Conclusions The radionuclide imaging and therapy technique for NPC hNIS-transfected cell lines can provide a new therapy strategy for monitoring and treatment of NPC. PMID:25615643

Origin and development of the germ line in sea stars

PubMed Central

Wessel, Gary M.; Fresques, Tara; Kiyomoto, Masato; Yajima, Mamiko; Zazueta, Vanesa

2014-01-01

This review summarizes and integrates our current understanding of how sea stars make gametes. Although little is known of the mechanism of germ line formation in these animals, recent results point to specific cells and to cohorts of molecules in the embryos and larvae that may lay the ground work for future research efforts. A coelomic outpocketing forms in the posterior of the gut in larvae, referred to as the posterior enterocoel (PE), that when removed, significantly reduces the number of germ cell later in larval growth. This same PE structure also selectively accumulates several germ-line associated factors – vasa, nanos, piwi – and excludes factors involved in somatic cell fate. Since its formation is relatively late in development, these germ cells may form by inductive mechanisms. When integrated into the morphological observations of germ cells and gonad development in larvae, juveniles, and adults, the field of germ line determination appears to have a good model system to study inductive germ line determination to complement the recent work on the molecular mechanisms in mice. We hope this review will also guide investigators interested in germ line determination and regulation of the germ line in how these animals can help in this research field. The review is not intended to be comprehensive – sea star reproduction has been studied over 100 years and many reviews are comprehensive in their coverage of, for example, seasonal growth of the gonads in response to light, nutrient, and temperature. Rather the intent of this review is to help the reader focus on new experimental results attached to the historical underpinnings of how the germ cell functions in sea stars with particular emphasis to clarify the important areas of priority for future research. PMID:24648114

SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

PubMed

Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

2010-04-20

To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

A flexible and qualitatively stable model for cell cycle dynamics including DNA damage effects.

PubMed

Jeffries, Clark D; Johnson, Charles R; Zhou, Tong; Simpson, Dennis A; Kaufmann, William K

2012-01-01

This paper includes a conceptual framework for cell cycle modeling into which the experimenter can map observed data and evaluate mechanisms of cell cycle control. The basic model exhibits qualitative stability, meaning that regardless of magnitudes of system parameters its instances are guaranteed to be stable in the sense that all feasible trajectories converge to a certain trajectory. Qualitative stability can also be described by the signs of real parts of eigenvalues of the system matrix. On the biological side, the resulting model can be tuned to approximate experimental data pertaining to human fibroblast cell lines treated with ionizing radiation, with or without disabled DNA damage checkpoints. Together these properties validate a fundamental, first order systems view of cell dynamics. Classification Codes: 15A68.

Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

PubMed Central

Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

2013-01-01

Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

Model-based strategy for cell culture seed train layout verified at lab scale.

PubMed

Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

2016-08-01

Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

Cell culture medium improvement by rigorous shuffling of components using media blending.

PubMed

Jordan, Martin; Voisard, Damien; Berthoud, Antoine; Tercier, Laetitia; Kleuser, Beate; Baer, Gianni; Broly, Hervé

2013-01-01

A novel high-throughput methodology for the simultaneous optimization of many cell culture media components is presented. The method is based on the media blending approach which has several advantages as it works with ready-to-use media. In particular it allows precise pH and osmolarity adjustments and eliminates the need of concentrated stock solutions, a frequent source of serious solubility issues. In addition, media blending easily generates a large number of new compositions providing a remarkable screening tool. However, media blending designs usually do not provide information on distinct factors or components that are causing the desired improvements. This paper addresses this last point by considering the concentration of individual medium components to fix the experimental design and for the interpretation of the results. The extended blending strategy was used to reshuffle the 20 amino acids in one round of experiments. A small set of 10 media was specifically designed to generate a large number of mixtures. 192 mixtures were then prepared by media blending and tested on a recombinant CHO cell line expressing a monoclonal antibody. A wide range of performances (titers and viable cell density) was achieved from the different mixtures with top titers significantly above our previous results seen with this cell line. In addition, information about major effects of key amino acids on cell densities and titers could be extracted from the experimental results. This demonstrates that the extended blending approach is a powerful experimental tool which allows systematic and simultaneous reshuffling of multiple medium components.

Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

DOE PAGES

Botcheva, Krassimira; McCorkle, Sean R.

2014-11-21

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

PubMed

Krajňáková, Jana; Sutela, Suvi; Aronen, Tuija; Gömöry, Dušan; Vianello, Angelo; Häggman, Hely

2011-08-01

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities. Copyright © 2011 Elsevier Inc. All rights reserved.

Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity

PubMed Central

Boudreau, Colton; Wee, Tse-Luen (Erika); Duh, Yan-Rung (Silvia); Couto, Melissa P.; Ardakani, Kimya H.; Brown, Claire M.

2016-01-01

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity. PMID:27485088

Excitation Light Dose Engineering to Reduce Photo-bleaching and Photo-toxicity.

PubMed

Boudreau, Colton; Wee, Tse-Luen Erika; Duh, Yan-Rung Silvia; Couto, Melissa P; Ardakani, Kimya H; Brown, Claire M

2016-08-03

It is important to determine the most effective method of delivering light onto a specimen for minimal light induced damage. Assays are presented to measure photo-bleaching of fluorophores and photo-toxicity to living cells under different illumination conditions. Turning the light off during part of the experimental time reduced photo-bleaching in a manner proportional to the time of light exposure. The rate of photo-bleaching of EGFP was reduced by 9-fold with light pulsing on the micro-second scale. Similarly, in living cells, rapid line scanning resulted in reduced cell stress as measured by mitochondrial potential, rapid cell protrusion and reduced cell retraction. This was achieved on a commercial confocal laser scanning microscope, without any compromise in image quality, by using rapid laser scan settings and line averaging. Therefore this technique can be implemented broadly without any software or hardware upgrades. Researchers can use the rapid line scanning option to immediately improve image quality on fixed samples, reduce photo-bleaching for large high resolution 3D datasets and improve cell health in live cell experiments. The assays developed here can be applied to other microscopy platforms to measure and optimize light delivery for minimal sample damage and photo-toxicity.

Computer-aided nanotoxicology: assessing cytotoxicity of nanoparticles under diverse experimental conditions by using a novel QSTR-perturbation approach

NASA Astrophysics Data System (ADS)

Luan, Feng; Kleandrova, Valeria V.; González-Díaz, Humberto; Ruso, Juan M.; Melo, André; Speck-Planche, Alejandro; Cordeiro, M. Natália D. S.

2014-08-01

Nowadays, the interest in the search for new nanomaterials with improved electrical, optical, catalytic and biological properties has increased. Despite the potential benefits that can be gathered from the use of nanoparticles, only little attention has been paid to their possible toxic effects that may affect human health. In this context, several assays have been carried out to evaluate the cytotoxicity of nanoparticles in mammalian cells. Owing to the cost in both resources and time involved in such toxicological assays, there has been a considerable increase in the interest towards alternative computational methods, like the application of quantitative structure-activity/toxicity relationship (QSAR/QSTR) models for risk assessment of nanoparticles. However, most QSAR/QSTR models developed so far have predicted cytotoxicity against only one cell line, and they did not provide information regarding the influence of important factors rather than composition or size. This work reports a QSTR-perturbation model aiming at simultaneously predicting the cytotoxicity of different nanoparticles against several mammalian cell lines, and also considering different times of exposure of the cell lines, as well as the chemical composition of nanoparticles, size, conditions under which the size was measured, and shape. The derived QSTR-perturbation model, using a dataset of 1681 cases (nanoparticle-nanoparticle pairs), exhibited an accuracy higher than 93% for both training and prediction sets. In order to demonstrate the practical applicability of our model, the cytotoxicity of different silica (SiO2), nickel (Ni), and nickel(ii) oxide (NiO) nanoparticles were predicted and found to be in very good agreement with experimental reports. To the best of our knowledge, this is the first attempt to simultaneously predict the cytotoxicity of nanoparticles under multiple experimental conditions by applying a single unique QSTR model.Nowadays, the interest in the search for new nanomaterials with improved electrical, optical, catalytic and biological properties has increased. Despite the potential benefits that can be gathered from the use of nanoparticles, only little attention has been paid to their possible toxic effects that may affect human health. In this context, several assays have been carried out to evaluate the cytotoxicity of nanoparticles in mammalian cells. Owing to the cost in both resources and time involved in such toxicological assays, there has been a considerable increase in the interest towards alternative computational methods, like the application of quantitative structure-activity/toxicity relationship (QSAR/QSTR) models for risk assessment of nanoparticles. However, most QSAR/QSTR models developed so far have predicted cytotoxicity against only one cell line, and they did not provide information regarding the influence of important factors rather than composition or size. This work reports a QSTR-perturbation model aiming at simultaneously predicting the cytotoxicity of different nanoparticles against several mammalian cell lines, and also considering different times of exposure of the cell lines, as well as the chemical composition of nanoparticles, size, conditions under which the size was measured, and shape. The derived QSTR-perturbation model, using a dataset of 1681 cases (nanoparticle-nanoparticle pairs), exhibited an accuracy higher than 93% for both training and prediction sets. In order to demonstrate the practical applicability of our model, the cytotoxicity of different silica (SiO2), nickel (Ni), and nickel(ii) oxide (NiO) nanoparticles were predicted and found to be in very good agreement with experimental reports. To the best of our knowledge, this is the first attempt to simultaneously predict the cytotoxicity of nanoparticles under multiple experimental conditions by applying a single unique QSTR model. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01285b

Development and Characterisation of a Human Chronic Skin Wound Cell Line—Towards an Alternative for Animal Experimentation

PubMed Central

Wall, Ivan B.; Peake, Matthew; Kipling, David; Giles, Peter; Thomas, David W.

2018-01-01

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening. PMID:29584680

Active chinese mistletoe lectin-55 enhances colon cancer surveillance through regulating innate and adaptive immune responses

PubMed Central

Ma, Yan-Hui; Cheng, Wei-Zhi; Gong, Fang; Ma, An-Lun; Yu, Qi-Wen; Zhang, Ji-Ying; Hu, Chao-Ying; Chen, Xue-Hua; Zhang, Dong-Qing

2008-01-01

AIM: To investigate the potential role of Active Chinese mistletoe lectin-55 (ACML-55) in tumor immune surveillance. METHODS: In this study, an experimental model was established by hypodermic inoculating the colon cancer cell line CT26 (5 × 105 cells) into BALB/c mice. The experimental treatment was orally administered with ACML-55 or PBS, followed by the inoculation of colon cancer cell line CT26. Intracellular cytokine staining was used to detect IFN-γ production by tumor antigen specific CD8+ T cells. FACS analysis was employed to profile composition and activation of CD4+, CD8+, γδ T and NK cells. RESULTS: Our results showed, compared to PBS treated mice, ACML-55 treatment significantly delayed colon cancer development in colon cancer -bearing Balb/c mice in vivo. Treatment with ACML-55 enhanced both Ag specific activation and proliferation of CD4+ and CD8+ T cells, and increased the number of tumor Ag specific CD8+ T cells. It was more important to increase the frequency of tumor Ag specific IFN-γ producing-CD8+ T cells. Interestingly, ACML-55 treatment also showed increased cell number of NK, and γδT cells, indicating the role of ACML-55 in activation of innate lymphocytes. CONCLUSION: Our results demonstrate that ACML-55 therapy can enhance function in immune surveillance in colon cancer-bearing mice through regulating both innate and adaptive immune responses. PMID:18785279

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics

PubMed Central

Haake, Scott M.; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio; Chen, Yian Ann; Koomen, John; Rathmell, W. Kimryn; Fishman, Mayer; Haura, Eric B.

2016-01-01

Purpose Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Experimental Design Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes. PMID:27220961

The availability of filament ends modulates actin stochastic dynamics in live plant cells

PubMed Central

Li, Jiejie; Staiger, Benjamin H.; Henty-Ridilla, Jessica L.; Abu-Abied, Mohamad; Sadot, Einat; Blanchoin, Laurent; Staiger, Christopher J.

2014-01-01

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament–filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls. PMID:24523291

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

PubMed

Cacci, E; Villa, A; Parmar, M; Cavallaro, M; Mandahl, N; Lindvall, O; Martinez-Serrano, A; Kokaia, Z

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

JNK-1 Inhibition Leads to Antitumor Activity in Ovarian Cancer

PubMed Central

Vivas-Mejia, Pablo; Benito, Juliana Maria; Fernandez, Ariel; Han, Hee-Dong; Mangala, Lingegowda; Rodriguez-Aguayo, Cristian; Chavez-Reyes, Arturo; Lin, Yvonne G.; Nick, Alpa M.; Stone, Rebecca L.; Kim, Hye Sun; Claret, Francois-Xavier; Bornmann, William; Hennessy, Bryan TJ.; Sanguino, Angela; Peng, Zhengong; Sood, Anil K.; Lopez-Berestein, Gabriel

2011-01-01

Purpose To demonstrate the functional, clinical and biological significance of JNK-1 in ovarian carcinoma. Experimental Design Analysis of the impact of JNK on 116 epithelial ovarian cancers was conducted. The role of JNK in vitro and in experimental models of ovarian cancer was assessed. We studied the role of WBZ_4, a novel JNK inhibitor redesigned from imatinib based on targeting wrapping defects, in cell lines and in experimental models of ovarian cancer. Results We found a significant association of pJNK with progression free survival in the 116 epithelial ovarian cancers obtained at primary debulking therapy. WBZ_4 led to cell growth inhibition and increased apoptosis in a dose dependent fashion in four ovarian cancer cell lines. In vivo, while imatinib had no effect on tumor growth, WBZ_4 inhibited tumor growth in orthotopic murine models of ovarian cancer. The anti-tumor effect was further increased in combination with docetaxel. Silencing of JNK-1 with systemically administered siRNA led to significantly reduced tumor weights as compared to non-silencing siRNA controls, indicating that indeed the antitumor effects observed were due to JNK-1 inhibition. Conclusions These studies identify JNK-1 as an attractive therapeutic target in ovarian carcinoma and that the re-designed WBZ_4 compound should be considered for further clinical development. PMID:20028751

Soft x-ray holography and microscopy of biological cells

NASA Astrophysics Data System (ADS)

Chen, Jianwen; Gao, Hongyi; Xie, Honglan; Li, Ruxin; Xu, Zhizhan

2003-10-01

Some experimental results on soft X-ray microscopy and holography imaging of biological specimens are presented in the paper. As we know, due to diffraction effects, there exists a resolution limit determined by wavelength λ and numerical aperture NA in conventional optical microscopy. In order to improve resolution, the num erical aperture should be made as large as possible and the wavelength as short as possible. Owing to the shorter wavelength, X-rays provide the potential of higher resolution in X-ray microscopy, holography image and allow for exam ination the interior structures of thicker specimens. In the experiments, we used synchrotron radiation source in Hefei as light source. Soft X-rays come from a bending magnet in 800 M eV electron storage ring with characteristic wavelength of 2.4 nm. The continuous X-ray spectrums are monochromatized by a zone-plate and a pinhole with 300 m diameter. The experimental set-up is typical contact microscopic system, its main advantage is simplicity and no special optical element is needed. The specimens used in the experiments of microscopic imaging are the colibacillus, the gingko vascular hundle and the fritillaries ovary karyon. The specimen for holographic imaging is the spider filam ents. The basic structures of plant cells such as the cell walls, the cytoplasm and the karyon especially the joint structures between the cells are observed clearly. An experimental study on a thick biological specimen that is a whole sporule w ith the thickness of about 30 μm is performed. In the holographic experiments, the experimental setup is typical Gabor in-line holography. The specimen is placed in line with X-ray source, which provides both the reference w aves and specimen illum ination. The specimen is some spider filament, which adhere to a Si3N4 film. The recording medium is PM M A, which is placed at recording distance of about 400 μm from the specimen. The hologram s were reconstructed by digital method with 300 nm resolutions. A novel method for recording in-line hologram is proposed which is called X-ray in-line holography with zone-plate magnification in this paper. The magnification factor of the micro zone plate imaging is about 103. The transverse resolution can be 48 nm in this method.

Low Level Laser Irradiation of Nerve Cells In Vitro

DTIC Science & Technology

1996-01-01

Advisor Michael Miloro, D.M.D., M.D. College of Dentistry ABSTRACT Low energy laser treatment of patients with nerve injuries has been reported to achieve...Isolation and Culture 15 vii Cell Lines 17 Cell Expansion 19 Cell Freezing 20 Experimental Design 20 GaA1As Laser Diode 22 Radiation Schedule 23...of 1 six well plate. Two groups served as controls. The remaining groups were irradiated with a 70 mW GaA1As laser diode , wavelength 820-830 nm

CD4 expression on EL4 cells as an epiphenomenon of retroviral transduction and selection.

PubMed

Logan, Grant J; Spinoulas, Afroditi; Alexander, Stephen I; Smythe, Jason A; Alexander, Ian E

2004-04-01

The EL4 murine tumour cell line, isolated from a chemically induced lymphoma over 50 years ago, has been extensively exploited in immunological research. The conclusions drawn from many of these studies have been based on the presumption that EL4 cells maintain a stable phenotype during experimental manipulation. To the contrary, we have observed 100-fold greater expression of cell surface CD4 (CD4(high)) on a subpopulation of EL4 cells following retroviral transduction and G418 selection when compared with unmodified populations. Although the mechanism responsible for this effect remains to be elucidated, the unexpected expression of CD4, a molecule that functions as both a coreceptor with the T-cell receptor and ligand for the pro-inflammatory cytokine IL-16, has the potential to influence experimental outcomes. Upregulation of CD4 should be excluded when EL4 cells are utilized in experiments requiring a consistent immuno-phenotype.

Interim report on intrathoracic radiotherapy of human small-cell lung carcinoma in nude mice with Re-188-RC-160, a radiolabeled somatostatin analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zamora, P.O.; Bender, H.; Biersack, H.J.

1995-07-01

The purpose of this study was to evaluate the therapeutic efficacy of Re-188-RC-160 in experimental models of human small cell lung carcinomas which mimic the clinical presentation. In the experimental model, cells from the human small cell lung carcinoma cell line NCI-H69 cells were inoculated into the thoracic cavity of athymic mice and rats. Subsequently, the biodistribution of Re-188-RC-160 after injection into the pleural cavity, a radiolabeled somatostatin analogue, was monitored as was the effect on the subsequent growth of tumors. The results presented here, and which are a part of a larger series of studies, suggest that Re-188-RC-160 canmore » be effectively used in this animal model to restrict the growth of small cell lung carcinoma in the thoracic cavity.« less

University of Texas Southwestern Medical Center (UTSW): Lung Cancer Oncogenotype-Selective Drug Target Discovery (Natural Products Focus) | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use small molecules and RNAi to functionally define subtypes of non-small cell lung cancer (NSCLC) using a panel of cell lines prepared and molecularly annotated by Drs. John Minna and Adi Gazdar. Experimental Approaches Lung Cancer Natural Products Screening/Chemical Library Screening

University of Texas Southwestern Medical Center: Lung Cancer Oncogenotype-Selective Drug Target Discovery (Natural Products Focus) | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use small molecules and RNAi to functionally define subtypes of non-small cell lung cancer (NSCLC) using a panel of cell lines prepared and molecularly annotated by Drs. John Minna and Adi Gazdar. Experimental Approaches Lung Cancer Natural Products Screening/Chemical Library Screening

Mathematical models of cytotoxic effects in endpoint tumor cell line assays: critical assessment of the application of a single parametric value as a standard criterion to quantify the dose-response effects and new unexplored proposal formats.

PubMed

Calhelha, Ricardo C; Martínez, Mireia A; Prieto, M A; Ferreira, Isabel C F R

2017-10-23

The development of convenient tools for describing and quantifying the effects of standard and novel therapeutic agents is essential for the research community, to perform more precise evaluations. Although mathematical models and quantification criteria have been exchanged in the last decade between different fields of study, there are relevant methodologies that lack proper mathematical descriptions and standard criteria to quantify their responses. Therefore, part of the relevant information that can be drawn from the experimental results obtained and the quantification of its statistical reliability are lost. Despite its relevance, there is not a standard form for the in vitro endpoint tumor cell lines' assays (TCLA) that enables the evaluation of the cytotoxic dose-response effects of anti-tumor drugs. The analysis of all the specific problems associated with the diverse nature of the available TCLA used is unfeasible. However, since most TCLA share the main objectives and similar operative requirements, we have chosen the sulforhodamine B (SRB) colorimetric assay for cytotoxicity screening of tumor cell lines as an experimental case study. In this work, the common biological and practical non-linear dose-response mathematical models are tested against experimental data and, following several statistical analyses, the model based on the Weibull distribution was confirmed as the convenient approximation to test the cytotoxic effectiveness of anti-tumor compounds. Then, the advantages and disadvantages of all the different parametric criteria derived from the model, which enable the quantification of the dose-response drug-effects, are extensively discussed. Therefore, model and standard criteria for easily performing the comparisons between different compounds are established. The advantages include a simple application, provision of parametric estimations that characterize the response as standard criteria, economization of experimental effort and enabling rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data fitted, the calculated parameters were always statistically significant, the equations proved to be consistent and the correlation coefficient of determination was, in most of the cases, higher than 0.98.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins.

PubMed

Debray, H; Dus, D; Hueso, P; Radzikowski, C; Montreuil, J

1990-01-01

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.

Influence of in vivo growth on human glioma cell line gene expression: Convergent profiles under orthotopic conditions

PubMed Central

Camphausen, Kevin; Purow, Benjamin; Sproull, Mary; Scott, Tamalee; Ozawa, Tomoko; Deen, Dennis F.; Tofilon, Philip J.

2005-01-01

Defining the molecules that regulate tumor cell survival is an essential prerequisite for the development of targeted approaches to cancer treatment. Whereas many studies aimed at identifying such targets use human tumor cells grown in vitro or as s.c. xenografts, it is unclear whether such experimental models replicate the phenotype of the in situ tumor cell. To begin addressing this issue, we have used microarray analysis to define the gene expression profile of two human glioma cell lines (U251 and U87) when grown in vitro and in vivo as s.c. or as intracerebral (i.c.) xenografts. For each cell line, the gene expression profile generated from tissue culture was significantly different from that generated from the s.c. tumor, which was significantly different from those grown i.c. The disparity between the i.c gene expression profiles and those generated from s.c. xenografts suggests that whereas an in vivo growth environment modulates gene expression, orthotopic growth conditions induce a different set of modifications. In this study the U251 and U87 gene expression profiles generated under the three growth conditions were also compared. As expected, the profiles of the two glioma cell lines were significantly different when grown as monolayer cultures. However, the glioma cell lines had similar gene expression profiles when grown i.c. These results suggest that tumor cell gene expression, and thus phenotype, as defined in vitro is affected not only by in vivo growth but also by orthotopic growth, which may have implications regarding the identification of relevant targets for cancer therapy. PMID:15928080

Analyses of the combination of 6-MP and dasatinib in cell culture

PubMed Central

KAUR, GURMEET; BEHRSING, HOLGER; PARCHMENT, RALPH E.; MILLIN, MYRTLE DAVIS; TEICHER, BEVERLY A.

2013-01-01

A major tenet of cancer therapeutics is that combinations of anticancer agents with different mechanisms of action and different toxicities may be effective treatment regimens. Evaluation of additivity/synergy in cell culture may be used to identify drug combination opportunities and to assess risk of additive/synergistic toxicity. The combination of 6-mercaptopurine and dasatinib was assessed for additivity/synergy using the combination index (CI) method and a response surface method in six human tumor cell lines including MCF-7 and MDA-MB-468 breast cancer, NCI-H23 and NCI-H460 non-small cell lung cancer, and A498 and 786-O renal cell cancer, based on two experimental end-points: ATP content and colony formation. Clonal colony formation by human bone marrow CFU-GM was used to assess risk of enhanced toxicity. The concentration ranges tested for each drug were selected to encompass the clinical Cmax concentrations. The combination regimens were found to be additive to sub-additive by both methods of data analysis, but synergy was not detected. The non-small cell lung cancer cell lines were the most responsive among the tumor lines tested and the renal cell carcinoma lines were the least responsive. The bone marrows CFU-GM were more sensitive to the combination regimens than were the tumor cell lines. Based upon these data, it appears that the possibility of enhanced efficacy from combining 6-mercaptopurine (6-MP) and dasatinib would be associated with increased risk of severe bone marrow toxicity, so the combination is unlikely to provide a therapeutic advantage for treating solid tumor patients where adequate bone marrow function must be preserved. PMID:23652925

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Cell sources for in vitro human liver cell culture models.

PubMed

Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-09-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

Characterization and comparison of perezone with some analogues. Experimental and theoretical study

NASA Astrophysics Data System (ADS)

Escobedo-González, Rene Gerardo; Bahena, Luis; Arias Tellez, José Luis; Hinojosa Torres, Jaime; Ruvalcaba, Rene Miranda; Aceves-Hernández, Juan Manuel

2015-10-01

Perezone had been used for centuries in the traditional Mexican medicine, it is useful and a handful of illness. Perezone and other derivatives also present activity against certain lines of cancer, such as the myeloblastoid leukemia cell line K-562 and carcinoma cell lines (PC-3 and SKLU-1) with IC50 <10 μM. Perezone and isoperezone have shown the major cytotoxic potency. Characterization of perezone was carried out by UV-Visible, IR, DSC, TGA and powder X-ray diffraction, as well as docking studies using caspase-3 structures as receptors. Theoretical studies for optimizing the geometry of perezone were carried out and the results compared with values of single crystal X-ray diffraction. The experimental values of atomic distances, angles and dihedral angles are in good agreement with the theoretical values. Interaction of perezone with the cysteine catalytic site with the caspase-3 was found in the docking studies. A docking study of perezone, with horminone, thymoquinone and isoperezone as ligands and the protein apoptein, caspase-3 as receptor, was carried to demonstrate that the hindrance steric factor, chemical structure and the functional groups are important in the biological activity of these natural products. The docking score energetic values are in good agreement with the experimental cytotoxic results obtained from the experiments when perezone and analogues were studied in different types of cancer.

Hepatocyte growth factor secreted by ovarian cancer cells stimulates peritoneal implantation via the mesothelial-mesenchymal transition of the peritoneum.

PubMed

Nakamura, Michihiko; Ono, Yoshihiro J; Kanemura, Masanori; Tanaka, Tomohito; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2015-11-01

A current working model for the metastatic process of ovarian carcinoma suggests that cancer cells are shed from the ovarian tumor into the peritoneal cavity and attach to the layer of mesothelial cells that line the inner surface of the peritoneum, and several studies suggest that hepatocyte growth factor (HGF) plays an important role in the dissemination of ovarian cancer. Our objectives were to evaluate the HGF expression of ovarian cancer using clinical data and assess the effect of HGF secreted from human ovarian cancer cells to human mesothelial cells. HGF expression was immunohistochemically evaluated in 165 epithelial ovarian cancer patients arranged as tissue microarrays. HGF expression in four ovarian cancer cell lines was evaluated by using semi-quantitative polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. The effect of ovarian cancer cell derived HGF to the human mesothelial cells was assessed by using morphologic analysis, Western blotting and cell invasion assay. The effect of HGF on ovarian cancer metastasis was assessed by using in vivo experimental model. The clinical data showed a significantly high correlation between the HGF expression and the cancer stage. The in vivo and in vitro experimental models revealed that HGF secreted by ovarian cancer cells induces the mesothelial-to-mesenchymal transition and stimulates the invasion of mesothelial cells. Furthermore, manipulating the HGF activity affected the degree of dissemination and ascite formation. We demonstrated that HGF secreted by ovarian cancer cells plays an important role in cancer peritoneal implantation. Copyright © 2015 Elsevier Inc. All rights reserved.

Gold Nano Popcorn Attached SWCNT Hybrid Nanomaterial for Targeted Diagnosis and Photothermal Therapy of Human Breast Cancer Cells

PubMed Central

Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

2011-01-01

Breast cancer presents greatest challenge in health care in today’s world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube’s D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 minutes of photothermal therapy treatment by 1.5 W/cm2 power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly. PMID:21842867

Gold nano-popcorn attached SWCNT hybrid nanomaterial for targeted diagnosis and photothermal therapy of human breast cancer cells.

PubMed

Beqa, Lule; Fan, Zhen; Singh, Anant Kumar; Senapati, Dulal; Ray, Paresh Chandra

2011-09-01

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.

Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression.

PubMed

Lochmann, Timothy L; Floros, Konstantinos V; Naseri, Mitra; Powell, Krista M; Cook, Wade; March, Ryan J; Stein, Giovanna T; Greninger, Patricia; Maves, Yuki Kato; Saunders, Laura R; Dylla, Scott J; Costa, Carlotta; Boikos, Sosipatros A; Leverson, Joel D; Souers, Andrew J; Krystal, Geoffrey W; Harada, Hisashi; Benes, Cyril H; Faber, Anthony C

2018-01-15

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2-expressing SCLC tumors in vivo BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2-expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2-expressing SCLCs. Clin Cancer Res; 24(2); 360-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Establishment of immortal multipotent rat salivary progenitor cell line toward salivary gland regeneration.

PubMed

Yaniv, Adi; Neumann, Yoav; David, Ran; Stiubea-Cohen, Raluca; Orbach, Yoav; Lang, Stephan; Rotter, Nicole; Dvir-Ginzberg, Mona; Aframian, Doron J; Palmon, Aaron

2011-01-01

Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6β1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.

A Targeted RNAi Screen of the Breast Cancer Genome Identifies KIF14 and TLN1 as Genes That Modulate Docetaxel Chemosensitivity in Triple-Negative Breast Cancer

PubMed Central

Singel, Stina Mui; Cornelius, Crystal; Batten, Kimberly; Fasciani, Gail; Wright, Woodring E.; Lum, Lawrence; Shay, Jerry W.

2015-01-01

Purpose To identify biomarkers within the breast cancer genome that may predict chemosensitivity in breast cancer. Experimental Design We conducted an RNA interference (RNAi) screen within the breast cancer genome for genes whose loss-of-function enhanced docetaxel chemosensitivity in an estrogen receptor–negative, progesterone receptor–negative, and Her2-negative (ER−, PR−, and Her2−, respectively) breast cancer cell line, MDA-MB-231. Top candidates were tested for their ability to modulate chemosensitivity in 8 breast cancer cell lines and to show in vivo chemosensitivity in a mouse xenograft model. Results From ranking chemosensitivity of 328 short hairpin RNA (shRNA) MDA-MB-231 cell lines (targeting 133 genes with known somatic mutations in breast cancer), we focused on the top two genes, kinesin family member 14 (KIF14) and talin 1 (TLN1). KIF14 and TLN1 loss-of-function significantly enhanced chemosensitivity in four triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, HCC38, HCC1937, and Hs478T) but not in three hormone receptor–positive cell lines (MCF7, T47D, and HCC1428) or normal human mammary epithelial cells (HMEC). Decreased expression of KIF14, but not TLN1, also enhanced docetaxel sensitivity in a Her2-amplified breast cancer cell line, SUM190PT. Higher KIF14 and TLN1 expressions are found in TNBCs compared with the other clinical subtypes. Mammary fat pad xenografts of KIF14- and TLN1-deficient MDA-MB-231 cells revealed reduced tumor mass compared with control MDA-MB-231 cells after chemotherapy. KIF14 expression is also prognostic of relapse-free and overall survival in representative breast cancer expression arrays. Conclusion KIF14 and TLN1 are modulators of response to docetaxel and potential therapeutic targets in TNBC. PMID:23479679

Expanding the role of 3-O sulfated heparan sulfate in herpes simplex virus type-1 entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Donnell, Christopher D., E-mail: codonn3@uic.ed; Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612; Kovacs, Maria, E-mail: marcsika101@yahoo.co

2010-02-20

Heparan sulfate (HS) proteoglycans are commonly exploited by multiple viruses for initial attachment to host cells. Herpes simplex virus-1 (HSV-1) is unique because it can use HS for both attachment and penetration, provided specific binding sites for HSV-1 envelope glycoprotein gD are present. The interaction with gD is mediated by specific HS moieties or 3-O sulfated HS (3-OS HS), which are generated by all but one of the seven isoforms of 3-O sulfotransferases (3-OSTs). Here we demonstrate that several common experimental cell lines express unique sets of 3-OST isoforms. While the isoforms 3-OST-3, -5 and -6 were most commonly expressed,more » isoforms 3-OST-2 and -4 were undetectable in the cell lines examined. Since most cell lines expressed multiple 3-OST isoforms, we addressed the significance of 3-OS HS in HSV-1 entry by down-regulating 2-O-sulfation, a prerequisite for 3-OS HS formation, by knocking down 2-OST expression by RNA interference (RNAi). 2-OST knockdown was verified by reverse-transcriptase PCR and Western blot analysis, while 3-OS HS knockdown was verified by immunofluorescence. Cells showed a significant decrease in viral entry, suggesting an important role for 3-OS HS. Implicating 3-OS HS further, cells knocked down for 2-OST expression also demonstrated decreased cell-cell fusion when cocultivated with effector cells transfected with HSV-1 glycoproteins. Our findings suggest that 3-OS HS may play an important role in HSV-1 entry into many different cell lines.« less

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Insulin receptor in mouse neuroblastoma cell line N18TG2: binding properties and visualization with colloidal gold.

PubMed

Sartori, C; Stefanini, S; Bernardo, A; Augusti-Tocco, G

1992-08-01

Insulin function in the nervous system is still poorly understood. Possible roles as a neuromodulator and as a growth factor have been proposed (Baskin et al., 1987, Ann. Rev. Physiol. 49, 335-347). Stable cell lines may provide an appropriate experimental system for the analysis of insulin action on the various cellular components of the central nervous system. We report here a study to investigate the presence and the properties of insulin specific binding sites in the murine neuroblastoma line, N18TG2, together with insulin action on cell growth and metabolism. Also, receptor internalization has been studied. Binding experiments, carried out in standard conditions at 20 degrees C, enabled us to demonstrate that these cells bind insulin in a specific manner, thus confirming previous findings on other cell lines. Saturation curves showed the presence of two binding sites with Kd 0.3 and 9.7 nM. Competition experiments with porcine and bovine insulin showed an IC50 of 1 and 10 nM, respectively. Competition did not occur in the presence of the unrelated hormones ACTH and FSH. Dissociation experiments indicated the existence of an internalization process of the ligand-receptor complex; this was confirmed by an ultrastructural study using gold conjugated insulin. As far as the insulin action in N18TG2 cells is concerned, physiological concentrations stimulate cell proliferation, whereas no stimulation of glucose uptake was observed, indicating that insulin action in these cells is not mediated by general metabolic effects. On the basis of these data, N18TG2 line appears to be a very suitable model for further studies of the neuronal type insulin receptors, and possibly insulin specific action on the nervous system.

A cytotoxic protein (BF-CT1) purified from Bungarus fasciatus venom acts through apoptosis, modulation of PI3K/AKT, MAPKinase pathway and cell cycle regulation.

PubMed

Bhattacharya, Shamik; Das, Tanaya; Biswas, Archita; Gomes, Aparna; Gomes, Antony; Dungdung, Sandhya Rekha

2013-11-01

BF-CT1, a 13 kDa protein isolated from Bungarus fasciatus snake venom through CM cellulose ion exchange chromatography at 0.02 M NaCl salt gradient showed cytotoxicity in in vitro and in vivo experimental models. In in vivo Ehrlich ascites carcinoma (EAC) induced BALB/c mice model, BF-CT1 treatment reduced EAC cell count significantly through apoptotic cell death pathway as evidenced by FACS analysis, increased caspase 3, 9 activity and altered pro, antiapoptotic protein expression. BF-CT1 treatment caused cell shrinkage, chromatin condensation and induced apoptosis through increased caspase 3, caspase 9 activity, PARP cleavage and down regulation of heat shock proteins in U937 leukemic cell line. Cytosolic cytochrome C production was increased after BF-CT1 treatment upon U937 cell line. BF-CT1 treated U937 cell showed cell cycle arrest at sub G1 phase through cyclin D and CDK down regulation with up regulation of p15 and p16. It also down regulated PI3K/AKT pathway and MAPkinase pathway and promoted apoptosis and regulated cell proliferation in U937 cells. BF-CT1 prevented angiogenesis in in vitro U937 cell line through decreased VEGF and TGF-β1 production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Decursin enhances TRAIL‐induced apoptosis through oxidative stress mediated‐ endoplasmic reticulum stress signalling in non‐small cell lung cancers

PubMed Central

Kim, Jaekwang; Yun, Miyong; Kim, Eun‐Ok; Jung, Deok‐Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon

2016-01-01

Background and Purpose The TNF‐related apoptosis‐inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL‐induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Experimental Approach Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Key Results Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL‐resistant non‐small‐cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer‐binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down‐regulated expression of survivin and Bcl‐xL in TRAIL‐resistant NSCLC cells. Conclusions and Implications ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL‐resistant NSCLC cell lines, partly via up‐regulation of DR5. PMID:26661339

Protein Corona Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and Nonphagocytic Cells in a Size-Dependent Manner.

PubMed

Cheng, Xiaju; Tian, Xin; Wu, Anqing; Li, Jianxiang; Tian, Jian; Chong, Yu; Chai, Zhifang; Zhao, Yuliang; Chen, Chunying; Ge, Cuicui

2015-09-23

The interaction at nanobio is a critical issue in designing safe nanomaterials for biomedical applications. Recent studies have reported that it is nanoparticle-protein corona rather than bare nanoparticle that determines the nanoparticle-cell interactions, including endocytic pathway and biological responses. Here, we demonstrate the effects of protein corona on cellular uptake of different sized gold nanoparticles in different cell lines. The experimental results show that protein corona significantly decreases the internalization of Au NPs in a particle size- and cell type-dependent manner. Protein corona exhibits much more significant inhibition on the uptake of large-sized Au NPs by phagocytic cell than that of small-sized Au NPs by nonphagocytic cell. The endocytosis experiment indicates that different endocytic pathways might be responsible for the differential roles of protein corona in the interaction of different sized Au NPs with different cell lines. Our findings can provide useful information for rational design of nanomaterials in biomedical application.

Microenvironmental independence associated with tumor progression.

PubMed

Anderson, Alexander R A; Hassanein, Mohamed; Branch, Kevin M; Lu, Jenny; Lobdell, Nichole A; Maier, Julie; Basanta, David; Weidow, Brandy; Narasanna, Archana; Arteaga, Carlos L; Reynolds, Albert B; Quaranta, Vito; Estrada, Lourdes; Weaver, Alissa M

2009-11-15

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments.

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Experimental evidence for killing the resistant cells and raising the efficacy and decreasing the toxicity of cytostatics and irradiation by mixtures of the agents of the passive antitumor defense system in the case of various tumor and normal cell lines in vitro.

PubMed

Kulcsár, Gyula

2009-02-01

Despite the substantial decline of the immune system in AIDS, only a few kinds of tumors increase in incidence. This shows that the immune system has no absolute role in the prevention of tumors. Therefore, the fact that tumors do not develop in the majority of the population during their lifetime indicates the existence of other defense system(s). According to our hypothesis, the defense is made by certain substances of the circulatory system. Earlier, on the basis of this hypothesis, we experimentally selected 16 substances of the circulatory system and demonstrated that the mixture of them (called active mixture) had a cytotoxic effect (inducing apoptosis) in vitro and in vivo on different tumor cell lines, but not on normal cells and animals. In this paper, we provide evidence that different cytostatic drugs or irradiation in combination with the active mixture killed significantly more cancer cells, compared with either treatments alone. The active mixture decreased, to a certain extent, the toxicity of cytostatics and irradiation on normal cells, but the most important result was that the active mixture destroyed the multidrug-resistant cells. Our results provide the possibility to improve the efficacy and reduce the side-effects of chemotherapy and radiation therapy and to prevent the relapse by killing the resistant cells.

Expression profile of undifferentiated cell transcription factor 1 in normal and cancerous human epithelia.

PubMed

Mouallif, Mustapha; Albert, Adelin; Zeddou, Mustapha; Ennaji, My Mustapha; Delvenne, Philippe; Guenin, Samuel

2014-08-01

Undifferentiated cell Transcription Factor 1 (UTF1) is a chromatin-bound protein involved in stem cell differentiation. It was initially reported to be restricted to stem cells or germinal tissues. However, recent work suggests that UTF1 is also expressed in somatic cells and that its expression may increase during carcinogenesis. To further clarify the expression profile of UTF1, we evaluated UTF1 expression levels immunohistochemically in eight normal human epithelia (from breast, prostate, endometrium, bladder, colon, oesophagus, lung and kidney) and their corresponding tumours as well as in several epithelial cell lines. We showed UTF1 staining in normal and tumour epithelial tissues, but with varying intensities according to the tissue location. In vitro analyses also revealed that UTF1 is expressed in somatic epithelial cell lines even in the absence of Oct4A and Sox2, its two main known regulators. The comparison of UTF1 levels in normal and tumoral tissues revealed significant overexpression in endometrial and prostatic adenocarcinomas, whereas lower intensity of the staining was observed in renal and colic tumours, suggesting a potential tissue-specific function of UTF1. Altogether, these results highlight a potential dual role for UTF1, acting either as an oncogene or as a tumour suppressor depending on the tissue. These findings also question its role as a specific marker for stem cells. © 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene.

PubMed

Gobeil, Stephane; Zhu, Xiaochun; Doillon, Charles J; Green, Michael R

2008-11-01

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

HIPdb: a database of experimentally validated HIV inhibiting peptides.

PubMed

Qureshi, Abid; Thakur, Nishant; Kumar, Manoj

2013-01-01

Besides antiretroviral drugs, peptides have also demonstrated potential to inhibit the Human immunodeficiency virus (HIV). For example, T20 has been discovered to effectively block the HIV entry and was approved by the FDA as a novel anti-HIV peptide (AHP). We have collated all experimental information on AHPs at a single platform. HIPdb is a manually curated database of experimentally verified HIV inhibiting peptides targeting various steps or proteins involved in the life cycle of HIV e.g. fusion, integration, reverse transcription etc. This database provides experimental information of 981 peptides. These are of varying length obtained from natural as well as synthetic sources and tested on different cell lines. Important fields included are peptide sequence, length, source, target, cell line, inhibition/IC(50), assay and reference. The database provides user friendly browse, search, sort and filter options. It also contains useful services like BLAST and 'Map' for alignment with user provided sequences. In addition, predicted structure and physicochemical properties of the peptides are also included. HIPdb database is freely available at http://crdd.osdd.net/servers/hipdb. Comprehensive information of this database will be helpful in selecting/designing effective anti-HIV peptides. Thus it may prove a useful resource to researchers for peptide based therapeutics development.

Logic-Based and Cellular Pharmacodynamic Modeling of Bortezomib Responses in U266 Human Myeloma Cells

PubMed Central

Chudasama, Vaishali L.; Ovacik, Meric A.; Abernethy, Darrell R.

2015-01-01

Systems models of biological networks show promise for informing drug target selection/qualification, identifying lead compounds and factors regulating disease progression, rationalizing combinatorial regimens, and explaining sources of intersubject variability and adverse drug reactions. However, most models of biological systems are qualitative and are not easily coupled with dynamical models of drug exposure-response relationships. In this proof-of-concept study, logic-based modeling of signal transduction pathways in U266 multiple myeloma (MM) cells is used to guide the development of a simple dynamical model linking bortezomib exposure to cellular outcomes. Bortezomib is a commonly used first-line agent in MM treatment; however, knowledge of the signal transduction pathways regulating bortezomib-mediated cell cytotoxicity is incomplete. A Boolean network model of 66 nodes was constructed that includes major survival and apoptotic pathways and was updated using responses to several chemical probes. Simulated responses to bortezomib were in good agreement with experimental data, and a reduction algorithm was used to identify key signaling proteins. Bortezomib-mediated apoptosis was not associated with suppression of nuclear factor κB (NFκB) protein inhibition in this cell line, which contradicts a major hypothesis of bortezomib pharmacodynamics. A pharmacodynamic model was developed that included three critical proteins (phospho-NFκB, BclxL, and cleaved poly (ADP ribose) polymerase). Model-fitted protein dynamics and cell proliferation profiles agreed with experimental data, and the model-predicted IC50 (3.5 nM) is comparable to the experimental value (1.5 nM). The cell-based pharmacodynamic model successfully links bortezomib exposure to MM cellular proliferation via protein dynamics, and this model may show utility in exploring bortezomib-based combination regimens. PMID:26163548

The apoptotic effects of silibinin on MDA-MB-231 and MCF-7 human breast carcinoma cells.

PubMed

Bayram, D; Çetin, E S; Kara, M; Özgöçmen, M; Candan, I A

2017-06-01

Silibinin is a bioactive flavonolignan extracted from milk thistle, known as Silybum marianum. Silibinin exerts strong antiproliferative, proapoptotic, and anti-inflammatory effects. Many studies have shown that silibinin inhibits experimentally induced malignancies of the liver, prostate, skin, and colon as well as promotes inhibition of the proliferation of cancer cell lines in vitro. This study aimed to investigate the effects of silibinin on the human breast carcinoma cell lines MDA-MB-231 and MCF-7 in monolayer and spheroid cultures. The MDA-MB-231 and MCF-7 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with silibinin at 24, 48, and 72 h of incubation. The 5-bromo-2'-deoxyuridine labeling index was used to determine the cells of the synthesis phase. Poly-ADP-ribose-polimerase immunohistochemical staining and the terminal deoxynucleotidyl transferase dUTP nick and labeling assay were used to determine the death of cells in both the monolayer and spheroid cultures. An half maximal inhibitory concentration dose of silibinin in MDA-MB-231 and MCF-7 cells was 100 µM/mL at 24, 48, and 72 h of incubation. Terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase were detected after treatment with silibinin in both the monolayer and spheroid cultures. The dead cell count was higher in the MDA-MB-231 and MCF-7 cell lines with silibinin applied than in the controls. Our study demonstrated that silibinin applications enhanced terminal deoxynucleotidyl transferase dUTP nick and labeling positive cells and active poly-ADP-ribose-polimerase in comparison to the control in both the monolayer and spheroid cultures.

Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected newly synthesized N-succinimide derivatives.

PubMed

Milosevic, Natasa P; Kojic, Vesna; Curcic, Jelena; Jakimov, Dimitar; Milic, Natasa; Banjac, Nebojsa; Uscumlic, Gordana; Kaliszan, Roman

2017-04-15

Design of a new drug entity is usually preceded by analysis of quantitative structure activity (properties) relationships, QSA(P)R. Six newly synthesized succinimide derivatives have been determined for (i) in silico physico-chemical descriptors, pharmacokinetic and toxicity predictors, (ii) in vitro biological activity on four different carcinoma cell lines and on normal fetal lung cells and (iii) lipophilicity on liquid chromatography. All compounds observed were predicted for good permeability and solubility, good oral absorption rate and moderate volume of distribution as well as for modest blood brain permeation, followed by acceptable observed toxicity. In silico determined lipophilicity, permeability through jejunum and aqueous solubility were correlated with experimentally obtained lipophilic constants (by use of high pressure liquid chromatography) and linear correlations were obtained. Absorption rate and volume of distribution were predicted by chromatographic lipophilicity measurements while permeation through blood bran barrier was predicted dominantly by molecular size defined with molecular weight. Five compounds have demonstrated antiproliferative activity toward cervix carcinoma HeLa cell lines; three were cytotoxic against breast carcinoma MCF-7 cells, while one inhibited proliferation of colon carcinoma HT-29 cell lines. Only one compound was cytotoxic toward normal cell lines, while other compounds were proven as safe. Antiproliferative potential against HeLa cells was described as exponential function of lipophilicity. Based on obtained results, lead compounds were selected. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of human cortical neurons from a new immortal fetal neural stem cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cacci, E.; Villa, A.; Parmar, M.

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markersmore » like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.« less

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells.

PubMed

Sluch, Valentin M; Chamling, Xitiz; Liu, Melissa M; Berlinicke, Cynthia A; Cheng, Jie; Mitchell, Katherine L; Welsbie, Derek S; Zack, Donald J

2017-11-01

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

PubMed

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Meta-Analysis and Experimental Validation Identified FREM2 and SPRY1 as New Glioblastoma Marker Candidates.

PubMed

Vidak, Marko; Jovcevska, Ivana; Samec, Neja; Zottel, Alja; Liovic, Mirjana; Rozman, Damjana; Dzeroski, Saso; Juvan, Peter; Komel, Radovan

2018-05-04

Glioblastoma (GB) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence. In order to find new GB/GSC marker candidates that would be cell surface proteins (CSP), we have performed meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, and three (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GB/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation. The selection identified four new CSP-encoding candidate genes— CD276 , FREM2 , SPRY1 , and SLC47A1 —and the bioinformatic validation confirmed these findings. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation. This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in GB stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GB biomarker and a putative biomarker of glioblastoma stem cells. Both FREM2 and SPRY1 are expressed on the surface of the GB cells, while SPRY1 alone was found overexpressed in the cytosol of non-malignant astrocytes.

Integrated analysis of breast cancer cell lines reveals unique signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised ofmore » 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.« less

Integrated analysis of breast cancer cell lines reveals unique signaling pathways.

PubMed

Heiser, Laura M; Wang, Nicholas J; Talcott, Carolyn L; Laderoute, Keith R; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L; Laquerre, Sylvie; Jackson, Jeffrey R; Wooster, Richard F; Kuo, Wen Lin; Gray, Joe W; Spellman, Paul T

2009-01-01

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

Decoupling of the PI3K pathway via mutation necessitates combinatorial treatment in HER2+ breast cancer

DOE PAGES

Korkola, James E.; Collisson, Eric A.; Heiser, Laura; ...

2015-07-16

We report here on experimental and theoretical efforts to determine how best to combine drugs that inhibit HER2 and AKT in HER2 + breast cancers. We accomplished this by measuring cellular and molecular responses to lapatinib and the AKT inhibitors (AKT i) GSK690693 and GSK2141795 in a panel of 22 HER2 + breast cancer cell lines carrying wild type or mutant PIK3CA. We observed that combinations of lapatinib plus AKT i were synergistic in HER2 /PIK3CA mut cell lines but not in HER2 +/PIK3CA wt cell lines. We measured changes in phospho-protein levels in 15 cell lines after treatment withmore » lapatinib, AKT i or lapatinib + AKT i to shed light on the underlying signaling dynamics. This revealed that p-S6RP levels were less well attenuated by lapatinib in HER2 +/PIK3CA mut cells compared to HER2 +/PIK3CA wt cells and that lapatinib + AKT i reduced p-S6RP levels to those achieved in HER2 +/PIK3CA wt cells with lapatinib alone. We also found that that compensatory up-regulation of p-HER3 and p-HER2 is blunted in PIK3CA mut cells following lapatinib + AKT i treatment. Responses of HER2 + SKBR3 cells transfected with lentiviruses carrying control or PIK3CA mut sequences were similar to those observed in HER2 +/PIK3CA mut cell lines but not in HER2 +/PIK3CA wt cell lines. We used a nonlinear ordinary differential equation model to support the idea that PIK3CA mutations act as downstream activators of AKT that blunt lapatinib inhibition of downstream AKT signaling and that the effects of PIK3CA mutations can be countered by combining lapatinib with an AKT i. This combination does not confer substantial benefit beyond lapatinib in HER2 +/PIK3CA wt cells.« less

Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

PubMed

Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

2017-01-01

Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types.

PubMed

Iliopoulos, Dimitrios; Hirsch, Heather A; Struhl, Kevin

2011-05-01

Metformin, the first-line drug for treating diabetes, selectively kills the chemotherapy resistant subpopulation of cancer stem cells (CSC) in genetically distinct types of breast cancer cell lines. In mouse xenografts, injection of metformin and the chemotherapeutic drug doxorubicin near the tumor is more effective than either drug alone in blocking tumor growth and preventing relapse. Here, we show that metformin is equally effective when given orally together with paclitaxel, carboplatin, and doxorubicin, indicating that metformin works together with a variety of standard chemotherapeutic agents. In addition, metformin has comparable effects on tumor regression and preventing relapse when combined with a four-fold reduced dose of doxorubicin that is not effective as a monotherapy. Finally, the combination of metformin and doxorubicin prevents relapse in xenografts generated with prostate and lung cancer cell lines. These observations provide further evidence for the CSC hypothesis for cancer relapse, an experimental rationale for using metformin as part of combinatorial therapy in a variety of clinical settings, and for reducing the chemotherapy dose in cancer patients.

A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease.

PubMed

Hazelbaker, Dane Z; Beccard, Amanda; Bara, Anne M; Dabkowski, Nicole; Messana, Angelica; Mazzucato, Patrizia; Lam, Daisy; Manning, Danielle; Eggan, Kevin; Barrett, Lindy E

2017-10-10

Scaling of CRISPR-Cas9 technology in human pluripotent stem cells (hPSCs) represents an important step for modeling complex disease and developing drug screens in human cells. However, variables affecting the scaling efficiency of gene editing in hPSCs remain poorly understood. Here, we report a standardized CRISPR-Cas9 approach, with robust benchmarking at each step, to successfully target and genotype a set of psychiatric disease-implicated genes in hPSCs and provide a resource of edited hPSC lines for six of these genes. We found that transcriptional state and nucleosome positioning around targeted loci was not correlated with editing efficiency. However, editing frequencies varied between different hPSC lines and correlated with genomic stability, underscoring the need for careful cell line selection and unbiased assessments of genomic integrity. Together, our step-by-step quantification and in-depth analyses provide an experimental roadmap for scaling Cas9-mediated editing in hPSCs to study psychiatric disease, with broader applicability for other polygenic diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cell wall composition and biomass recalcitrance differences within a genotypically diverse set of Brachypodium distachyon inbred lines

DOE PAGES

Cass, Cynthia L.; Lavell, Anastasiya A.; Santoro, Nicholas; ...

2016-05-26

Brachypodium distachyon ( Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences andmore » recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. Lastly, the availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.« less

Cell wall composition and biomass recalcitrance differences within a genotypically diverse set of Brachypodium distachyon inbred lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cass, Cynthia L.; Lavell, Anastasiya A.; Santoro, Nicholas

Brachypodium distachyon ( Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences andmore » recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. Lastly, the availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.« less

Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion.

PubMed

Li, Yangyang; Wang, Cheng; Liu, Xinye; Song, Jian; Li, Hongjian; Sui, Zhipeng; Zhang, Ming; Fang, Shuang; Chu, Jinfang; Xin, Mingming; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

2016-04-01

Heterosis has been widely used in agriculture, but the underlying molecular principles are still largely unknown. During seed germination, we observed that maize (Zea mays) hybrid B73/Mo17 was less sensitive than its parental inbred lines to exogenous abscisic acid (ABA), and endogenous ABA content in hybrid embryos decreased more rapidly than in the parental inbred lines. ZmABA8ox1b, an ABA inactivation gene, was consistently more highly up-regulated in hybrid B73/Mo17 than in its parental inbred lines at early stages of seed germination. Moreover, ectopic expression of ZmABA8ox1b obviously promoted seed germination in Arabidopsis Remarkably, microscopic observation revealed that cell expansion played a major role in the ABA-mediated maize seed germination heterosis, which could be attributed to the altered expression of cell wall-related genes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231.

PubMed

Tenorio, María J; Ross, Breyan H; Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V; Ehrenfeld, Pamela; Mardones, Gonzalo A

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

PubMed Central

Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V.; Ehrenfeld, Pamela; Mardones, Gonzalo A.

2016-01-01

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

Determination of the intracellular Ca2+ concentration in the N1E-115 neuronal cell line in perspective of its use for peripheric nerve regeneration.

PubMed

Rodrigues, J M; Luís, A L; Lobato, J V; Pinto, M V; Lopes, M A; Freitas, M; Geuna, S; Santos, J D; Maurício, A C

2005-01-01

Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which was capable of growing, differentiating and producing locally nerve growth factors that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a gap in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for various cellular processes, such as growth and differentiation. It is also known that can be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca2+]i in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca2+]i was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

BIANCA, a biophysical model of cell survival and chromosome damage by protons, C-ions and He-ions at energies and doses used in hadrontherapy

NASA Astrophysics Data System (ADS)

Pietro Carante, Mario; Aimè, Chiara; Tello Cajiao, John James; Ballarini, Francesca

2018-04-01

An upgraded version of the BIANCA II biophysical model, which describes more realistically interphase chromosome organization and the link between chromosome aberrations and cell death, was applied to V79 and AG01522 cells exposed to protons, C-ions and He-ions over a wide LET interval (0.6–502 keV µm‑1), as well as proton-irradiated U87 cells. The model assumes that (i) ionizing radiation induces DNA ‘cluster lesions’ (CLs), where by definition each CL produces two independent chromosome fragments; (ii) fragment (distance-dependent) mis-rejoining, or un-rejoining, produces chromosome aberrations; (iii) some aberrations lead to cell death. The CL yield, which mainly depends on radiation quality but is also modulated by the target cell, is an adjustable parameter. The fragment un-rejoining probability, f, is the second, and last, parameter. The value of f, which is assumed to depend on the cell type but not on radiation quality, was taken from previous studies, and only the CL yield was adjusted in the present work. Good agreement between simulations and experimental data was obtained, suggesting that BIANCA II is suitable for calculating the biological effectiveness of hadrontherapy beams. For both V79 and AG01522 cells, the mean number of CLs per micrometer was found to increase with LET in a linear-quadratic fashion before the over-killing region, where a less rapid increase, with a tendency to saturation, was observed. Although the over-killing region deserves further investigation, the possibility of fitting the CL yields is an important feature for hadrontherapy, because it allows performing predictions also at LET values where experimental data are not available. Finally, an approach was proposed to predict the ion-response of the cell line(s) of interest from the ion-response of a reference cell line and the photon response of both. A pilot study on proton-irradiated AG01522 and U87 cells, taking V79 cells as a reference, showed encouraging results.

Single-frequency TEA CO2 laser with a bleaching spectral intracavity filter

NASA Astrophysics Data System (ADS)

Sorochenko, V. R.

2017-02-01

The regime of single-frequency operation is realised in a TEA CO2 laser with a spectral filter inside the cavity (a cell filled with SF6) on P(12)-P(24) lines of the 10P band. The minimal scatter of the peak powers of the laser pulses in a series of ‘shots’ and the maximal ratio of the output energies in the single-frequency and free running regimes (greater than 0.84) are obtained on the P(16) line at an optimal SF6 pressure in the cell. Experimental results qualitatively agree with the absorption spectrum of SF6 calculated from the SPECTRA information-analytical system. It is shown that the high ratio of energies in two regimes is achived due to gas bleaching in the cell.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

PubMed

Furihata, Tomomi; Ito, Ryo; Kamiichi, Atsuko; Saito, Kosuke; Chiba, Kan

2016-01-01

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions. © 2015 International Society for Neurochemistry.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

PubMed

Ishibashi, Osamu; Akagi, Ichiro; Ogawa, Yota; Inui, Takashi

2018-05-11

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Epigenetic Inactivation of GALR1 in Head and Neck Cancer

PubMed Central

Misawa, Kiyoshi; Ueda, Yo; Kanazawa, Takeharu; Misawa, Yuki; Jang, Ilwhan; Brenner, John Chadwick; Ogawa, Tetsuya; Takebayashi, Satoru; Grenman, Reidar A.; Herman, James G.; Mineta, Hiroyuki; Carey, Thomas E.

2011-01-01

Purpose One copy of the GALR1 locus on 18q is often deleted and expression is absent in some head and neck squamous cell carcinoma (HNSCC) cell lines. To determine if LOH and hypermethylation might silence the GALR1 gene, promoter methylation status and gene expression were assessed in a large panel of HNSCC cell lines and tumors. Experimental Design Promoter methylation of GALR1 in 72 cell lines and 100 primary tumor samples was analyzed using methylation-specific PCR (MSP). GALR1 expression and methylation status were analyzed further by real-time PCR and bisulfite sequencing analysis. Results The GALR1 promoter was fully or partially methylated in 38 of 72 HNSCC cell lines (52.7%) but not in the majority 18/20 (90.0%) of non-malignant lines. GALR1 methylation was also found in 38/100 (38%) primary tumor specimens. Methylation correlated with decreased GALR1 expression. In tumors methylation was significantly correlated with increased tumor size (P=0.0036), lymph-node status (P=0.0414), tumor stage (P=0.0037), cyclin D1 expression (P=0.0420), and p16 methylation (P=0.0494) and survival (P=0.045). Bisulfite sequencing of 36 CpG sites upstream of the transcription start site revealed that CpG methylation within transcription factor binding sites correlated with complete suppression of GALR1 mRNA. Treatment with TSA and 5-azacytidine restored GALR1 expression. In UM-SCC-23 cells that have total silencing of GALR1, exogenous GALR1 expression and stimulation with galanin suppressed cell proliferation. Conclusions Frequent promoter hypermethylation, gene silencing, association with prognosis, and growth suppression after re-expression support the hypothesis that GALR1 is a tumor suppressor gene in HNSCC. PMID:19047085

TU-H-BRA-07: Design, Construction, and Installation of An Experimental Beam Line for the Development of MRI-Linac Compatible Electron Accelerator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whelan, B; Keall, P; Holloway, L

Purpose: MRI guided radiation therapy (MRIgRT) is a rapidly growing field; however, Linac operation in MRI fringe fields represents an ongoing challenge. We have previously shown in-silico that Linacs could be redesigned to function in the in-line orientation with no magnetic shielding by adopting an RF-gun configuration. Other authors have also published insilico studies of Linac operation in magnetic fields; however to date no experimental validation data is published. This work details the design, construction, and installation of an experimental beam line to validate our in-silico results. Methods: An RF-gun comprising 1.5 accelerating cells and capable of generating electron energiesmore » up to 3.2MeV is used. The experimental apparatus was designed to monitor both beam current (toroid current monitor), spot size (two phosphor screens with viewports), and generate peak magnetic fields of at least 1000G (three variable current electromagnetic coils). Thermal FEM simulations were developed to ensure coil temperature remained within 100degC. Other design considerations included beam disposal, vacuum maintenance, radiation shielding, earthquake safety, and machine protection interlocks. Results: The beam line has been designed, built, and installed in a radiation shielded bunker. Water cooling, power supplies, thermo-couples, cameras, and radiation shielding have been successfully connected and tested. Interlock testing, vacuum processing, and RF processing have been successfully completed. The first beam on is expected within weeks. The coil heating simulations show that with care, peak fields of up to 1200G (320G at cathode) can be produced using 40A current, which is well within the fields expected for MRI-Linac systems. The maximum coil temperature at this current was 84degC after 6 minutes. Conclusion: An experimental beam line has been constructed and installed at SLAC in order to experimentally characterise RF gun performance in in-line magnetic fields, validate in-silico design work, and provide the first published experimental data relating to accelerator functionality for MRIgRT.« less

Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

PubMed

Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

2012-06-01

Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells

PubMed Central

Shimizu, Akira; Nakatani, Yoko; Nakamura, Takako; Jinno-Oue, Atsushi; Ishikawa, Osamu; Boeke, Jef D.; Takeuchi, Yasuhiro; Hoshino, Hiroo

2014-01-01

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements. PMID:24875540

Synthesis and photodynamic activities of modified corrole derivatives on nasopharyngeal carcinoma cells

NASA Astrophysics Data System (ADS)

Chang, Chi K.; Kong, Pak-Wing; Liu, Hai-Yang; Yeung, Lam-Lung; Koon, Ho-Kee; Mak, Nai-Ki

2006-02-01

Ten trans-A2B and A3-type corrole photosensitizers carrying functional groups were synthesized and screened for PDT activities. Photocytotoxicity was measured by the MTT cell reduction assay on a cultured human nasopharyngeal carcinoma (NPC) cell line (HONE-1). Experimental results indicated that corroles containing a single hydroxyphenyl substituent (3, 4 and 5) exhibit the highest activity among the corrole derivatives investigated. Confocal microscopy revealed that the site of cellular localization of the photosensitizers is predominantly at mitochondria. Also, nuclear staining detected apoptotic cell death.

Life span prediction from the rate of age-related DNA demethylation in normal and cancer cell lines.

PubMed

Mazin, A L

1995-01-01

A method has been proposed for the Hayflick Limit prediction by the analysis of the 5-methylcytosine content in DNA at earlier and later cell passages. The following facts were used as the basis of the method: (i) the rate of m5C loss from DNA remains approximately constant during cell divisions and it does not depend on the cell donor age; (ii) this rate is inversely proportional to the Hayflick Limit as well as to the life span of cell donor species; (iii) the period corresponded to loss of all m5C residues from the genome coincides with or somewhat exceeds the Hayflick Limit of normal cells. The prognosis of the Hayflick Limit has usually been found in good agreement with the experimental evidences for various human, hamster, and mouse cell lines. The method proposed may be used for early detection of precrisis and cancer cells. The age-related m5C loss may result from accumulation of the m5C-->T+C transitions occurring with DNA methylation in every cell division.

Separation of CHO cells using hydrocyclones.

PubMed

Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

2008-01-01

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

PubMed

Okayasu, H; Ishihara, M; Satoh, K; Sakagami, H

2001-01-01

Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

Characterizing steady states of genome-scale metabolic networks in continuous cell cultures.

PubMed

Fernandez-de-Cossio-Diaz, Jorge; Leon, Kalet; Mulet, Roberto

2017-11-01

In the continuous mode of cell culture, a constant flow carrying fresh media replaces culture fluid, cells, nutrients and secreted metabolites. Here we present a model for continuous cell culture coupling intra-cellular metabolism to extracellular variables describing the state of the bioreactor, taking into account the growth capacity of the cell and the impact of toxic byproduct accumulation. We provide a method to determine the steady states of this system that is tractable for metabolic networks of arbitrary complexity. We demonstrate our approach in a toy model first, and then in a genome-scale metabolic network of the Chinese hamster ovary cell line, obtaining results that are in qualitative agreement with experimental observations. We derive a number of consequences from the model that are independent of parameter values. The ratio between cell density and dilution rate is an ideal control parameter to fix a steady state with desired metabolic properties. This conclusion is robust even in the presence of multi-stability, which is explained in our model by a negative feedback loop due to toxic byproduct accumulation. A complex landscape of steady states emerges from our simulations, including multiple metabolic switches, which also explain why cell-line and media benchmarks carried out in batch culture cannot be extrapolated to perfusion. On the other hand, we predict invariance laws between continuous cell cultures with different parameters. A practical consequence is that the chemostat is an ideal experimental model for large-scale high-density perfusion cultures, where the complex landscape of metabolic transitions is faithfully reproduced.

Characterizing steady states of genome-scale metabolic networks in continuous cell cultures

PubMed Central

Leon, Kalet; Mulet, Roberto

2017-01-01

In the continuous mode of cell culture, a constant flow carrying fresh media replaces culture fluid, cells, nutrients and secreted metabolites. Here we present a model for continuous cell culture coupling intra-cellular metabolism to extracellular variables describing the state of the bioreactor, taking into account the growth capacity of the cell and the impact of toxic byproduct accumulation. We provide a method to determine the steady states of this system that is tractable for metabolic networks of arbitrary complexity. We demonstrate our approach in a toy model first, and then in a genome-scale metabolic network of the Chinese hamster ovary cell line, obtaining results that are in qualitative agreement with experimental observations. We derive a number of consequences from the model that are independent of parameter values. The ratio between cell density and dilution rate is an ideal control parameter to fix a steady state with desired metabolic properties. This conclusion is robust even in the presence of multi-stability, which is explained in our model by a negative feedback loop due to toxic byproduct accumulation. A complex landscape of steady states emerges from our simulations, including multiple metabolic switches, which also explain why cell-line and media benchmarks carried out in batch culture cannot be extrapolated to perfusion. On the other hand, we predict invariance laws between continuous cell cultures with different parameters. A practical consequence is that the chemostat is an ideal experimental model for large-scale high-density perfusion cultures, where the complex landscape of metabolic transitions is faithfully reproduced. PMID:29131817

Comparative theoretical and experimental study on novel tri-quinoline system and its anticancer studies

NASA Astrophysics Data System (ADS)

Gayathri, Kasirajan; Radhika, Ramachandran; Shankar, Ramasamy; Malathi, Mahalingam; Savithiri, Krishnaswamy; Sparkes, Hazel A.; Howard, Judith A. K.; Mohan, Palathurai Subramaniam

2017-04-01

A novel compound 2-chloro-3,6-bis-(quinolin-8-yloxymethyl)-quinoline 3 bearing a tri-quinoline moiety has been synthesized from 2-chloro-3,6-dimethyl quinoline 1 and 8-hydroxy quinoline 2 using dry acetone and K2CO3 as a base. 3 has been characterized by using FT-IR, FT-Raman, UV-Vis, 1H NMR, 13C NMR spectra and single crystal X-ray diffraction methods. We have also made a combined experimental and theoretical study on the molecular structure, vibrational spectra, NMR, FT-IR, FT-Raman and UV-Vis spectra of 2-chloro-3,6-bis-(quinolin-8-yloxymethyl)-quinoline. The theoretical studies of the title compound have been evaluated by using density functional theory calculations using B3LYP/6-31+G(d,p) and M06-2X/6-31+G(d,p) level of theories. The calculated theoretical values were found to be in good agreement with the experimental findings. The single crystal structure 3 crystallized in the orthorhombic space group Pna21. The compound 3 exhibits higher cytotoxicity in human cervical cancer cell lines (HeLa) than human breast cancer cell lines (MCF7).

Metal Free Graphene Oxide (GO) Nanosheets and Pristine-Single Wall Carbon Nanotubes (p-SWCNTs) Biocompatibility Investigation: A Comparative Study in Different Human Cell Lines.

PubMed

Valentini, Federica; Mari, Emanuela; Zicari, Alessandra; Calcaterra, Andrea; Talamo, Maurizio; Scioli, Maria Giovanna; Orlandi, Augusto; Mardente, Stefania

2018-04-28

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests.

Quinazolinones-Phenylquinoxaline hybrids with unsaturation/saturation linkers as novel anti-proliferative agents.

PubMed

Palem, Jyothsna Devi; Alugubelli, Gopi Reddy; Bantu, Rajashaker; Nagarapu, Lingaiah; Polepalli, Sowjanya; Jain, S Nishanth; Bathini, Raju; Manga, Vijjulatha

2016-07-01

A new series of novel quinazolinones with allylphenyl quinoxaline hybrids 9a-n were efficiently synthesized in good yields by the reaction of 3-allyl-2-methylquinazolin-4(3H)-one (5a-n) with bromophenyl)quinoxaline (8) utilizing Pd catalyzed Heck-cross coupling and evaluated for anti-proliferative activity against four cancer cell lines such as HeLa (cervical), MIAPACA (pancreatic), MDA-MB-231 (breast) and IMR32 (neuroblastoma). Compounds 9a, 9e, 9g and 9h exhibited promising anti-proliferative activity with GI50 values ranging from 0.06 to 0.2μM against four cell lines, while compounds 9e and 9k showed significant activity against HeLa and MIAPACA cell lines and compounds 9b, 9d, 9h and 9j showed selective potency against IMR32 and MDA-MB-231 cell lines. This is the first report on the synthesis and in vitro anti-proliferative evaluation of E-2-(4-substituted)-3-(3-(4-(quinoxalin-2-yl)phenyl)allyl)quinazolin-4(3H)-ones (9a-n). Docking results indicate a sign of good correlation between experimental activity and calculated binding affinity (dock score), suggesting that these compounds could act as promising DNA intercalates. Copyright © 2016 Elsevier Ltd. All rights reserved.

A physical sciences network characterization of non-tumorigenic and metastatic cells

PubMed Central

Agus, David B.; Alexander, Jenolyn F.; Arap, Wadih; Ashili, Shashanka; Aslan, Joseph E.; Austin, Robert H.; Backman, Vadim; Bethel, Kelly J.; Bonneau, Richard; Chen, Wei-Chiang; Chen-Tanyolac, Chira; Choi, Nathan C.; Curley, Steven A.; Dallas, Matthew; Damania, Dhwanil; Davies, Paul C. W.; Decuzzi, Paolo; Dickinson, Laura; Estevez-Salmeron, Luis; Estrella, Veronica; Ferrari, Mauro; Fischbach, Claudia; Foo, Jasmine; Fraley, Stephanie I.; Frantz, Christian; Fuhrmann, Alexander; Gascard, Philippe; Gatenby, Robert A.; Geng, Yue; Gerecht, Sharon; Gillies, Robert J.; Godin, Biana; Grady, William M.; Greenfield, Alex; Hemphill, Courtney; Hempstead, Barbara L.; Hielscher, Abigail; Hillis, W. Daniel; Holland, Eric C.; Ibrahim-Hashim, Arig; Jacks, Tyler; Johnson, Roger H.; Joo, Ahyoung; Katz, Jonathan E.; Kelbauskas, Laimonas; Kesselman, Carl; King, Michael R.; Konstantopoulos, Konstantinos; Kraning-Rush, Casey M.; Kuhn, Peter; Kung, Kevin; Kwee, Brian; Lakins, Johnathon N.; Lambert, Guillaume; Liao, David; Licht, Jonathan D.; Liphardt, Jan T.; Liu, Liyu; Lloyd, Mark C.; Lyubimova, Anna; Mallick, Parag; Marko, John; McCarty, Owen J. T.; Meldrum, Deirdre R.; Michor, Franziska; Mumenthaler, Shannon M.; Nandakumar, Vivek; O’Halloran, Thomas V.; Oh, Steve; Pasqualini, Renata; Paszek, Matthew J.; Philips, Kevin G.; Poultney, Christopher S.; Rana, Kuldeepsinh; Reinhart-King, Cynthia A.; Ros, Robert; Semenza, Gregg L.; Senechal, Patti; Shuler, Michael L.; Srinivasan, Srimeenakshi; Staunton, Jack R.; Stypula, Yolanda; Subramanian, Hariharan; Tlsty, Thea D.; Tormoen, Garth W.; Tseng, Yiider; van Oudenaarden, Alexander; Verbridge, Scott S.; Wan, Jenny C.; Weaver, Valerie M.; Widom, Jonathan; Will, Christine; Wirtz, Denis; Wojtkowiak, Jonathan; Wu, Pei-Hsun

2013-01-01

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis. PMID:23618955

A physical sciences network characterization of non-tumorigenic and metastatic cells.

PubMed

Agus, David B; Alexander, Jenolyn F; Arap, Wadih; Ashili, Shashanka; Aslan, Joseph E; Austin, Robert H; Backman, Vadim; Bethel, Kelly J; Bonneau, Richard; Chen, Wei-Chiang; Chen-Tanyolac, Chira; Choi, Nathan C; Curley, Steven A; Dallas, Matthew; Damania, Dhwanil; Davies, Paul C W; Decuzzi, Paolo; Dickinson, Laura; Estevez-Salmeron, Luis; Estrella, Veronica; Ferrari, Mauro; Fischbach, Claudia; Foo, Jasmine; Fraley, Stephanie I; Frantz, Christian; Fuhrmann, Alexander; Gascard, Philippe; Gatenby, Robert A; Geng, Yue; Gerecht, Sharon; Gillies, Robert J; Godin, Biana; Grady, William M; Greenfield, Alex; Hemphill, Courtney; Hempstead, Barbara L; Hielscher, Abigail; Hillis, W Daniel; Holland, Eric C; Ibrahim-Hashim, Arig; Jacks, Tyler; Johnson, Roger H; Joo, Ahyoung; Katz, Jonathan E; Kelbauskas, Laimonas; Kesselman, Carl; King, Michael R; Konstantopoulos, Konstantinos; Kraning-Rush, Casey M; Kuhn, Peter; Kung, Kevin; Kwee, Brian; Lakins, Johnathon N; Lambert, Guillaume; Liao, David; Licht, Jonathan D; Liphardt, Jan T; Liu, Liyu; Lloyd, Mark C; Lyubimova, Anna; Mallick, Parag; Marko, John; McCarty, Owen J T; Meldrum, Deirdre R; Michor, Franziska; Mumenthaler, Shannon M; Nandakumar, Vivek; O'Halloran, Thomas V; Oh, Steve; Pasqualini, Renata; Paszek, Matthew J; Philips, Kevin G; Poultney, Christopher S; Rana, Kuldeepsinh; Reinhart-King, Cynthia A; Ros, Robert; Semenza, Gregg L; Senechal, Patti; Shuler, Michael L; Srinivasan, Srimeenakshi; Staunton, Jack R; Stypula, Yolanda; Subramanian, Hariharan; Tlsty, Thea D; Tormoen, Garth W; Tseng, Yiider; van Oudenaarden, Alexander; Verbridge, Scott S; Wan, Jenny C; Weaver, Valerie M; Widom, Jonathan; Will, Christine; Wirtz, Denis; Wojtkowiak, Jonathan; Wu, Pei-Hsun

2013-01-01

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

Derivation of the open-circuit voltage of organic solar cells

NASA Astrophysics Data System (ADS)

Staple, Douglas B.; Oliver, Patricia A. K.; Hill, Ian G.

2014-05-01

Organic photovoltaic cells have improved in efficiency from 1% two decades ago to over 10% today. Continued improvement necessitates a theoretical understanding of the factors determining efficiency. Organic photovoltaic efficiency can be parameterized in terms of open-circuit voltage, short-circuit current, and fill factor. Here we present a theory that explains the dependencies of open-circuit voltage on semiconductor energy levels, light intensity, solar cell and light-source temperatures, charge-carrier recombination, and external fluorescence efficiency. The present theory also explains why recombination at the donor-acceptor heterointerface is a dominant process in heterojunction-based cells. Furthermore, the Carnot efficiency appears, highlighting the connection to basic thermodynamics. The theory presented here is consistent with and builds on the experimental and theoretical observations already in the literature. Crucially, the present theory can be straightforwardly derived in a line-by-line fashion using standard tools from statistical physics.

High-speed video capillaroscopy method for imaging and evaluation of moving red blood cells

NASA Astrophysics Data System (ADS)

Gurov, Igor; Volkov, Mikhail; Margaryants, Nikita; Pimenov, Aleksei; Potemkin, Andrey

2018-05-01

The video capillaroscopy system with high image recording rate to resolve moving red blood cells with velocity up to 5 mm/s into a capillary is considered. Proposed procedures of the recorded video sequence processing allow evaluating spatial capillary area, capillary diameter and central line with high accuracy and reliability independently on properties of individual capillary. Two-dimensional inter frame procedure is applied to find lateral shift of neighbor images in the blood flow area with moving red blood cells and to measure directly the blood flow velocity along a capillary central line. The developed method opens new opportunities for biomedical diagnostics, particularly, due to long-time continuous monitoring of red blood cells velocity into capillary. Spatio-temporal representation of capillary blood flow is considered. Experimental results of direct measurement of blood flow velocity into separate capillary as well as capillary net are presented and discussed.

A physical sciences network characterization of non-tumorigenic and metastatic cells

NASA Astrophysics Data System (ADS)

Physical Sciences-Oncology Centers Network; Agus, David B.; Alexander, Jenolyn F.; Arap, Wadih; Ashili, Shashanka; Aslan, Joseph E.; Austin, Robert H.; Backman, Vadim; Bethel, Kelly J.; Bonneau, Richard; Chen, Wei-Chiang; Chen-Tanyolac, Chira; Choi, Nathan C.; Curley, Steven A.; Dallas, Matthew; Damania, Dhwanil; Davies, Paul C. W.; Decuzzi, Paolo; Dickinson, Laura; Estevez-Salmeron, Luis; Estrella, Veronica; Ferrari, Mauro; Fischbach, Claudia; Foo, Jasmine; Fraley, Stephanie I.; Frantz, Christian; Fuhrmann, Alexander; Gascard, Philippe; Gatenby, Robert A.; Geng, Yue; Gerecht, Sharon; Gillies, Robert J.; Godin, Biana; Grady, William M.; Greenfield, Alex; Hemphill, Courtney; Hempstead, Barbara L.; Hielscher, Abigail; Hillis, W. Daniel; Holland, Eric C.; Ibrahim-Hashim, Arig; Jacks, Tyler; Johnson, Roger H.; Joo, Ahyoung; Katz, Jonathan E.; Kelbauskas, Laimonas; Kesselman, Carl; King, Michael R.; Konstantopoulos, Konstantinos; Kraning-Rush, Casey M.; Kuhn, Peter; Kung, Kevin; Kwee, Brian; Lakins, Johnathon N.; Lambert, Guillaume; Liao, David; Licht, Jonathan D.; Liphardt, Jan T.; Liu, Liyu; Lloyd, Mark C.; Lyubimova, Anna; Mallick, Parag; Marko, John; McCarty, Owen J. T.; Meldrum, Deirdre R.; Michor, Franziska; Mumenthaler, Shannon M.; Nandakumar, Vivek; O'Halloran, Thomas V.; Oh, Steve; Pasqualini, Renata; Paszek, Matthew J.; Philips, Kevin G.; Poultney, Christopher S.; Rana, Kuldeepsinh; Reinhart-King, Cynthia A.; Ros, Robert; Semenza, Gregg L.; Senechal, Patti; Shuler, Michael L.; Srinivasan, Srimeenakshi; Staunton, Jack R.; Stypula, Yolanda; Subramanian, Hariharan; Tlsty, Thea D.; Tormoen, Garth W.; Tseng, Yiider; van Oudenaarden, Alexander; Verbridge, Scott S.; Wan, Jenny C.; Weaver, Valerie M.; Widom, Jonathan; Will, Christine; Wirtz, Denis; Wojtkowiak, Jonathan; Wu, Pei-Hsun

2013-04-01

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

Proliferation and performance of hybridoma cells in microgravity (7-IML-1)

NASA Technical Reports Server (NTRS)

Cogoli, Augusto

1992-01-01

The purpose of this experiment is to study how cell performance (biosynthesis and secretion) is altered by altered gravity conditions. Hybridoma cells are obtained by fusion of an activated B-lymphocyte with a myeloma cell. Activated B-lymphocytes, derived from a human or an animal, carry the information required to produce antibodies of a certain specificity and can survive only a few days in culture. Myeloma cells are tumor cells which can grow indefinitely in culture. Therefore, the product of the fusion is an immortal cell line capable of producing homogeneous antibodies (monoclonal antibodies). Experimental procedures are explained in some detail.

Varicocele-caused progressive damage in bilateral testis and sertoli cell-only syndrome in homolateral testis in rats.

PubMed

Liu, Jianjun; Ding, Degang; Liu, Jie

2014-10-14

We aimed to investigate whether varicocele (VC) in rats can cause Sertoli cell-only syndrome (SCOS). Forty adolescent SD rats were randomly divided into 4 groups: 4-weeks control group, 4-weeks experimental group, 12-weeks control group, and 12-weeks experimental group. Left varicocele models were introduced by partially ligating left kidney veins for the experimental groups, and the sham surgery groups as controls were executed with exactly the same surgery as in the experimental groups except for the ligation. Rats in control and experimental groups for 4 and 12 weeks were killed after laparotomy at 4 and 12 weeks, respectively, the testes were taken out and fixed in fixative containing 4% polyformaldehyde, then were stained by hematoxylin and eosin (HE). The density and viability of sperm were analyzed by computer-aided sperm analysis. Compared with rats in 4-weeks and 12-weeks control group, histological structures of bilateral testes in both experimental groups were impaired, most of them showing as focal focuses. The pathological changes of testes in rats of the 12-weeks experimental group were bilateral, and included atrophy of seminiferous tubules, turbulence of spermatogenic cells in seminiferous tubules, defluvium of most spermatogenic cells, abortion of spermatogenesis, and degradation of spermatogenic epithelia. One rat in the 12-weeks experimental group was shown having SCOS, with the spermatogenic cells in seminiferous tubules completely flaked, degraded, or absent, and only Sertoli cells lined the seminiferous tubules. Laboratory VC caused progressive impairment of homolateral testes, and SCOS could be induced when the damage was severe. Our results indicate that asthenozoospermia, azoospermia, and SCOS can be prevented by the earlier treatment of VC.

Neural cells derived from adult bone marrow and umbilical cord blood.

PubMed

Sanchez-Ramos, Juan R

2002-09-15

Under experimental conditions, tissue-specific stem cells have been shown to give rise to cell lineages not normally found in the organ or tissue of residence. Neural stem cells from fetal brain have been shown to give rise to blood cell lines and conversely, bone marrow stromal cells have been reported to generate skeletal and cardiac muscle, oval hepatocytes, as well as glia and neuron-like cells. This article reviews studies in which cells from postnatal bone marrow or umbilical cord blood were induced to proliferate and differentiate into glia and neurons, cellular lineages that are not their normal destiny. The review encompasses in vitro and in vivo studies with focus on experimental variables, such as the source and characterization of cells, cell-tracking methods, and markers of neural differentiation. The existence of stem/progenitor cells with previously unappreciated proliferation and differentiation potential in postnatal bone marrow and in umbilical cord blood opens up the possibility of using stem cells found in these tissues to treat degenerative, post-traumatic and hereditary diseases of the central nervous system. Copyright 2002 Wiley-Liss, Inc.

Development of a TDLAS sensor for temperature and concentration of H2 O in high speed and high temperature flows

NASA Astrophysics Data System (ADS)

Sheehe, Suzanne; O'Byrne, Sean

2017-06-01

The development of a sensor for simultaneous temperature concentration of H2 O and temperature in high speed flows is presented. H2 O is a desirable target sensing species because it is a primary product in combustion systems; both temperature and concentration profiles can be used to assess both the extent of the combustion and the flow field characteristics. Accurate measurements are therefore highly desirable. The sensor uses a vertical-cavity surface emitting laser (VCSEL) scanned at 50 kHz from 7172 to 7186 cm-1. Temperatures and concentrations are extracted from the spectra by fitting theoretical spectra to the experimental data. The theoretical spectra are generated using GENSPECT in conjunction with line parameters from the HITRAN 2012 database. To validate the theoretical spectra, experimental spectra of H2 O were obtained at known temperatures (290-550 K) and pressures (30 torr) in a heated static gas cell. The results show that some theoretical lines deviate from the experimental lines. New line-strengths are calculated assuming that the line assignments and broadening parameters in HITRAN are correct. This data is essential for accurate H2 O concentration and temperature measurements at low pressure and high temperature conditions. US Air Force Asian Office of Aerospace Research and Development Grant FA2386-16-1-4092.

Chloride transporting capability of Calu-3 epithelia following persistent knockdown of the cystic fibrosis transmembrane conductance regulator, CFTR

PubMed Central

MacVinish, L J; Cope, G; Ropenga, A; Cuthbert, A W

2007-01-01

Background and purpose: Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available. Experimental approach: A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability. Key results: All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent. Conclusions and implications: It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity. PMID:17339840

DIAC object recognition system

NASA Astrophysics Data System (ADS)

Buurman, Johannes

1992-03-01

This paper describes the object recognition system used in an intelligent robot cell. It is used to recognize and estimate pose and orientation of parts as they enter the cell. The parts are mostly metal and consist of polyhedral and cylindrical shapes. The system uses feature-based stereo vision to acquire a wireframe of the observed part. Features are defined as straight lines and ellipses, which lead to a wireframe of straight lines and circular arcs (the latter using a new algorithm). This wireframe is compared to a number of wire frame models obtained from the CAD database. Experimental results show that image processing hardware and parallelization may add considerably to the speed of the system.

In Vitro Co-Delivery Evaluation of Novel Pegylated Nano-Liposomal Herbal Drugs of Silibinin and Glycyrrhizic Acid (Nano-Phytosome) to Hepatocellular Carcinoma Cells

PubMed Central

Ochi, Mohammad Mahdi; Amoabediny, Ghasem; Rezayat, Seyed Mahdi; Akbarzadeh, Azim; Ebrahimi, Bahman

2016-01-01

Objective This study aimed to evaluate a co-encapsulated pegylated nano-liposome system based on two herbal anti-tumor drugs, silibinin and glycyrrhizic acid, for delivery to a hepatocellular carcinoma (HCC) cell line (HepG2). Materials and Methods In this experimental study, co-encapsulated nano-liposomes by the thin layer film hydration method with HEPES buffer and sonication at 60% amplitude. Liposomes that co-encapsulated silibinin and glycyrrhizic acid were prepared with a specified molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), and methoxy-polyethylene glycol 2000 (PEG2000)–derived distearoyl phosphatidylethanolamine (mPEG2000-DSPE). We used the MTT technique to assess cytotoxicity for various concentrations of co-encapsulated nano-liposomes, free silibinin (25% w/v) and glycyrrhizic acid (75% w/v) on HepG2 and fibroblast cell lines over a 48-hour period. Results Formulation of pegylated nano-liposomes showed a narrow size distribution with an average diameter of 46.3 nm. The encapsulation efficiency (EE) for silibinin was 24.37%, whereas for glycyrrhizic acid it was 68.78%. Results of in vitro cytotoxicity showed significantly greater co-encapsulated nano-liposomes on the HepG2 cell line compared to the fibroblast cell line. The half maximal inhibitory concentration (IC50) for co-encapsulated pegylated nanoliposomal herbal drugs was 48.68 µg/ml and free silibinin with glycyrrhizic acid was 485.45 µg/ml on the HepG2 cell line. Conclusion This in vitro study showed that nano-liposome encapsulation of silibinin with glycyrrhizic acid increased the biological activity of free drugs, increased the stability of silibinin, and synergized the therapeutic effect of silibinin with glycyrrhizic acid. The IC50 of the co-encapsulated nano-liposomes was lower than the combination of free silibinin and glycyrrhizic acid on the HepG2 cell line. PMID:27540518

Nanoindentation characterisation of human colorectal cancer cells considering cell geometry, surface roughness and hyperelastic constitutive behaviour

NASA Astrophysics Data System (ADS)

Boccaccio, Antonio; Uva, Antonio E.; Papi, Massimiliano; Fiorentino, Michele; De Spirito, Marco; Monno, Giuseppe

2017-01-01

Characterisation of the mechanical behaviour of cancer cells is an issue of crucial importance as specific cell mechanical properties have been measured and utilized as possible biomarkers of cancer progression. Atomic force microscopy certainly occupies a prominent place in the field of the mechanical characterisation devices. We developed a hybrid approach to characterise different cell lines (SW620 and SW480) of the human colon carcinoma submitted to nanoindentation measurements. An ad hoc algorithm was written that compares the force-indentation curves experimentally retrieved with those predicted by a finite element model that simulates the nanoindentation process and reproduces the cell geometry and the surface roughness. The algorithm perturbs iteratively the values of the cell mechanical properties implemented in the finite element model until the difference between the experimental and numerical force-indentation curves reaches the minimum value. The occurrence of this indicates that the implemented material properties are very close to the real ones. Different hyperelastic constitutive models, such as Arruda-Boyce, Mooney-Rivlin and Neo-Hookean were utilized to describe the structural behaviour of indented cells. The algorithm was capable of separating, for all the cell lines investigated, the mechanical properties of cell cortex and cytoskeleton. Material properties determined via the algorithm were different with respect to those obtained with the Hertzian contact theory. This demonstrates that factors such as: the cell geometry/anatomy and the hyperelastic constitutive behaviour, which are not contemplated in the Hertz’s theory hypotheses, do affect the nanoindentation measurements. The proposed approach represents a powerful tool that, only on the basis of nanoindentation measurements, is capable of characterising material at the subcellular level.

[Systematic review and Meta-analysis of Shenqi Fuzheng injection combined with first-line chemotherapy for non-small cell lung cancer].

PubMed

Hao, Teng-teng; Xie, Yan-ming; Liao, Xing; Wang, Jing

2015-10-01

The paper is to systematically evaluate the effect and safety of Shenqi Fuzheng injection (SFI) combined with first-line chemotherapy for non-small cell lung cancer (NSCLC). Randomized controlled trials (RCTs) on Shenqi Fuzheng injection (SFI) combined with first-line chemotherapy (experiment group) and chemotherapy alone group ( control group) were electronically retrieved from Medline, EMbase, Clinical Trials, Cochrane Library, CBM, CNKI, VIP, and Wanfang Data base. All trials were assessed for quality according to the Cochrane Reviewer's Handbook for Systematic Reviews of Intervention and then Meta-analysis was performed withRevMan5. 2 Software. A total of 43 RCTs (3433 patients) were included after screening and selecting. Results of Meta-analysis showed that: Objective remission rate (ORR): ORR of experimental group was about 20% higher than that of control group [RR = 1.23, 95% CI (1.11,1.35), P < 0.0001]. Disease control rate (DCR):DCR of SFI combined with first-line chemotherapy was 11% higher than that of first-line chemotherapy alone [RR = 1.11, 95% CI (1.07, 1.16), P < 0.000 01]. Life quality evaluated by Kosovan performance status (KPS) showed that: life quality improvement rate of experimental group was about twice of that in control group [RR = 2.02, 95% CI (1.81, 2.26), P < 0.000 01]. Toxic and side reaction analysis showed that: the incidence of side reactions in experimental group was about 50% lower than that in control group [RR = 0.59, 95% CI (0.53, 0.66), P < 0.000 01]. Immune function test showed that: the function of experimental group was 3.2 (standard deviations) times greater than that of control group [MD = 3.23, 95% CI (2.86, 3.60), P < 0.000 01]. We can see that SFI combined with first-line chemotherapy for NSCLC can increase objective efficacy, improve life quality, decrease toxic and side reactionsinduced by chemotherapy, and improve the immune functions. As most of the included studies in this systematic evaluation had poor quality, the evidence to support conclusion was weak, so it was necessary to conduct more multi-center clinical trials with high quality methods and rigorous design.

Effect of Berry Extracts and Bioactive Compounds on Fulvestrant (ICI 182,780) Sensitive and Resistant Cell Lines.

PubMed

Woode, Denzel R; Aiyer, Harini S; Sie, Nicole; Zwart, Alan L; Li, Liya; Seeram, Navindra P; Clarke, Robert

2012-01-01

Fulvestrant (ICI 182,780; ICI) is approved for the treatment of advanced metastatic breast cancer that is unresponsive to other endocrine therapies. Berries are frequently consumed for their antioxidant, anti-inflammatory, and anticancer potential. In this study, we tested the efficacy of two berry extracts (Jamun-EJAE and red raspberry-RRE) and their bioactive compounds (Delphinidin-Del and Ellagic acid-EA) to inhibit cell proliferation with or without a sublethal dose of ICI in various breast cancer cell lines. ICI-sensitive (LCC1, ZR75-1, and BT474) and -resistant (LCC9, ZR75-1R) cells were subjected to treatment with berry extracts alone (0.1-100 μg/mL) or with a sub-lethal dose of ICI ( 1). EA, in doses tested, did not have any significant effects on any of the cell lines. Finally, we found that the extracts were more effective at lower, physiologically relevant concentrations than at higher experimental doses.

Effect of Berry Extracts and Bioactive Compounds on Fulvestrant (ICI 182,780) Sensitive and Resistant Cell Lines

PubMed Central

Woode, Denzel R.; Aiyer, Harini S.; Sie, Nicole; Zwart, Alan L.; Li, Liya; Seeram, Navindra P.; Clarke, Robert

2012-01-01

Fulvestrant (ICI 182,780; ICI) is approved for the treatment of advanced metastatic breast cancer that is unresponsive to other endocrine therapies. Berries are frequently consumed for their antioxidant, anti-inflammatory, and anticancer potential. In this study, we tested the efficacy of two berry extracts (Jamun-EJAE and red raspberry-RRE) and their bioactive compounds (Delphinidin-Del and Ellagic acid-EA) to inhibit cell proliferation with or without a sublethal dose of ICI in various breast cancer cell lines. ICI-sensitive (LCC1, ZR75-1, and BT474) and -resistant (LCC9, ZR75-1R) cells were subjected to treatment with berry extracts alone (0.1–100 μg/mL) or with a sub-lethal dose of ICI ( 1). EA, in doses tested, did not have any significant effects on any of the cell lines. Finally, we found that the extracts were more effective at lower, physiologically relevant concentrations than at higher experimental doses. PMID:23346406

Experimental Spectroscopic Studies of Carbon Monoxide (CO) Fluorescence at High Temperatures and Pressures.

PubMed

Carrivain, Olivier; Orain, Mikael; Dorval, Nelly; Morin, Celine; Legros, Guillaume

2017-10-01

Two-photon excitation laser-induced fluorescence of carbon monoxide (CO-LIF) is investigated experimentally in order to determine the applicability of this technique for imaging CO concentration in aeronautical combustors. Experiments are carried out in a high temperature, high-pressure test cell, and in a laminar premixed CH 4 /air flame. Influence of temperature and pressure on CO-LIF spectra intensity and shape is reported. The experimental results show that as pressure increases, the CO-LIF excitation spectrum becomes asymmetric. Additionally, the spectrum strongly shifts to the red with a quadratic dependence of the collisional shift upon pressure, which is different from the classical behavior where the collisional shift is proportional to pressure. Moreover, pressure line broadening cannot be reproduced by a Lorenztian profile in the temperature range investigated here (300-1750 K) and, therefore, an alternative line shape is suggested.

Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

PubMed

Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

2017-07-01

Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels drives accumulation of immunostimulatory ATP versus immunosuppressive adenosine within the tumor microenvironment. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

PubMed Central

Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

2017-01-01

Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Yang, E-mail: muyang@nwsuaf.edu.cn; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture of the People's Republic of China, No. 22 Xinong Road, Yangling, Shaanxi 712100; Li, Liangliang, E-mail: lifeiyang2007@126.com

Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5{sup Δ84-96} (aa 84-96 deletion), and GP5{sup Δ97-119} (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5{sup Δ97-119}, but not full-length or GP5{sup Δ84-96}, induced a cell cycle arrest at the G2/M phasemore » resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5{sup Δ84-96} inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5{sup Δ97-119} expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5{sup Δ84-96} was significantly inhibited.« less

Central projections of the lateral line and eighth nerves in the bowfin, Amia calva.

PubMed

McCormick, C A

1981-03-20

The first-order connections of the anterior and posterior lateral line nerves and of the eighth nerve were determined in the bowfin, Amia calva, using experimental degeneration and anterograde HRP transport techniques. The termination sites of these nerves define a dorsal lateralis cell column and a ventral octavus cell column. The anterior and posterior lateralis nerves distribute ipsilaterally to two medullary nuclei-nucleus medialis and nucleus caudalis. Nucleus medialis comprises the rostral two-thirds of the lateralis column and contains large, Purkinje-like cells dorsally and polygonal, granule, and fusiform cells ventrally. Nucleus caudalis is located posterior to nucleus medialis and consists of small, granule cells. Anterior lateralis fibers terminate ventrally to ventromedially in both nucleus medialis and nucleus caudalis. Posterior lateralis fibers terminate dorsally to dorsolaterally within these two nuclei. A sparse anterior lateralis input may also be present on the dendrites of one of the nuclei within the octavus cell column, nucleus magnocellularis. In contrast, the anterior and posterior rami of the eighth nerve each terminate within four medullary nuclei which comprise the octavus cell column: the anterior, magnocellular, descending, and posterior octavus nuclei. An eighth nerve projection to the medial reticular formation is also present. Some fibers of the lateralis and eighth nerves terminate within the ipsilateral eminentia granularis of the cerebellum. Lateralis fibers distribute to approximately the lateral half of this structure with posterior lateral line fibers terminating laterally and anterior lateral line fibers terminating medially. Eighth nerve fibers distribute to the medial half of the eminentia granularis.

Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

PubMed

Rozzo, Carla; Sanna, Daniele; Garribba, Eugenio; Serra, Maria; Cantara, Alessio; Palmieri, Giuseppe; Pisano, Marina

2017-09-01

In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [V IV O(dhp) 2 ] where dhp - is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [V IV O(mpp) 2 ] where mpp - is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [V IV O(ppp) 2 ] where ppp - is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC 50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC 50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

Artificial fish skin of self-powered micro-electromechanical systems hair cells for sensing hydrodynamic flow phenomena.

PubMed

Asadnia, Mohsen; Kottapalli, Ajay Giri Prakash; Miao, Jianmin; Warkiani, Majid Ebrahimi; Triantafyllou, Michael S

2015-10-06

Using biological sensors, aquatic animals like fishes are capable of performing impressive behaviours such as super-manoeuvrability, hydrodynamic flow 'vision' and object localization with a success unmatched by human-engineered technologies. Inspired by the multiple functionalities of the ubiquitous lateral-line sensors of fishes, we developed flexible and surface-mountable arrays of micro-electromechanical systems (MEMS) artificial hair cell flow sensors. This paper reports the development of the MEMS artificial versions of superficial and canal neuromasts and experimental characterization of their unique flow-sensing roles. Our MEMS flow sensors feature a stereolithographically fabricated polymer hair cell mounted on Pb(Zr(0.52)Ti(0.48))O3 micro-diaphragm with floating bottom electrode. Canal-inspired versions are developed by mounting a polymer canal with pores that guide external flows to the hair cells embedded in the canal. Experimental results conducted employing our MEMS artificial superficial neuromasts (SNs) demonstrated a high sensitivity and very low threshold detection limit of 22 mV/(mm s(-1)) and 8.2 µm s(-1), respectively, for an oscillating dipole stimulus vibrating at 35 Hz. Flexible arrays of such superficial sensors were demonstrated to localize an underwater dipole stimulus. Comparative experimental studies revealed a high-pass filtering nature of the canal encapsulated sensors with a cut-off frequency of 10 Hz and a flat frequency response of artificial SNs. Flexible arrays of self-powered, miniaturized, light-weight, low-cost and robust artificial lateral-line systems could enhance the capabilities of underwater vehicles. © 2015 The Author(s).

Artificial fish skin of self-powered micro-electromechanical systems hair cells for sensing hydrodynamic flow phenomena

PubMed Central

Asadnia, Mohsen; Kottapalli, Ajay Giri Prakash; Miao, Jianmin; Warkiani, Majid Ebrahimi; Triantafyllou, Michael S.

2015-01-01

Using biological sensors, aquatic animals like fishes are capable of performing impressive behaviours such as super-manoeuvrability, hydrodynamic flow ‘vision’ and object localization with a success unmatched by human-engineered technologies. Inspired by the multiple functionalities of the ubiquitous lateral-line sensors of fishes, we developed flexible and surface-mountable arrays of micro-electromechanical systems (MEMS) artificial hair cell flow sensors. This paper reports the development of the MEMS artificial versions of superficial and canal neuromasts and experimental characterization of their unique flow-sensing roles. Our MEMS flow sensors feature a stereolithographically fabricated polymer hair cell mounted on Pb(Zr0.52Ti0.48)O3 micro-diaphragm with floating bottom electrode. Canal-inspired versions are developed by mounting a polymer canal with pores that guide external flows to the hair cells embedded in the canal. Experimental results conducted employing our MEMS artificial superficial neuromasts (SNs) demonstrated a high sensitivity and very low threshold detection limit of 22 mV/(mm s−1) and 8.2 µm s−1, respectively, for an oscillating dipole stimulus vibrating at 35 Hz. Flexible arrays of such superficial sensors were demonstrated to localize an underwater dipole stimulus. Comparative experimental studies revealed a high-pass filtering nature of the canal encapsulated sensors with a cut-off frequency of 10 Hz and a flat frequency response of artificial SNs. Flexible arrays of self-powered, miniaturized, light-weight, low-cost and robust artificial lateral-line systems could enhance the capabilities of underwater vehicles. PMID:26423435

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9.

PubMed

Yumlu, Saniye; Stumm, Jürgen; Bashir, Sanum; Dreyer, Anne-Kathrin; Lisowski, Pawel; Danner, Eric; Kühn, Ralf

2017-05-15

Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones. Copyright © 2017 Elsevier Inc. All rights reserved.

Computational Biology Methods for Characterization of Pluripotent Cells.

PubMed

Araúzo-Bravo, Marcos J

2016-01-01

Pluripotent cells are a powerful tool for regenerative medicine and drug discovery. Several techniques have been developed to induce pluripotency, or to extract pluripotent cells from different tissues and biological fluids. However, the characterization of pluripotency requires tedious, expensive, time-consuming, and not always reliable wet-lab experiments; thus, an easy, standard quality-control protocol of pluripotency assessment remains to be established. Here to help comes the use of high-throughput techniques, and in particular, the employment of gene expression microarrays, which has become a complementary technique for cellular characterization. Research has shown that the transcriptomics comparison with an Embryonic Stem Cell (ESC) of reference is a good approach to assess the pluripotency. Under the premise that the best protocol is a computer software source code, here I propose and explain line by line a software protocol coded in R-Bioconductor for pluripotency assessment based on the comparison of transcriptomics data of pluripotent cells with an ESC of reference. I provide advice for experimental design, warning about possible pitfalls, and guides for results interpretation.

Downregulated TIPE2 is associated with poor prognosis and promotes cell proliferation in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yuexia; Li, Xiaohui; Liu, Gang

2015-01-30

Highlights: • TIPE2 is down-regulated in NSCLC tissues. • TIPE2 inhibits NSCLC cell proliferation, colony formation and invasion. • TIPE2 reduces the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. - Abstract: The present study aims to investigate the expression pattern of TIPE2 protein and its clinical significance in human non-small cell lung cancer (NSCLC). We investigated the expression levels of TIPE2 in 96 NSCLC tumor samples by immunohistochemistry and then analyzed its clinical significance. Furthermore, the role of TIPE2 on the biological properties of the NSCLC cell line H1299 and A549 was experimentally tested in vitro and in vivo.more » We found that the expression level of TIPE2 was significantly higher in normal lung tissues compared with NSCLC tissues (P < 0.001), and TIPE2 downregulation was significantly correlated with advanced TNM stage (P = 0.006). TIPE2 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TIPE2 plasmid was performed in H1299 and A549 cells. TIPE2 overexpression inhibited lung cancer cell proliferation, colony formation and cell invasive in vitro, and prevented lung tumor growth in vivo. In addition, TIPE2 transfection reduced the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. Taken together, our results demonstrate that TIPE2 might serve as a tumor suppressor in NSCLC progression.« less

NMR-driven identification of anti-amyloidogenic compounds in green and roasted coffee extracts.

PubMed

Ciaramelli, Carlotta; Palmioli, Alessandro; De Luigi, Ada; Colombo, Laura; Sala, Gessica; Riva, Chiara; Zoia, Chiara Paola; Salmona, Mario; Airoldi, Cristina

2018-06-30

To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aβ molecules in natural edible matrices. We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents. Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aβ on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line. Copyright © 2018 Elsevier Ltd. All rights reserved.

OGEE v2: an update of the online gene essentiality database with special focus on differentially essential genes in human cancer cell lines.

PubMed

Chen, Wei-Hua; Lu, Guanting; Chen, Xiao; Zhao, Xing-Ming; Bork, Peer

2017-01-04

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Induction of apoptosis in human multiple myeloma cell lines by ebselen via enhancing the endogenous reactive oxygen species production.

PubMed

Zhang, Liang; Zhou, Liwei; Du, Jia; Li, Mengxia; Qian, Chengyuan; Cheng, Yi; Peng, Yang; Xie, Jiayin; Wang, Dong

2014-01-01

Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM) cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS) accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC) completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

PubMed

Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

2016-01-01

We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

In vitro downregulated hypoxia transcriptome is associated with poor prognosis in breast cancer.

PubMed

Abu-Jamous, Basel; Buffa, Francesca M; Harris, Adrian L; Nandi, Asoke K

2017-06-15

Hypoxia is a characteristic of breast tumours indicating poor prognosis. Based on the assumption that those genes which are up-regulated under hypoxia in cell-lines are expected to be predictors of poor prognosis in clinical data, many signatures of poor prognosis were identified. However, it was observed that cell line data do not always concur with clinical data, and therefore conclusions from cell line analysis should be considered with caution. As many transcriptomic cell-line datasets from hypoxia related contexts are available, integrative approaches which investigate these datasets collectively, while not ignoring clinical data, are required. We analyse sixteen heterogeneous breast cancer cell-line transcriptomic datasets in hypoxia-related conditions collectively by employing the unique capabilities of the method, UNCLES, which integrates clustering results from multiple datasets and can address questions that cannot be answered by existing methods. This has been demonstrated by comparison with the state-of-the-art iCluster method. From this collection of genome-wide datasets include 15,588 genes, UNCLES identified a relatively high number of genes (>1000 overall) which are consistently co-regulated over all of the datasets, and some of which are still poorly understood and represent new potential HIF targets, such as RSBN1 and KIAA0195. Two main, anti-correlated, clusters were identified; the first is enriched with MYC targets participating in growth and proliferation, while the other is enriched with HIF targets directly participating in the hypoxia response. Surprisingly, in six clinical datasets, some sub-clusters of growth genes are found consistently positively correlated with hypoxia response genes, unlike the observation in cell lines. Moreover, the ability to predict bad prognosis by a combined signature of one sub-cluster of growth genes and one sub-cluster of hypoxia-induced genes appears to be comparable and perhaps greater than that of known hypoxia signatures. We present a clustering approach suitable to integrate data from diverse experimental set-ups. Its application to breast cancer cell line datasets reveals new hypoxia-regulated signatures of genes which behave differently when in vitro (cell-line) data is compared with in vivo (clinical) data, and are of a prognostic value comparable or exceeding the state-of-the-art hypoxia signatures.

Infrared Spectrum of Methanol: A First-Year Student Experiment.

ERIC Educational Resources Information Center

Boehm, Garth; Dwyer, Mark

1981-01-01

Describes an experiment providing an experimental introduction to vibrational spectroscopy and experience in using an elementary vacuum line. The experiment, using a gas cell charged with methanol, is completed in a three-hour laboratory period and is directed toward understanding vibrational spectroscopy rather than the diagnostic value of the…

Cell wall composition and biomass recalcitrance differences within a set of Brachypodium distachyon inbred lines

USDA-ARS?s Scientific Manuscript database

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species, owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. We assessed the extent of natural variation for tr...

Evaluating the effect of four extracts of avocado fruit on esophageal squamous carcinoma and colon adenocarcinoma cell lines in comparison with peripheral blood mononuclear cells.

PubMed

Vahedi Larijani, Laleh; Ghasemi, Maryam; AbedianKenari, Saeid; Naghshvar, Farshad

2014-01-01

Most patients with gastrointestinal cancers refer to the health centers at advanced stages of the disease and conventional treatments are not significantly effective for these patients. Therefore, using modern therapeutic approaches with lower toxicity bring higher chance for successful treatment and reduced adverse effects in such patients. The aim of this study is to evaluate the effect of avocado fruit extracts on inhibition of the growth of cancer cells in comparison with normal cells. In an experimental study, ethanol, chloroform, ethyl acetate, and petroleum extracts of avocado (Persea americana) fruit were prepared. Then, the effects if the extracts on the growth of esophageal squamous cell carcinoma and colon adenocarcinoma cell lines were evaluated in comparison with the control group using the MTT test in the cell culture medium. Effects of the four extracts of avocado fruit on three cells lines of peripheral blood mononuclear cells, esophageal squamous cell carcinoma, and colon adenocarcinoma were tested. The results showed that avocado fruit extract is effective in inhibition of cancer cell growth in comparison with normal cells (P<0.05). Avocado fruit is rich in phytochemicals, which play an important role in inhibition of growth of cancer cells. The current study for the first time demonstrates the anti-cancer effect of avocado fruit extracts on two cancers common in Iran. Therefore, it is suggested that the fruit extracts can be considered as appropriate complementary treatments in treatment of esophageal and colon cancers.

Effect of duct shape, Mach number, and lining construction on measured suppressor attenuation and comparison with theory

NASA Technical Reports Server (NTRS)

Olsen, W. A.; Krejsa, E. A.; Coats, J. W.

1972-01-01

Noise attenuation was measured for several types of cylindrical suppressors that use a duct lining composed of honeycomb cells covered with a perforated plate. The experimental technique used gave attenuation data that were repeatable and free of noise floors and other sources of error. The suppressor length, the effective acoustic diameter, suppressor shape and flow velocity were varied. The agreement among the attenuation data and two widely used analytical models was generally satisfactory. Changes were also made in the construction of the acoustic lining to measure their effect on attenuation. One of these produced a very broadband muffler.

Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris.

PubMed

Sweat JMDunigan, D D; Wright, S D

2001-06-01

The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.

A comparison of the in vitro cytotoxicity of conventional and resin modified glass ionomer cements

PubMed Central

Selimović-Dragaš, Mediha; Huseinbegović, Amina; Kobašlija, Sedin; Hatibović-Kofman, Šahza

2012-01-01

To evaluate cytotoxicity of experimental conventional and resin modified glass-ionomer cements on UMR-106 osteoblast cell cultures and cell cultures of NIH3T3 mouse fibroblasts specimens were prepared for every experimental material and divided into: group 1. Conventional glass-ionomer cements: GC Fuji IX GP Fast, GC Fuji Triage and Ketac Silver; group 2. Resin modified glass-ionomer cements: GC Fuji II LC, GC Fuji Plus and Vitrebond; group 3. Positive control was presented by specimens of composite Vit-l-ecence® and negative control-group 4. was presented by α-minimum essential medium for UMR-106 – osteoblast-like cells and Dulbecco’s Modified Eagle’s Medium for NIH3T3 mouse fibroblast cells. Both cell cultures were exposed to 10% of eluate of each single specimen of each experimental material. Experimental dishes were incubated for 24 h. Cell metabolism was evaluated using methyltetrazolium assay. Kruskal-Wallis test and Tukey-Kramer post hoc test for the materials evaluated on NIH3T3 mouse fibroblast cells, as well as UMR-106 osteoblast-like cells showed significantly more cytotoxicity of RMGICs, predominantly Vitrebond to both GICs and composite-Vit-l-ecence®. The lowest influence on cell’s metabolism on UMR-106 osteoblas-like cells was shown by Ketac Silver and the lowest influence on cell’s metabolism on NIH3T3 mouse fibroblast cells was shown by Fuji IX GP Fast. Statistical evaluation of sensitivity of cell lines UMR-106 osteoblast-like cells and NIH3T3 mouse fibroblast cells, using Mann-Whitney test, showed that NIH3T3 mouse fibroblast cells were more sensitive for the evaluation of cytotoxicity of dental materials. PMID:23198945

Cellular dosimetry of (111)In using monte carlo N-particle computer code: comparison with analytic methods and correlation with in vitro cytotoxicity.

PubMed

Cai, Zhongli; Pignol, Jean-Philippe; Chan, Conrad; Reilly, Raymond M

2010-03-01

Our objective was to compare Monte Carlo N-particle (MCNP) self- and cross-doses from (111)In to the nucleus of breast cancer cells with doses calculated by reported analytic methods (Goddu et al. and Farragi et al.). A further objective was to determine whether the MCNP-predicted surviving fraction (SF) of breast cancer cells exposed in vitro to (111)In-labeled diethylenetriaminepentaacetic acid human epidermal growth factor ((111)In-DTPA-hEGF) could accurately predict the experimentally determined values. MCNP was used to simulate the transport of electrons emitted by (111)In from the cell surface, cytoplasm, or nucleus. The doses to the nucleus per decay (S values) were calculated for single cells, closely packed monolayer cells, or cell clusters. The cell and nucleus dimensions of 6 breast cancer cell lines were measured, and cell line-specific S values were calculated. For self-doses, MCNP S values of nucleus to nucleus agreed very well with those of Goddu et al. (ratio of S values using analytic methods vs. MCNP = 0.962-0.995) and Faraggi et al. (ratio = 1.011-1.024). MCNP S values of cytoplasm and cell surface to nucleus compared fairly well with the reported values (ratio = 0.662-1.534 for Goddu et al.; 0.944-1.129 for Faraggi et al.). For cross doses, the S values to the nucleus were independent of (111)In subcellular distribution but increased with cluster size. S values for monolayer cells were significantly different from those of single cells and cell clusters. The MCNP-predicted SF for monolayer MDA-MB-468, MDA-MB-231, and MCF-7 cells agreed with the experimental data (relative error of 3.1%, -1.0%, and 1.7%). The single-cell and cell cluster models were less accurate in predicting the SF. For MDA-MB-468 cells, relative error was 8.1% using the single-cell model and -54% to -67% using the cell cluster model. Individual cell-line dimensions had large effects on S values and were needed to estimate doses and SF accurately. MCNP simulation compared well with the reported analytic methods in the calculation of subcellular S values for single cells and cell clusters. Application of a monolayer model was most accurate in predicting the SF of breast cancer cells exposed in vitro to (111)In-DTPA-hEGF.

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudzok, S., E-mail: susanne.rudzok@ufz.d; Schlink, U., E-mail: uwe.schlink@ufz.d; Herbarth, O., E-mail: olf.herbarth@medizin.uni-leipzig.d

2010-05-01

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-likemore » enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.« less

Cell reprogramming modelled as transitions in a hierarchy of cell cycles

NASA Astrophysics Data System (ADS)

Hannam, Ryan; Annibale, Alessia; Kühn, Reimer

2017-10-01

We construct a model of cell reprogramming (the conversion of fully differentiated cells to a state of pluripotency, known as induced pluripotent stem cells, or iPSCs) which builds on key elements of cell biology viz. cell cycles and cell lineages. Although reprogramming has been demonstrated experimentally, much of the underlying processes governing cell fate decisions remain unknown. This work aims to bridge this gap by modelling cell types as a set of hierarchically related dynamical attractors representing cell cycles. Stages of the cell cycle are characterised by the configuration of gene expression levels, and reprogramming corresponds to triggering transitions between such configurations. Two mechanisms were found for reprogramming in a two level hierarchy: cycle specific perturbations and a noise induced switching. The former corresponds to a directed perturbation that induces a transition into a cycle-state of a different cell type in the potency hierarchy (mainly a stem cell) whilst the latter is a priori undirected and could be induced, e.g. by a (stochastic) change in the cellular environment. These reprogramming protocols were found to be effective in large regimes of the parameter space and make specific predictions concerning reprogramming dynamics which are broadly in line with experimental findings.

Immunomodulation of Experimental Autoimmune Encephalomyelitis by Oral Administration of Copolymer 1

NASA Astrophysics Data System (ADS)

Teitelbaum, Dvora; Arnon, Ruth; Sela, Michael

1999-03-01

The activity of copolymer 1 (Cop 1, Copax-one, glatiramer acetate) in suppressing experimental autoimmune encephalomyelitis (EAE) and in the treatment of multiple sclerosis patients when injected parenterally has been extensively demonstrated. In the present study we addressed the question of whether Cop 1 can induce oral tolerance to EAE similar to myelin basic protein (MBP). We now have demonstrated that oral Cop 1 inhibited EAE induction in both rats and mice. Furthermore, oral Cop 1 was more effective than oral MBP in suppressing EAE in rats. The beneficial effect of oral Cop 1 was found to be associated with specific inhibition of the proliferative and Th1 cytokine secretion responses to MBP of spleen cells from Cop 1-fed mice and rats. In all of these assays, oral Cop 1 was more effective than oral MBP. The tolerance induced by Cop 1 could be adoptively transferred with spleen cells from Cop 1-fed animals. Furthermore, Cop 1-specific T cell lines, which inhibit EAE induction in vivo, could be isolated from the above spleen cells. These T cell lines secrete the anti-inflammatory cytokines IL-10 and transforming growth factor type β , but not IL-4, in response to both Cop 1 and MBP. In conclusion, oral Cop 1 has a beneficial effect on the development of EAE that is associated with down-regulation of T cell immune responses to MBP and is mediated by Th2/3 type regulatory cells. These results suggest that oral administration of Cop 1 may modulate multiple sclerosis as well.

Probing cytoskeleton organisation of neuroblastoma cells with single-cell force spectroscopy.

PubMed

Mescola, Andrea; Vella, Serena; Scotto, Marco; Gavazzo, Paola; Canale, Claudio; Diaspro, Alberto; Pagano, Aldo; Vassalli, Massimo

2012-05-01

Single-cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life-threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells. Copyright © 2012 John Wiley & Sons, Ltd.

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells

PubMed Central

Bressan, Raul Bardini; Dewari, Pooran Singh; Kalantzaki, Maria; Gangoso, Ester; Matjusaitis, Mantas; Garcia-Diaz, Claudia; Blin, Carla; Grant, Vivien; Bulstrode, Harry; Gogolok, Sabine; Skarnes, William C.

2017-01-01

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis. PMID:28096221

Steroids from the rhizome of Anemarrhena asphodeloides and their cytotoxic activities.

PubMed

Sun, Yu; Wu, Jie; Sun, Xue; Huang, Xiaoxiao; Li, Lingzhi; Liu, Qingbo; Song, ShaoJiang

2016-07-01

Cancer remains a major killer worldwide. To search for novel naturally occurring compounds that are cytotoxic to cancer cells to be used as lead structures for drug development, five new steroids (1-5) along with seven known ones (6-12) were isolated from the rhizome of Anemarrhena asphodeloides Bge. Their structures were established by detailed spectral studies, including 1D-NMR, 2D-NMR, HR-ESI-MS and by comparison with literature data. These compounds exhibited different levels of growth inhibition against A549, HepG2, Hep3B, Bcap37 and MCF7 cell lines in vitro. Compounds 9, 10 and 11 showed potent inhibitory against all the tested cell lines with IC50 values ranging from 0.35±0.15 to 25.53±0.31μM. The three compounds displayed stronger inhibitory activities against A549, HepG2 and Hep3B cell lines compared with the positive control 5-fluorouracil. The experimental data obtained permit us to identify the roles of the sugar moieties, hydroxyl group, double bond and F-ring with regard to their cytotoxic activities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Experimental Procedure for Determination of the Dielectric Properties of Biological Samples in the 2-50 GHz Range

PubMed Central

Odelstad, Elias; Raman, Sujith; Rydberg, Anders

2014-01-01

The objective of this paper was to test and evaluate an experimental procedure for providing data on the complex permittivity of different cell lines in the 2–50-GHz range at room temperature, for the purpose of future dosimetric studies. The complex permittivity measurements were performed on cells suspended in culture medium using an open-ended coaxial probe. Maxwell’s mixture equation then allows the calculation of the permittivity profiles of the cells from the difference in permittivity between the cell suspensions and pure culture medium. The open-ended coaxial probe turned out to be very sensitive to disturbances affecting the measurements, resulting in poor precision. Permittivity differences were not large in relation to the spread of the measurements and repeated measurements were performed to improve statistics. The 95% confidence intervals were computed for the arithmetic means of the measured permittivity differences in order to test the statistical significance. The results showed that for bone cells at the lowest tested concentration (33 500/ml), there were significance in the real part of the permittivity at frequencies above 30 GHz, and no significance in the imaginary part. For the second lowest concentration (67 000/ml) there was no significance at all. For a medium concentration of bone cells (135 000/ml) there was no significance in the real part, but there was significance in the imaginary part at frequencies below about 25 GHz. The cell suspension with a concentration of 1 350 000/ml had significance in the real part for both high (above 30 GHz) and low (below 15 GHz) frequencies. The imaginary part showed significance for frequencies below 25 GHz. In the case of an osteosarcoma cell line with a concentration of 2 700 000/ml, only the imaginary part showed significance, and only for frequencies below 15 GHz. For muscle cells at a concentration of 743 450/ml, there was only significance in the imaginary part for frequencies below 5 GHz. The experimental data indicated that the complex permittivity of the culture medium may be used for modeling of cell suspensions. PMID:27170886

Differences in Chlamydia trachomatis Serovar E Growth Rate in Polarized Endometrial and Endocervical Epithelial Cells Grown in Three-Dimensional Culture▿

PubMed Central

Guseva, Natalia V.; Dessus-Babus, Sophie; Moore, Cheryl G.; Whittimore, Judy D.; Wyrick, Priscilla B.

2007-01-01

In vitro studies of obligate intracellular chlamydia biology and pathogenesis are highly dependent on the use of experimental models and growth conditions that mimic the mucosal architecture and environment these pathogens encounter during natural infections. In this study, the growth of Chlamydia trachomatis genital serovar E was monitored in mouse fibroblast McCoy cells and compared to more relevant host human epithelial endometrium-derived HEC-1B and cervix-derived HeLa cells, seeded and polarized on collagen-coated microcarrier beads, using a three-dimensional culture system. Microscopy analysis of these cell lines prior to infection revealed morphological differences reminiscent of their in vivo architecture. Upon infection, early chlamydial inclusion distribution was uniform in McCoy cells but patchy in both epithelial cell lines. Although no difference in chlamydial attachment to or entry into the two genital epithelial cell lines was noted, active bacterial genome replication and transcription, as well as initial transformation of elementary bodies to reticulate bodies, were detected earlier in HEC-1B than in HeLa cells, suggesting a faster growth, which led to higher progeny counts and titers in HEC-1B cells upon completion of the developmental cycle. Chlamydial development in the less relevant McCoy cells was very similar to that in HeLa cells, although higher progeny counts were obtained. In conclusion, this three-dimensional bead culture system represents an improved model for harvesting large quantities of infectious chlamydia progeny from their more natural polarized epithelial host cells. PMID:17088348

Microwave magnetic field detection based on Cs vapor cell in free space

NASA Astrophysics Data System (ADS)

Liu, Xiaochi; Jiang, Zhiyuan; Qu, Jifeng; Hou, Dong; Huang, Xianhe; Sun, Fuyu

2018-06-01

In this study, we demonstrate the direct measurement of a microwave (MW) magnetic field through the detection of atomic Rabi resonances with Cs vapor cells in a free-space low-Q cavity. The line shape (amplitude and linewidth) of detected Rabi resonances is investigated versus several experimental parameters such as the laser intensity, cell buffer gas pressure, and cell length. The specially designed low-Q cavity creates a suitable MW environment allowing easy testing of different vapor cells with distinct properties. Obtained results are analyzed to optimize the performances of a MW magnetic field sensor based on the present atom-based detection technique.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

PubMed

Olivier, Emmanuel N; Qiu, Caihong; Velho, Michelle; Hirsch, Rhoda Elison; Bouhassira, Eric E

2006-12-01

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Wall effects in continuous microfluidic magneto-affinity cell separation.

PubMed

Wu, Liqun; Zhang, Yong; Palaniapan, Moorthi; Roy, Partha

2010-05-01

Continuous microfluidic magneto-affinity cell separator combines unique microscale flow phenomenon with advantageous nanobead properties, to isolate cells with high specificity. Owing to the comparable size of the cell-bead complexes and the microchannels, the walls of the microchannel exert a strong influence on the separation of cells by this method. We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid shear forces. Separation of a human colon carcinoma cell line from a mixture of red blood cells, with folic acid conjugated 1 microm and 200 nm beads, is reported.

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

PubMed

El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

2018-02-01

Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Experimental results using 3-bromopyruvate in mesothelioma: in vitro and in vivo studies.

PubMed

Icard, Philippe; Philippe, Icard; Zhang, Xiao-Dong; Xiao-Dong, Zhang; Lemoisson, Edwige; Edwige, Lemoisson; Louis, Marie-Hélène; Marie-Hélène, Louis; Allouche, Stéphane; Stéphane, Allouche; Lincet, Hubert; Hubert, Lincet; Poulain, Laurent; Laurent, Poulain

2012-02-01

Over many years we have taken advantage of the special metabolism of cancer cells involving an increased consumption of glucose associated with lactic acid production even in the presence of oxygen, a phenomenon referred to as the "Warburg effect", to counteract cancer cell growth. We have tested 3-bromopyruvate (3-BrPA), an inhibitor of pyruvate-associated reactions. Firstly, we tested this agent, in vitro, in two mesothelioma cell lines. Cellular response would appear to depend on the mode of administration (immediately or 24 h after seeding). Depending on the line, 3-BrPA induced a cytostatic or cytotoxic effect. This effect was accompanied by cell death induction even in cells highly refractory to cisplatin. Mitochondrial apoptotic death appeared to involve both lines; however, a different death pathway such as necrosis cannot be excluded. Interestingly, 3-BrPA leads to a diminution of the expression of the anti-apotptoic protein Mcl-1. We then tested 3-BrPA in vivo. Survival of nude mice bearing human mesothelioma was significantly prolonged (p < 0.0001). Toxicity and clinical studies should be performed to test 3- BrPA as local therapy for patients suffering from pleural or peritoneal mesothelioma. Association with cisplatin should be particularly considered.

Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts.

PubMed

Bigović, Dubravka; Savikin, Katarina; Janković, Teodora; Menković, Nebojsa; Zdunić, Gordana; Stanojković, Tatjana; Djurić, Zorica

2011-06-01

Flowers of Helichrysum plicatum were extracted under different experimental conditions, and their antioxidant activity was determined by DPPH radical scavenging assay. Extracts obtained with higher concentration of ethyl acetate (90% or 100%) were found to contain the greatest amount of total phenolics (> 250 mg gallic acid equivalents/g of dried extract), and high correlation between total phenolic content and antiradical activity was observed (r = -0.79). Based on the total phenolic content and antiradical activity, some extracts were selected for investigation of cytotoxic activity toward PC3, HeLa and K562 human cancer cell lines in vitro. All tested extracts exhibited moderate activity against HeLa cells (41.9-42.1 microg/mL), whereas the extract obtained with 100% ethyl acetate was the most active against K562 and PC3 cell lines (25.9 and 39.2 microg/mL, respectively). Statistical analysis revealed significant correlation between total phenolic content and cytotoxic activity against PC3 and K562 cells. HPLC identification of phenolic compounds from the extracts indicated the presence of apigenin, naringenin and kaempferol as free aglycones, and glycosides of apigenin, naringenin, quercetin and kaempferol. Among aglycones, kaempferol displayed moderate cytostatic activity against all cell lines (24.8-64.7 microM).

A Carotenoid Extract from a Southern Italian Cultivar of Pumpkin Triggers Nonprotective Autophagy in Malignant Cells

PubMed Central

Spagnuolo, Carmela; Durante, Miriana; Mita, Giovanni; Aquino, Rita Patrizia

2017-01-01

Carotenoids, including β-carotene, lycopene, and derivatives, such as retinoic acid, have been studied for their significant antiproliferative and differentiating activity on cancer cells in experimental models and in clinics. We are presenting here data on the mechanism of action of a carotenoid-enriched extract obtained from the pumpkin Cucurbita moschata, variety “long of Naples,” on two malignant human cell lines, Caco-2 and SAOs, derived from a colon adenocarcinoma and an osteosarcoma, respectively. The carotenoid extract has been obtained from pumpkin pulp and seeds by supercritical CO2 extraction and employed to prepare oil-in-water nanoemulsions. The nanoemulsions, applied at a final carotenoid concentration of 200–400 μg/ml, were not cytotoxic, but induced a delay in cell growth of about 40% in both SAOs and Caco-2 cell lines. This effect was associated with the activation of a “nonprotective” form of autophagy and, in SAOs cells, to the induction of cell differentiation via a mechanism that involved AMPK activation. Our data suggest the presence of a pool of bioactive compounds in the carotenoid-enriched extract, acting additively, or synergistically, to delay cell growth in cancer cells. PMID:29430284

Thyroid Hormone Differentially Modulates Warburg Phenotype in Breast Cancer Cells

PubMed Central

Suhane, Sonal; Ramanujan, V Krishnan

2011-01-01

Sustenance of cancer cells in vivo critically depends on a variety of genetic and metabolic adaptations. Aerobic glycolysis or Warburg effect has been a defining biochemical hallmark of transformed cells for more than five decades although a clear molecular basis of this observation is emerging only in recent years. In this study, we present our findings that thyroid hormone exerts its non-genomic and genomic actions in two model human breast cancer cell lines differentially. By laying a clear foundation for experimentally monitoring the Warburg phenotype in living cancer cells, we demonstrate that thyroid hormone-induced modulation of bioenergetic profiles in these two model cell lines depends on the degree of Warburg phenotype that they display. Further we also show that thyroid hormone can sensitize mitochondria in aggressive, triple-negative breast cancer cells favorably to increase the chemotherapeutic efficacy in these cells. Even though the role of thyroid hormone in modulating mitochondrial metabolism has been known, the current study accentuates the critical role it plays in modulating Warburg phenotype in breast cancer cells. The clinical significance of this finding is the possibility to devise strategies for metabolically modulating aggressive triple-negative tumors so as to enhance their chemosensitivity in vivo. PMID:21945435

Failure of matrix metalloproteinase-9 dimer induction by phorbol 12-myristate 13-acetate in normal human cell lines.

PubMed

Waheed Roomi, Mohd; Kalinovsky, Tatiana; Rath, Matthias; Niedzwiecki, Aleksandra

2015-06-01

Increasing experimental and clinical data has identified an association between increased levels of matrix metalloproteinase (MMP)-9 and shortened patient survival, cancer progression and metastasis. MMP-9 has a significant role in tumor cell invasion and metastasis, as it digests the basement membrane and components of the extracellular matrix. MMP-9 is secreted in either a monomeric or dimeric form. Although limited evidence exists concerning MMP-9 dimers, certain studies have demonstrated that the dimer is associated with aggressive tumor progression. This is believed to be due to the fact that cellular migration depends upon the MMP-9 dimer, and not the monomer. Our previous study revealed that cancer cell MMP-9 dimer secretion patterns could be divided into different categories, and that high MMP-9 and MMP-9 dimer secretion levels were correlated with the most aggressive cancer cell lines. It has been established that signal transduction pathways and cytokines, including those activated by phorbol 12-myristate 13-acetate (PMA), regulate the expression of MMPs. The aim of the present study was to analyze the expression patterns of MMP-2, MMP-9 and MMP-9 dimer in normal human cells from a number of tissues treated with PMA. Muscle, epithelial and connective tissues were selected for use in the present study, since adenosarcomas, carcinomas and sarcomas are derived from these tissue types, respectively. The cell lines were first cultured in 24-well tissue culture plates containing recommended media that was supplemented with 10% fetal bovine serum and antibiotics. When at confluency, the cells were washed and fresh medium was added. In addition, a parallel set of cultures was treated with PMA. Subsequent to a 24-h incubation period, the media were collected and analyzed using gelatinase zymography for the expression of MMP-2 and MMP-9 monomer and dimer forms. The results revealed that the cellular expression of MMP-2 and MMP-9 was dependent upon the primary tissue subtype. All cell lines, regardless of tissue origin, expressed MMP-2. PMA induced the expression of MMP-9 in muscle tissue, glandular epithelia and supportive connective tissue cell lines. By contrast, cell lines of endothelial origin and proper connective tissue were insensitive to treatment with PMA. MMP-9 dimer secretion was not observed in any of the cell lines, which indicated that cellular migration is not supported by these cells.

Design, synthesis and in vitro evaluation of 18β-glycyrrhetinic acid derivatives for anticancer activity against human breast cancer cell line MCF-7.

PubMed

Yadav, Dharmendra Kumar; Kalani, Komal; Singh, Abhishek K; Khan, Feroz; Srivastava, Santosh K; Pant, Aditya B

2014-01-01

In the present work, QSAR model was derived by multiple linear regression method for the prediction of anticancer activity of 18β-glycyrrhetinic acid derivatives against the human breast cancer cell line MCF-7. The QSAR model for anti-proliferative activity against MCF-7 showed high correlation (r(2)=0.90 and rCV(2)=0.83) and indicated that chemical descriptors namely, dipole moment (debye), steric energy (kcal/mole), heat of formation (kcal/mole), ionization potential (eV), LogP, LUMO energy (eV) and shape index (basic kappa, order 3) correlate well with activity. The QSAR virtually predicted that active derivatives were first semi-synthesized and characterized on the basis of their (1)H and (13)C NMR spectroscopic data and then were in-vitro tested against MCF-7 cancer cell line. In particular, octylamide derivative of glycyrrhetinic acid GA-12 has marked cytotoxic activity against MCF-7 similar to that of standard anticancer drug paclitaxel. The biological assays of active derivative selected by virtual screening showed significant experimental activity.

The miR-21/PTEN/Akt signaling pathway is involved in the anti-tumoral effects of zoledronic acid in human breast cancer cell lines.

PubMed

Fragni, M; Bonini, S A; Bettinsoli, P; Bodei, S; Generali, D; Bottini, A; Spano, P F; Memo, M; Sigala, S

2016-05-01

Preclinical data indicate a direct anti-tumor effect of zoledronic acid (ZA) outside the skeleton, but its molecular mechanism is still not completely clarified. The aim of this study was to investigate the anti-cancer effects of ZA in human breast cancer cell lines, suggesting that they may in part be mediated via the miR-21/PTEN/Akt signaling pathway. The effect of ZA on cell viability was measured by MTT assay, and cell death induction was analyzed using either a double AO/EtBr staining and M30 ELISA assay. A Proteome Profiler Human Apoptosis Array was executed to evaluate the molecular basis of ZA-induced apoptosis. Cell cycle analysis was executed by flow cytometry. The effect of ZA on miR-21 expression was quantified by qRT-PCR, and the amount of PTEN protein and its targets were analyzed by Western blot. ZA inhibited cell growth in a concentration- and time-dependent manner, through the activation of cell death pathways and arrest of cell cycle progression. ZA downregulated the expression of miR-21, resulting in dephosphorilation of Akt and Bad and in a significant increase of p21 and p27 proteins expression. These results were observed also in MDA-MB-231 cells, commonly used as an experimental model of bone metastasis of breast cancer. This study revealed, for the first time, an involvement of the miR-21/PTEN/Akt signaling pathway in the mechanism of ZA anti-cancer actions in breast cancer cells. We would like to underline that this pathway is present both in the hormone responsive BC cell line (MCF-7) as well as in a triple negative cell line (MDA-MB-231). Taken together these results reinforce the use of ZA in clinical practice, suggesting the role of miR-21 as a possible mediator of its therapeutic efficacy.

Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy.

PubMed

Maurer, Gabriele D; Brucker, Daniel P; Bähr, Oliver; Harter, Patrick N; Hattingen, Elke; Walenta, Stefan; Mueller-Klieser, Wolfgang; Steinbach, Joachim P; Rieger, Johannes

2011-07-26

Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Overcoming IGF1R/IR Resistance Through Inhibition of MEK Signaling in Colorectal Cancer Models

PubMed Central

Flanigan, Sara A.; Pitts, Todd M.; Newton, Timothy P.; Kulikowski, Gillian N.; Tan, Aik Choon; McManus, Martine C.; Spreafico, Anna; Kachaeva, Maria I.; Selby, Heather M.; Tentler, John J.; Eckhardt, S. Gail; Leong, Stephen

2013-01-01

Purpose Results from clinical trials involving resistance to molecularly targeted therapies have revealed the importance of rational single agent and combination treatment strategies. In this study, we tested the efficacy of a type 1 insulin-like growth factor receptor (IGF1R)/insulin receptor (IR) tyrosine kinase inhibitor (TKI), OSI-906, in combination with a MEK 1/2 inhibitor based on evidence that the MAPK pathway was upregulated in colorectal cancer (CRC) cell lines that were resistant to OSI-906. Experimental Design The antiproliferative effects of OSI-906 and the MEK 1/2 inhibitor U0126, were analyzed both as single agents and in combination in 13 CRC cell lines in vitro. Apoptosis, downstream effector proteins, and cell cycle were also assessed. Additionally, the efficacy of OSI-906 combined with the MEK 1/2 inhibitor selumetinib (AZD6244, ARRY-142886), was evaluated in vivo using human CRC xenograft models. Results The combination of OSI-906 and U0126 resulted in synergistic effects in 11 out of 13 CRC cell lines tested. This synergy was variably associated with apoptosis or cell cycle arrest in addition to molecular effects on pro-survival pathways. The synergy was also reflected in the in vivo xenograft studies following treatment with the combination of OSI-906 and selumetinib. Conclusions Results from this study demonstrate synergistic antiproliferative effects in response to the combination of OSI-906 with a MEK 1/2 inhibitor in CRC cell line models both in vitro and in vivo, which supports the rational combination of OSI-906 with a MEK inhibitor in patients with CRC. PMID:24045180

Apoptotic activity of 5-fluorouracil in breast cancer cells transformed by low doses of ionizing α-particle radiation.

PubMed

Ponce-Cusi, Richard; Calaf, Gloria M

2016-02-01

Globally, breast cancer in women is the leading cause of cancer death. This fact has generated an interest to obtain insight into breast tumorigenesis and also to develop drugs to control the disease. Ras is a proto-oncogene that is activated as a response to extracellular signals. As a member of the Ras GTPase superfamily, Rho-A is an oncogenic and a critical component of signaling pathways leading to downstream gene regulation. In chemotherapy, apoptosis is the predominant mechanism by which cancer cells die. However, even when the apoptotic machinery remains intact, survival signaling may antagonize the cell death by signals. The aim of this study was to evaluate 5-fluorouracil (5-FU) in cells transformed by low doses of ionizing α-particle radiation, in breast cancer cell lines on these genes, as well as apoptotic activity. We used two cell lines from an in vitro experimental breast cancer model. The MCF-10F and Tumor2 cell lines. MCF-10F was exposed to low doses of high linear energy transfer (LET) α-particles radiation (150 keV/µm). Tumor2, is a malignant and tumorigenic cell line obtained from Alpha5 (60cGy+E/60cGy+E) injected into the nude mice. Results indicated that 5-FU decreased H-ras, Rho-A, p53, Stat1 and increased Bax gene expression in Tumor2 and decreased Rac1, Rho-A, NF-κB and increased Bax and caspase-3 protein expression in Tumor2. 5-FU decreased H-ras, Bcl-xL and NF-κB and increased Bax gene expression. 5-FU decreased Rac1, Rho-A protein expression and increased Bax and caspase-3 protein expression in MDA-MB-231. Flow cytometry indicated 21.5% of cell death in the control MCF-10F and 80% in Tumor2 cell lines. It can be concluded that 5-FU may exert apoptotic activity in breast cancer cells transformed by low doses of ionizing α-particles in vitro regulating genes of Ras family and related to apoptosis such as Bax, Bcl-xL and NF-κB expression.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

PubMed

Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

An Îto stochastic differential equations model for the dynamics of the MCF-7 breast cancer cell line treated by radiotherapy.

PubMed

Oroji, Amin; Omar, Mohd; Yarahmadian, Shantia

2016-10-21

In this paper, a new mathematical model is proposed for studying the population dynamics of breast cancer cells treated by radiotherapy by using a system of stochastic differential equations. The novelty of the model is essentially in capturing the concept of the cell cycle in the modeling to be able to evaluate the tumor lifespan. According to the cell cycle, each cell belongs to one of three subpopulations G, S, or M, representing gap, synthesis and mitosis subpopulations. Cells in the M subpopulation are highly radio-sensitive, whereas cells in the S subpopulation are highly radio-resistant. Therefore, in the process of radiotherapy, cell death rates of different subpopulations are not equal. In addition, since flow cytometry is unable to detect apoptotic cells accurately, the small changes in cell death rate in each subpopulation during treatment are considered. Subsequently, the proposed model is calibrated using experimental data from previous experiments involving the MCF-7 breast cancer cell line. Consequently, the proposed model is able to predict tumor lifespan based on the number of initial carcinoma cells. The results show the effectiveness of the radiation under the condition of stability, which describes the decreasing trend of the tumor cells population. Copyright © 2016 Elsevier Ltd. All rights reserved.

On-line experimental validation of a model-based diagnostic algorithm dedicated to a solid oxide fuel cell system

NASA Astrophysics Data System (ADS)

Polverino, Pierpaolo; Esposito, Angelo; Pianese, Cesare; Ludwig, Bastian; Iwanschitz, Boris; Mai, Andreas

2016-02-01

In the current energetic scenario, Solid Oxide Fuel Cells (SOFCs) exhibit appealing features which make them suitable for environmental-friendly power production, especially for stationary applications. An example is represented by micro-combined heat and power (μ-CHP) generation units based on SOFC stacks, which are able to produce electric and thermal power with high efficiency and low pollutant and greenhouse gases emissions. However, the main limitations to their diffusion into the mass market consist in high maintenance and production costs and short lifetime. To improve these aspects, the current research activity focuses on the development of robust and generalizable diagnostic techniques, aimed at detecting and isolating faults within the entire system (i.e. SOFC stack and balance of plant). Coupled with appropriate recovery strategies, diagnosis can prevent undesired system shutdowns during faulty conditions, with consequent lifetime increase and maintenance costs reduction. This paper deals with the on-line experimental validation of a model-based diagnostic algorithm applied to a pre-commercial SOFC system. The proposed algorithm exploits a Fault Signature Matrix based on a Fault Tree Analysis and improved through fault simulations. The algorithm is characterized on the considered system and it is validated by means of experimental induction of faulty states in controlled conditions.

Amide I SFG Spectral Line Width Probes the Lipid-Peptide and Peptide-Peptide Interactions at Cell Membrane In Situ and in Real Time.

PubMed

Zhang, Baixiong; Tan, Junjun; Li, Chuanzhao; Zhang, Jiahui; Ye, Shuji

2018-06-13

The balance of lipid-peptide and peptide-peptide interactions at cell membrane is essential to a large variety of cellular processes. In this study, we have experimentally demonstrated for the first time that sum frequency generation vibrational spectroscopy can be used to probe the peptide-peptide and lipid-peptide interactions in cell membrane in situ and in real time by determination of the line width of amide I band of protein backbone. Using a "benchmark" model of α-helical WALP23, it is found that the dominated lipid-peptide interaction causes a narrow line width of the amide I band, whereas the peptide-peptide interaction can markedly broaden the line width. When WALP23 molecules insert into the lipid bilayer, a quite narrow line width of the amide I band is observed because of the lipid-peptide interaction. In contrast, when the peptide lies down on the bilayer surface, the line width of amide I band becomes very broad owing to the peptide-peptide interaction. In terms of the real-time change in the line width, the transition from peptide-peptide interaction to lipid-peptide interaction is monitored during the insertion of WALP23 into 1,2-dipalmitoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DPPG) lipid bilayer. The dephasing time of a pure α-helical WALP23 in 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) and DPPG bilayer is determined to be 2.2 and 0.64 ps, respectively. The peptide-peptide interaction can largely accelerate the dephasing time.

Probing battery chemistry with liquid cell electron energy loss spectroscopy

DOE PAGES

Unocic, Raymond R.; Baggetto, Loic; Veith, Gabriel M.; ...

2015-09-15

Electron energy loss spectroscopy (EELS) was used to determine the chemistry and oxidation state of LiMn 2O 4 and Li 4Ti 5O 12 thin film battery electrodes in liquid cells for in situ scanning/transmission electron microscopy (S/TEM). Using the L2,3 white line intensity ratio method we determine the oxidation state of Mn and Ti in a liquid electrolyte solvent and discuss experimental parameters that influence measurement sensitivity.

Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds.

PubMed

Koníčková, Renata; Vaňková, Kateřina; Vaníková, Jana; Váňová, Kateřina; Muchová, Lucie; Subhanová, Iva; Zadinová, Marie; Zelenka, Jaroslav; Dvořák, Aleš; Kolář, Michal; Strnad, Hynek; Rimpelová, Silvie; Ruml, Tomáš; J Wong, Ronald; Vítek, Libor

2014-01-01

Spirulina platensis is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of S. platensis and S. platensis-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of S. platensis and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines in vitro in a dose-dependent manner (from 0.16 g•L-1 [S. platensis], 60 μM [PCB], and 125 μM [chlorophyllin], p<0.05). The anti-proliferative effects of S. platensis were also shown in vivo, where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (p < 0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (p = 0.0006; 0.016; and 0.006 for S. platensis, PCB, and chlorophyllin, respectively). In conclusion, S. platensis and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.

Hurdles to clinical translation of human induced pluripotent stem cells

PubMed Central

Neofytou, Evgenios; O’Brien, Connor Galen; Couture, Larry A.; Wu, Joseph C.

2015-01-01

Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development. PMID:26132109

Hurdles to clinical translation of human induced pluripotent stem cells.

PubMed

Neofytou, Evgenios; O'Brien, Connor Galen; Couture, Larry A; Wu, Joseph C

2015-07-01

Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

PubMed Central

Rahman, Saifur; Quann, Kevin; Pandya, Devanshi; Singh, Shruti; Khan, Zafar K.; Jain, Pooja

2012-01-01

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type. PMID:22496815

Lean Big Data integration in systems biology and systems pharmacology.

PubMed

Ma'ayan, Avi; Rouillard, Andrew D; Clark, Neil R; Wang, Zichen; Duan, Qiaonan; Kou, Yan

2014-09-01

Data sets from recent large-scale projects can be integrated into one unified puzzle that can provide new insights into how drugs and genetic perturbations applied to human cells are linked to whole-organism phenotypes. Data that report how drugs affect the phenotype of human cell lines and how drugs induce changes in gene and protein expression in human cell lines can be combined with knowledge about human disease, side effects induced by drugs, and mouse phenotypes. Such data integration efforts can be achieved through the conversion of data from the various resources into single-node-type networks, gene-set libraries, or multipartite graphs. This approach can lead us to the identification of more relationships between genes, drugs, and phenotypes as well as benchmark computational and experimental methods. Overall, this lean 'Big Data' integration strategy will bring us closer toward the goal of realizing personalized medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines.

PubMed

Kocanova, Silvia; Goiffon, Isabelle; Bystricky, Kerstin

2018-06-01

Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb-300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis, molecular docking and anticancer studies of peptides and iso-peptides.

PubMed

Jabeen, Farukh; Panda, Siva S; Kondratyuk, Tamara P; Park, Eun-Jung; Pezzuto, John M; Ihsan-ul-Haq; Hall, C Dennis; Katritzky, Alan R

2015-08-01

Chiral peptides and iso-peptides were synthesized in excellent yield by using benzotriazole mediated solution phase synthesis. Benzotriazole acted both as activating and leaving group, eliminating frequent use of protection and subsequent deprotection. The procedure was based on the hypothesis that epimerization should be suppressed in solution due to a faster coupling rate than SPPS. All the synthesized peptides complied with Lipinski's Ro5 except for the rotatable bonds. Inhibition of cell proliferation of cancer cell lines is one of the most commonly used methods to study the effectiveness of any anticancer agents. Synthesized peptides and iso-peptides were tested against three cancer cell lines (MCF-7, MDA-MB 231) to determine their anti-proliferative potential. NFkB was also determined. Molecular docking studies were also carried out to complement the experimental results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional roles of CSPG4/NG2 in chondrosarcoma.

PubMed

Jamil, Nuor S M; Azfer, Asim; Worrell, Harrison; Salter, Donald M

2016-04-01

CSPG4/NG2 is a multifunctional transmembrane protein with limited distribution in adult tissues including articular cartilage. The purpose of this study was to investigate the possible roles of CSPG4/NG2 in chondrosarcomas and to establish whether this molecule may have potential for targeted therapy. Stable knock-down of CSPG4/NG2 in the JJ012 chondrosarcoma cell line by shRNA resulted in decreased cell proliferation and migration as well as a decrease in gene expression of the MMP (matrix metalloproteinase) 3 protease and ADAMTS4 (aggrecanase). Chondrosarcoma cells in which CSPG4/NG2 was knocked down were more sensitive to doxorubicin than wild-type cells. The results indicate that CSPG4/NG2 has roles in regulating chondrosarcoma cell function in relation to growth, spread and resistance to chemotherapy and that anti-CSPG4/NG2 therapies may have potential in the treatment of surgically unresectable chondrosarcoma. © 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.

Causative ehrlichial organisms in Potomac horse fever.

PubMed Central

Rikihisa, Y; Perry, B D

1985-01-01

An ehrlichia was consistently isolated from the peripheral blood leukocyte fraction of ponies that had been experimentally infected with Potomac horse fever by whole blood transfusion from naturally infected horses. The organism was propagated in a human histiocyte cell line for 3 to 5 weeks and then inoculated intravenously or intradermally into healthy adult ponies. Clinical signs of Potomac horse fever, which varied in the degree of severity, occurred 9 to 14 days post-inoculation in all of the ponies. One pony died 20 days post-inoculation. The ehrlichial organism was reisolated in the human histiocyte cell line from the blood leukocyte fraction of all of the experimental ponies on each day that samples were examined (days 9, 10, 11, 19, and 39). These organisms were identical to those originally detected in the wall of the intestine of ponies with clinically diagnosed Potomac horse fever when compared by light and electron microscopy and an immunofluorescence labeling technique. The immunofluorescent antibody titer became positive in a pony at 20 days postinjection. These results indicate that the ehrlichial organisms is the causative agent of Potomac horse fever. Images PMID:4030092

Causative ehrlichial organisms in Potomac horse fever.

PubMed

Rikihisa, Y; Perry, B D

1985-09-01

An ehrlichia was consistently isolated from the peripheral blood leukocyte fraction of ponies that had been experimentally infected with Potomac horse fever by whole blood transfusion from naturally infected horses. The organism was propagated in a human histiocyte cell line for 3 to 5 weeks and then inoculated intravenously or intradermally into healthy adult ponies. Clinical signs of Potomac horse fever, which varied in the degree of severity, occurred 9 to 14 days post-inoculation in all of the ponies. One pony died 20 days post-inoculation. The ehrlichial organism was reisolated in the human histiocyte cell line from the blood leukocyte fraction of all of the experimental ponies on each day that samples were examined (days 9, 10, 11, 19, and 39). These organisms were identical to those originally detected in the wall of the intestine of ponies with clinically diagnosed Potomac horse fever when compared by light and electron microscopy and an immunofluorescence labeling technique. The immunofluorescent antibody titer became positive in a pony at 20 days postinjection. These results indicate that the ehrlichial organisms is the causative agent of Potomac horse fever.

Anti-tumor effects of osthole on ovarian cancer cells in vitro.

PubMed

Jiang, Guoqiang; Liu, Jia; Ren, Baoyin; Tang, Yawei; Owusu, Lawrence; Li, Man; Zhang, Jing; Liu, Likun; Li, Weiling

2016-12-04

Cnidium monnieri (L.) Cusson is a commonly used traditional Chinese medicine to treat gynecological disease in some countries. Osthole, an active O-methylated coumadin isolated from Cnidium monnieri (L.) Cusson, has been shown to induce various beneficial biochemical effects such as anti-seizure and anti-inflammatory effects. However, the anti-tumor mechanism of osthole is not well known. Here, we show that osthole inhibited the proliferation and migration of two widely used ovarian cancer cell lines, A2780 and OV2008 cells, in a dose-dependent manner. The study investigated the molecular mechanisms underlying ovarian cancer cells proliferation, apoptosis, cell cycle arrest and migration triggered by osthole. Ovarian cancer cell lines A2780, OV2008 and normal ovarian cell line IOSE80 were used as experimental model. MTT assay was employed to evaluate cell viability. Flow cytometry assays were performed to confirm apoptosis and cell cycle. We employed wound healing and transwell assays to delineate invasive and migratory potential triggered by osthole. MTT assays indicated that cell viability significantly decreased in ovarian cancer cells treated with osthole without effect on normal ovarian cells. Flow cytometric analysis revealed that osthole suppressed cells proliferation by promoting G2/M arrest and inducing apoptosis. The underlying mechanisms involved were regulation of the relative apoptotic protein Bcl-2, Bax and Caspase 3/9. In addition, wound healing and transwell assays revealed that the migratory potential and activity of matrix metalloproteinase MMP-2 and MMP-9 were markedly inhibited when cells were exposed to osthole. Our findings suggested that osthole has the potential to be used in novel anti-cancer therapeutic formulations for ovarian cancer treatment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Modeling universal dynamics of cell spreading on elastic substrates.

PubMed

Fan, Houfu; Li, Shaofan

2015-11-01

A three-dimensional (3D) multiscale moving contact line model is combined with a soft matter cell model to study the universal dynamics of cell spreading over elastic substrates. We have studied both the early stage and the late stage cell spreading by taking into account the actin tension effect. In this work, the cell is modeled as an active nematic droplet, and the substrate is modeled as a St. Venant Kirchhoff elastic medium. A complete 3D simulation of cell spreading has been carried out. The simulation results show that the spreading area versus spreading time at different stages obeys specific power laws, which is in good agreement with experimental data and theoretical prediction reported in the literature. Moreover, the simulation results show that the substrate elasticity may affect force dipole distribution inside the cell. The advantage of this approach is that it combines the hydrodynamics of actin retrograde flow with moving contact line model so that it can naturally include actin tension effect resulting from actin polymerization and actomyosin contraction, and thus it might be capable of simulating complex cellular scale phenomenon, such as cell spreading or even crawling.

Laboratory measurements of white dwarf photospheric spectral lines: Hβ

DOE PAGES

Falcon, Ross Edward; Rochau, Gregory A.; Bailey, James E.; ...

2015-06-18

We spectroscopically measure multiple hydrogen Balmer line profiles from laboratory plasmas to investigate the theoretical line profiles used in white dwarf (WD) atmosphere models. X-ray radiation produced at the Z Pulsed Power Facility at Sandia National Laboratories initiates plasma formation in a hydrogen-filled gas cell, replicating WD photospheric conditions. We also present time-resolved measurements of Hβ and fit this line using different theoretical line profiles to diagnose electron density, n e, and n = 2 level population, n 2. Aided by synthetic tests, we characterize the validity of our diagnostic method for this experimental platform. During a single experiment, wemore » infer a continuous range of electron densities increasing from n e ~ 4 to ~30 × 10 16 cm -3 throughout a 120-ns evolution of our plasma. Also, we observe n 2 to be initially elevated with respect to local thermodynamic equilibrium (LTE); it then equilibrates within ~55 ns to become consistent with LTE. This also supports our electron-temperature determination of T e ~ 1.3 eV (~15,000 K) after this time. At n e≲ 10 17 cm -3, we find that computer-simulation-based line-profile calculations provide better fits (lower reduced χ 2) than the line profiles currently used in the WD astronomy community. The inferred conditions, however, are in good quantitative agreement. Lastly, this work establishes an experimental foundation for the future investigation of relative shapes and strengths between different hydrogen Balmer lines.« less

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344.

PubMed

Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D; Townsend, Danyelle M; Tew, Kenneth D

2016-08-01

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Multiwalled carbon nanotubes effect on the bioavailability of artemisinin and its cytotoxity to cancerous cells

NASA Astrophysics Data System (ADS)

Rezaei, Behzad; Majidi, Najmeh; Noori, Shokoofe; Hassan, Zuhair M.

2011-12-01

Artemisinin regarded as one of the most promising anticancer drugs can bind to DNA with a binding constant of 1.04 × 104 M-1. The electrochemical experiments indicated that for longer incubation time periods, the reduction peak current of artemisinin on carbon nanotube modified electrode increases. Therefore, the uptake of drug molecules from a solution into CNTs will be achieved automatically by adsorption of 88.7% of artemisinin onto carbon nanotubes surface without alteration in drug properties. Hence, capability of carbon nanotubes to have synergistic effect on the bioavailability of artemisinin was investigated. Experimental tests on K562 cancer cell lines growth by MTT assay proved that multi-walled carbon nanotubes can enhance the cytotoxity of artemisinin to the targeted cancer cells with unprecedented accuracy and efficiency. The IC50 values were 65 and 35 μM for artemisinin and artemisinin loaded on multi-walled carbon nanotubes, respectively; demonstrating that artemisinin loaded on multi-walled carbon nanotubes is more effective in inhibition of cancer cell lines growth.

Integrated Genomic Analysis of Diverse Induced Pluripotent Stem Cells from the Progenitor Cell Biology Consortium.

PubMed

Salomonis, Nathan; Dexheimer, Phillip J; Omberg, Larsson; Schroll, Robin; Bush, Stacy; Huo, Jeffrey; Schriml, Lynn; Ho Sui, Shannan; Keddache, Mehdi; Mayhew, Christopher; Shanmukhappa, Shiva Kumar; Wells, James; Daily, Kenneth; Hubler, Shane; Wang, Yuliang; Zambidis, Elias; Margolin, Adam; Hide, Winston; Hatzopoulos, Antonis K; Malik, Punam; Cancelas, Jose A; Aronow, Bruce J; Lutzko, Carolyn

2016-07-12

The rigorous characterization of distinct induced pluripotent stem cells (iPSC) derived from multiple reprogramming technologies, somatic sources, and donors is required to understand potential sources of variability and downstream potential. To achieve this goal, the Progenitor Cell Biology Consortium performed comprehensive experimental and genomic analyses of 58 iPSC from ten laboratories generated using a variety of reprogramming genes, vectors, and cells. Associated global molecular characterization studies identified functionally informative correlations in gene expression, DNA methylation, and/or copy-number variation among key developmental and oncogenic regulators as a result of donor, sex, line stability, reprogramming technology, and cell of origin. Furthermore, X-chromosome inactivation in PSC produced highly correlated differences in teratoma-lineage staining and regulator expression upon differentiation. All experimental results, and raw, processed, and metadata from these analyses, including powerful tools, are interactively accessible from a new online portal at https://www.synapse.org to serve as a reusable resource for the stem cell community. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

NASA Astrophysics Data System (ADS)

Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

2018-02-01

Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

PIC simulations of conical magnetically insulated transmission line with LTD generator: Transition from self-limited to load-limited flow

NASA Astrophysics Data System (ADS)

Liu, Laqun; Wang, Huihui; Guo, Fan; Zou, Wenkang; Liu, Dagang

2017-04-01

Based on the 3-dimensional Particle-In-Cell (PIC) code CHIPIC3D, with a new circuit boundary algorithm we developed, a conical magnetically insulated transmission line (MITL) with a 1.0-MV linear transformer driver (LTD) is explored numerically. The values of switch jitter time of LTD are critical parameters for the system, which are difficult to be measured experimentally. In this paper, these values are obtained by comparing the PIC results with experimental data of large diode-gap MITL. By decreasing the diode gap, we find that all PIC results agree well with experimental data only if MITL works on self-limited flow no matter how large the diode gap is. However, when the diode gap decreases to a threshold, the self-limited flow would transfer to a load-limited flow. In this situation, PIC results no longer agree with experimental data anymore due to the anode plasma expansion in the diode load. This disagreement is used to estimate the plasma expansion speed.

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

PubMed

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

PubMed Central

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-01-01

Background CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. Design and Methods We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d+ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. Results The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d+ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d+ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Conclusions Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d+ hematologic malignancies. PMID:19454494

Human embryonic stem cell lines model experimental human cytomegalovirus latency.

PubMed

Penkert, Rhiannon R; Kalejta, Robert F

2013-05-28

Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-04-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed Central

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-01-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel experimental model for further investigation. Images Figure 4 Figure 6 Figure 7 Figure 8 PMID:9176104

Perturbation Biology: Inferring Signaling Networks in Cellular Systems

PubMed Central

Miller, Martin L.; Gauthier, Nicholas P.; Jing, Xiaohong; Kaushik, Poorvi; He, Qin; Mills, Gordon; Solit, David B.; Pratilas, Christine A.; Weigt, Martin; Braunstein, Alfredo; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

2013-01-01

We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology. PMID:24367245

[Experimental study on TCRbeta idiotypic antigenic determinants DNA vaccine to induce anti-lymphoma antibodies].

PubMed

Zhang, Yeping; Zhu, Ping; Shi, Yongjin; Liu, Jihua; Pu, Dingfang; Cao, Xianghong; Zhu, Qiang; Wang, Yijia; Ma, Mingxin; Yu, Jiren

2002-02-01

To investigate the anti-human CEM lymphoma cell activities induced by TCR idiotypic DNA vaccine containing different antigen determinants in BALB/c mice. The specific rearranged gene fragment encoding TCRVbeta region of CEM cell line was obtained by RT-PCR technique. The PCR product was cloned into eukaryocytic expression vector pcDNA3, which was used as DNA vaccine and template for PCR amplifying different antigen determinant. Gene fragments encoding different antigen determinant were amplified and cloned into pcDNA3, separately. The experimental mice were immunized by intramuscular injection of the DNA vaccines. The specific anti-idiotype antibodies were detected by indirect immunofluorescence assay. TCRbetaV of CEM cell line contains five antigen determinants. Specific anti-idiotype antibody was detected in all of the six mice immunized with DNA vaccine containing all the five determinants (the highest titer was 1:480). Although the antibody could also be detected in four of the six mice immunized with DNA vaccine containing four of the five antigen determinants, the antibody titer was lower (the highest titer was 1:80). DNA vaccine containing two of the five determinants could not induce the specific antibody. The idiotypic DNA vaccine containing the whole TCRbetaV five antigen determinants could induce the specific anti-lymphoma idiotypic antibody in BALB/c mice.

Short-Term Grafting of Human Neural Stem Cells: Electrophysiological Properties and Motor Behavioral Amelioration in Experimental Parkinsons Disease.

PubMed

Martnez-Serrano, Alberto; Pereira, Marta P; Avaliani, Natalia; Nelke, Anna; Kokaia, Merab; Ramos-Moreno, Tania

2016-12-13

Cell replacement therapy in Parkinsons disease (PD) still lacks a study addressing the acquisition of electrophysiological properties of human grafted neural stem cells and their relation with the emergence of behavioral recovery after transplantation in the short term. Here we study the electrophysiological and biochemical profiles of two ventral mesencephalic human neural stem cell (NSC) clonal lines (C30-Bcl-XL and C32-Bcl-XL) that express high levels of Bcl-XL to enhance their neurogenic capacity, after grafting in an in vitro parkinsonian model. Electrophysiological recordings show that the majority of the cells derived from the transplants are not mature at 6 weeks after grafting, but 6.7% of the studied cells showed mature electrophysiological profiles. Nevertheless, parallel in vivo behavioral studies showed a significant motor improvement at 7 weeks postgrafting in the animals receiving C30-Bcl-XL, the cell line producing the highest amount of TH+ cells. Present results show that, at this postgrafting time point, behavioral amelioration highly correlates with the spatial dispersion of the TH+ grafted cells in the caudate putamen. The spatial dispersion, along with a high number of dopaminergic-derived cells, is crucial for behavioral improvements. Our findings have implications for long-term standardization of stem cell-based approaches in Parkinsons disease.

Garlic-derived compound S-allylmercaptocysteine (SAMC) is active against anaplastic thyroid cancer cell line 8305C (HPACC).

PubMed

Liu, Yuexin; Yan, Jinyin; Han, Xiaochen; Hu, Wanning

2015-01-01

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic have anticancer activity. In this study, the apoptotic effects of Garlic-derived compound S-allylmercaptocysteine (SAMC) were investigated in 8305C human anaplastic thyroid carcinoma cells. The cell line 8305C (HPACC) were treated with SAMC and the MTT assay, flow cytometry (FCM), electron microscope method were used to test cell cycle, inhibitory rate and morphologic changes respectively. HPACC-8305C cells were suppressed after exposure to SAMC of 0.02 mg/ml, 0.06 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P< 0.05). SAMC could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G2/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P< 0.05). After exposure to SAMC at 0.02 mg/ml for 24 hours, HPACC-8305C cells showed typical morphologic change. SAMC inhibits the growth of HPACC-8305C cells by induction of apoptotic cell death and inhibit telomerase activity, which appears to account for its anti-cancer activity.

In vitro fluoride release from a different kind of conventional and resin modified glass-ionomer cements.

PubMed

Selimović-Dragaš, Mediha; Hasić-Branković, Lajla; Korać, Fehim; Đapo, Nermin; Huseinbegović, Amina; Kobašlija, Sedin; Lekić, Meliha; Hatibović-Kofman, Šahza

2013-08-01

Fluoride release is important characteristic of glass-ionomer cements. Quantity of fluoride ions released from the glass-ionomer cements has major importance in definition of their biological activity. The objectives of this study were to define the quantity of fluoride ions released from the experimental glass-ionomer cements and to define the effect of fluoride ions released from the experimental glass-ionomer cements on their cytotoxicity. Concentrations of the fluoride ions released in the evaluated glass-ionomer cements were measured indirectly, by the fluoride-selective WTW, F500 electrode potential, combined with reference R503/D electrode. Statistical analyses of F-ion concentrations released by all glass-ionomers evaluated at two time points, after 8 and after 24 hours, show statistically higher fluoride releases from RMGICs: Vitrebond, Fuji II LC and Fuji Plus, when compared to conventional glass-ionomer cements: Fuji Triage, Fuji IX GP Fast and Ketac Silver, both after 8 and after 24 hours. Correlation coefficient between concentrations of fluoride ion released by evaluated glass-ionomer cements and cytotoxic response of UMR-106 osteoblast cell-line are relatively high, but do not reach levels of biological significance. Correlation between concentrations of fluoride ion released and cytotoxic response of NIH3T3 mouse fibroblast cell line after 8 hours is high, positive and statistically significant for conventional GICs, Fuji Triage and Fuji IX GP Fast, and RMGIC, Fuji II LC. Statistically significant Correlation coefficient between concentrations of fluoride ion released and cytotoxic response of NIH3T3 cell line after 24 hours is defined for RMGIC Fuji II LC only.

NPInter v3.0: an upgraded database of noncoding RNA-associated interactions

PubMed Central

Hao, Yajing; Wu, Wei; Li, Hui; Yuan, Jiao; Luo, Jianjun; Zhao, Yi; Chen, Runsheng

2016-01-01

Despite the fact that a large quantity of noncoding RNAs (ncRNAs) have been identified, their functions remain unclear. To enable researchers to have a better understanding of ncRNAs’ functions, we updated the NPInter database to version 3.0, which contains experimentally verified interactions between ncRNAs (excluding tRNAs and rRNAs), especially long noncoding RNAs (lncRNAs) and other biomolecules (proteins, mRNAs, miRNAs and genomic DNAs). In NPInter v3.0, interactions pertaining to ncRNAs are not only manually curated from scientific literature but also curated from high-throughput technologies. In addition, we also curated lncRNA–miRNA interactions from in silico predictions supported by AGO CLIP-seq data. When compared with NPInter v2.0, the interactions are more informative (with additional information on tissues or cell lines, binding sites, conservation, co-expression values and other features) and more organized (with divisions on data sets by data sources, tissues or cell lines, experiments and other criteria). NPInter v3.0 expands the data set to 491,416 interactions in 188 tissues (or cell lines) from 68 kinds of experimental technologies. NPInter v3.0 also improves the user interface and adds new web services, including a local UCSC Genome Browser to visualize binding sites. Additionally, NPInter v3.0 defined a high-confidence set of interactions and predicted the functions of lncRNAs in human and mouse based on the interactions curated in the database. NPInter v3.0 is available at http://www.bioinfo.org/NPInter/. Database URL: http://www.bioinfo.org/NPInter/ PMID:27087310

Electrochemical oxidation coupled with liquid chromatography and mass spectrometry to study the oxidative stability of active pharmaceutical ingredients in solution: A comparison of off-line and on-line approaches.

PubMed

Torres, Susana; Brown, Roland; Zelesky, Todd; Scrivens, Garry; Szucs, Roman; Hawkins, Joel M; Taylor, Mark R

2016-11-30

Stability studies of pharmaceutical drug products and pharmaceutical active substances are important to research and development in order to fully understand and maintain product quality and safety throughout its shelf-life. Oxidative forced degradation studies are among the different types of stability studies performed by the pharmaceutical industry in order to understand the intrinsic stability of drug molecules. We have been comparing the use of electrochemistry as an alternative oxidative forced degradation method to traditional forced degradation and accelerated stability studies. Using the electrochemical degradation approach the substrate oxidation takes place in a commercially available electrochemical cell and the effluent of the cell can be either a) directly infused into the mass spectrometer or b) injected in a chromatographic column for separation of the different products formed prior to the mass spectrometry analysis. To enable the study of large numbers of different experimental conditions and molecules we developed a new dual pump automated electrochemical screening platform. This system used a HPLC pump and autosampler to load and wash the electrochemical cell and deliver the oxidized sample plug to a second injection loop. This system enabled the automatic sequential analyses of large numbers of different solutions under varied experimental conditions without need for operator intervention during the run sequence. Here we describe the system and evaluate its performance using a test molecule with well characterized stability and compare results to those obtained using an off-line electrochemistry approach. Copyright © 2016 Elsevier B.V. All rights reserved.

0.Measurements of H216O Linestrengths and Air-Induced Broadenings and Shifts in the 815-nm Spectral Region

NASA Technical Reports Server (NTRS)

Ponsardin, Patrick L.; Browell, Edward V.

1997-01-01

The linestrengths for 40 absorption lines of H2 16-O water vapor that were located between 813 and 820 nm were measured; most of these lines were selected for their potential usefulness in laser remote measurements of atmospheric humidity using the differential absorption lidar technique. The air-induced pressure-broadening coefficients were also measured for 32 of these lines and the air-induced pressure shift coefficients were measured for 29 lines. These spectroscopic parameters were derived from spectra obtained with an AlGaAs diode laser and two long-path absorption cells. Collisional narrowing effects were observed and were accurately described by a Galatry profile. Comparisons were made with previous experimental work or theoretical calculations as available.

Lining seam elimination algorithm and surface crack detection in concrete tunnel lining

NASA Astrophysics Data System (ADS)

Qu, Zhong; Bai, Ling; An, Shi-Quan; Ju, Fang-Rong; Liu, Ling

2016-11-01

Due to the particularity of the surface of concrete tunnel lining and the diversity of detection environments such as uneven illumination, smudges, localized rock falls, water leakage, and the inherent seams of the lining structure, existing crack detection algorithms cannot detect real cracks accurately. This paper proposed an algorithm that combines lining seam elimination with the improved percolation detection algorithm based on grid cell analysis for surface crack detection in concrete tunnel lining. First, check the characteristics of pixels within the overlapping grid to remove the background noise and generate the percolation seed map (PSM). Second, cracks are detected based on the PSM by the accelerated percolation algorithm so that the fracture unit areas can be scanned and connected. Finally, the real surface cracks in concrete tunnel lining can be obtained by removing the lining seam and performing percolation denoising. Experimental results show that the proposed algorithm can accurately, quickly, and effectively detect the real surface cracks. Furthermore, it can fill the gap in the existing concrete tunnel lining surface crack detection by removing the lining seam.

Cell biology experiments conducted in space

NASA Technical Reports Server (NTRS)

Taylor, G. R.

1977-01-01

A review of cell biology experiments conducted during the first two decades of space flight is provided. References are tabulated for work done with six types of living test system: isolated viruses, bacteriophage-host, bacteria, yeasts and filamentous fungi, protozoans, and small groups of cells (such as hamster cell tissue and fertilized frog eggs). The general results of studies involving the survival of cells in space, the effect of space flight on growing cultures, the biological effects of multicharged high-energy particles, and the effects of space flight on the genetic apparatus of microorganisms are summarized. It is concluded that cell systems remain sufficiently stable during space flight to permit experimentation with models requiring a fixed cell line during the space shuttle era.

Experimental arthritis and uveitis in rats associated with Mycobacterium butyricum.

PubMed

Petty, R E; Hunt, D W; Mathers, D M; McCormick, A Q; Barker, H; Southwood, T R; Corson, L

1994-08-01

To determine if the anterior uveitis associated with adjuvant arthritis (AA) in the rat can be passively transferred with arthritis to syngeneic recipients using spleen cells or T cell lines prepared from animals given complete Freund's adjuvant (CFA) and Mycobacterium butyricum (M. butyricum) in incomplete Freund's adjuvant (IFA). Spleen cells from Lewis or Lewis SsN rats given IFA, CFA, type I collagen in IFA (CI-IFA), or type II collagen in IFA (CII-IFA) were administered to naive rats or rats treated with pertussis toxin or bacterial endotoxin. Three CD4+ T cell lines, propagated from CFA injected rats and maintained in vitro with M. butyricum (M-1), bovine proteoglycan (PR-1) or an extract of M. butyricum (MBE-1) were administered to naive or immunosuppressed rats. The arthritogenic and uveitogenic properties of these cell preparations and intradermal MBE-IFA, CII-IFA and intraperitoneal (ip) M. butyricum without adjuvant were evaluated. Uveitis was observed in 15/69 (22%) arthritic rats given CFA. Spleen cells prepared from CFA injected rats caused arthritis in 55 (82%) and uveitis in 2 (3%) of 67 cell recipients. Uveitis occurred in 2/6 cell recipients pretreated with bacterial endotoxin. Neither uveitis nor arthritis was observed in rats given IFA (0/6) or spleen cells prepared from rats given IFA (0/27), CI-IFA (0/6), or CII-IFA (0/28). CII-IFA produced polyarthritis in 5/6 rats, but no uveitis. CII-IFA induced arthritis associated uveitis in 1/15 animals receiving spleen cells from rats given CII-IFA, but not those given CI-IFA (0/3) or IFA (0/13). Uveitis was observed in one recipient of the M-1 T cell line and in 2 recipients of the PR-1 T cell line. Immunization with 400 micrograms of MBE-IFA induced uveitis but not arthritis in 3/11 animals. The MBE specific T cell line was neither arthritogenic nor uveitogenic. A high frequency (5/6) of uveitis accompanied arthritis in male Lewis rats given ip M. butyricum. Arthritis occurred in 4/10 female Lewis rats given ip M. butyricum and 2 arthritic animals also developed uveitis. Uveitis occurs infrequently in arthritic rats given spleen cells from CFA injected animals. The ip administration of M. butyricum constitutes a novel disease model in which the immunopathological relationships between arthritis and uveitis may be more reliably studied.

Autophagy is the predominant process induced by arsenite in human lymphoblastoid cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolt, Alicia M.; Byrd, Randi M.; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.ed

2010-05-01

Arsenic is a widespread environmental toxicant with a diverse array of molecular targets and associated diseases, making the identification of the critical mechanisms and pathways of arsenic-induced cytotoxicity a challenge. In a variety of experimental models, over a range of arsenic exposure levels, apoptosis is a commonly identified arsenic-induced cytotoxic pathway. Human lymphoblastoid cell lines (LCL) have been used as a model system in arsenic toxicology for many years, but the exact mechanism of arsenic-induced cytotoxicity in LCL is still unknown. We investigated the cytotoxicity of sodium arsenite in LCL 18564 using a set of complementary markers for cell deathmore » pathways. Markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase inhibition) were uniformly negative in arsenite exposed cells. Interestingly, electron microscopy, acidic vesicle fluorescence, and expression of LC3 in LCL 18564 identified autophagy as an arsenite-induced process that was associated with cytotoxicity. Autophagy, a cellular programmed response that is associated with both cellular stress adaptation as well as cell death appears to be the predominant process in LCL cytotoxicity induced by arsenite. It is unclear, however, whether LCL autophagy is an effector mechanism of arsenite cytotoxicity or alternatively a cellular compensatory mechanism. The ability of arsenite to induce autophagy in lymphoblastoid cell lines introduces a potentially novel mechanistic explanation of the well-characterized in vitro and in vivo toxicity of arsenic to lymphoid cells.« less

WE-EF-BRA-05: Experimental Design for High-Throughput In-Vitro RBE Measurements Using Protons, Helium and Carbon Ions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guan, F; Titt, U; Patel, D

2015-06-15

Purpose: To design and validate experimental setups for investigation of dose and LET effects in cell kill for protons, helium and carbon ions, in high throughput and high accuracy cell experiments. Methods: Using the Geant4 Monte Carlo toolkit, we designed 3 custom range compensators to simultaneously expose cancer cells to different doses and LETs from selected portions of pristine ion beams from the entrance to points just beyond the Bragg peak. To minimize the spread of LET, we utilized mono-energetic uniformly scanned beams at the HIT facility with support from the DKFZ. Using different entrance doses and LETs, a matrixmore » of cell survival data was acquired leading to a specific RBE matrix. We utilized the standard clonogenic assay for H460 and H1437 lung-cancer cell lines grown in 96-well plates. Using these plates, the data could be acquired in a small number of exposures. The ion specific compensators were located in a horizontal beam, designed to hold two 96-wells plates (12 columns by 8 rows) at an angle of 30o with respect to the beam direction. Results: Using about 20 hours of beam time, a total of about 11,000 wells containing cancer cells could be irradiated. The H460 and H1437 cell lines exhibited a significant dependence on LET when they were exposed to comparable doses. The results were similar for each of the investigated ion species, and indicate the need to incorporate RBE into the ion therapy planning process. Conclusion: The experimental design developed is a viable approach to rapidly acquire large amounts of accurate in-vitro RBE data. We plan to further improve the design to achieve higher accuracy and throughput, thereby facilitating the irradiation of multiple cell types. The results are indicative of the possibility to develop a new degree of freedom (variable RBE) for future clinical ion therapy optimization. Work supported by the Sister Institute Network Fund (SINF), University of Texas MD Anderson Cancer Center.« less

Identification of antipsychotic drug fluspirilene as a potential p53-MDM2 inhibitor: a combined computational and experimental study

NASA Astrophysics Data System (ADS)

Patil, Sachin P.; Pacitti, Michael F.; Gilroy, Kevin S.; Ruggiero, John C.; Griffin, Jonathan D.; Butera, Joseph J.; Notarfrancesco, Joseph M.; Tran, Shawn; Stoddart, John W.

2015-02-01

The inhibition of tumor suppressor p53 protein due to its direct interaction with oncogenic murine double minute 2 (MDM2) protein, plays a central role in almost 50 % of all human tumor cells. Therefore, pharmacological inhibition of the p53-binding pocket on MDM2, leading to p53 activation, presents an important therapeutic target against these cancers expressing wild-type p53. In this context, the present study utilized an integrated virtual and experimental screening approach to screen a database of approved drugs for potential p53-MDM2 interaction inhibitors. Specifically, using an ensemble rigid-receptor docking approach with four MDM2 protein crystal structures, six drug molecules were identified as possible p53-MDM2 inhibitors. These drug molecules were then subjected to further molecular modeling investigation through flexible-receptor docking followed by Prime/MM-GBSA binding energy analysis. These studies identified fluspirilene, an approved antipsychotic drug, as a top hit with MDM2 binding mode and energy similar to that of a native MDM2 crystal ligand. The molecular dynamics simulations suggested stable binding of fluspirilene to the p53-binding pocket on MDM2 protein. The experimental testing of fluspirilene showed significant growth inhibition of human colon tumor cells in a p53-dependent manner. Fluspirilene also inhibited growth of several other human tumor cell lines in the NCI60 cell line panel. Taken together, these computational and experimental data suggest a potentially novel role of fluspirilene in inhibiting the p53-MDM2 interaction. It is noteworthy here that fluspirilene has a long history of safe human use, thus presenting immediate clinical potential as a cancer therapeutic. Furthermore, fluspirilene could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53-MDM2 inhibitors against several types of cancer. Importantly, the combined computational and experimental screening protocol presented in this study may also prove useful for screening other commercially-available compound databases for identification of novel, small molecule p53-MDM2 inhibitors.

31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

PubMed

Lyng, H; Olsen, D R; Petersen, S B; Rofstad, E K

1995-04-01

The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of in vitro growth and arrest in the G0/G1 phase of HCT8 line human colon cancer cells by kaempferide triglycoside from Dianthus caryophyllus.

PubMed

Martineti, Valentina; Tognarini, Isabella; Azzari, Chiara; Carbonell Sala, Silvia; Clematis, Francesca; Dolci, Marcello; Lanzotti, Virginia; Tonelli, Francesco; Brandi, Maria Luisa; Curir, Paolo

2010-09-01

The effects of phytoestrogens have been studied in the hypothalamic-pituitary-gonadal axis and in various non-gonadal targets. Epidemiologic and experimental evidence indicates a protective effect of phytoestrogens also in colorectal cancer. The mechanism through which estrogenic molecules control colorectal cancer tumorigenesis could possibly involve estrogen receptor beta, the predominantly expressed estrogen receptor subtype in colon mucosa.To validate this hypothesis, we therefore used an engineered human colon cancer cell line induced to overexpress estrogen receptor beta, beside its native cell line, expressing very low levels of ERbeta and not expressing ERalpha; as a phytoestrogenic molecule, we used kaempferide triglycoside, a glycosylated flavonol from a Dianthus caryophyllus cultivar. The inhibitory properties of this molecule toward vegetal cell growth have been previously demonstrated: however, no data on its activity on animal cell or information about the mechanism of this activity are available. Kaempferide triglycoside proved to inhibit the proliferation of native and estrogen receptor beta overexpressing colon cancer cells through a mechanism not mediated by ligand binding dependent estrogen receptor activation. It affected HCT8 cell cycle progression by increasing the G(0)/G(1) cell fraction and in estrogen receptor beta overexpressing cells increased two antioxidant enzymes. Interestingly, the biological effects of this kaempferide triglycoside were strengthened by the presence of high levels of estrogen receptor beta.Pleiotropic molecular effects of phytoestrogens may explain their protective activity against colorectal cancer and may represent an interesting area for future investigation with potential clinical applications. Copyright 2010 John Wiley & Sons, Ltd.

Autophagy diminishes the early interferon-β response to influenza A virus resulting in differential expression of interferon-stimulated genes.

PubMed

Perot, Brieuc P; Boussier, Jeremy; Yatim, Nader; Rossman, Jeremy S; Ingersoll, Molly A; Albert, Matthew L

2018-05-10

Influenza A virus (IAV) infection perturbs metabolic pathways such as autophagy, a stress-induced catabolic pathway that crosstalks with cellular inflammatory responses. However, the impact of autophagy perturbation on IAV gene expression or host cell responses remains disputed. Discrepant results may be a reflection of in vivo studies using cell-specific autophagy-related (Atg) gene-deficient mouse strains, which do not delineate modification of developmental programmes from more proximal effects on inflammatory response. In vitro experiments can be confounded by gene expression divergence in wild-type cultivated cell lines, as compared to those experiencing long-term absence of autophagy. With the goal to investigate cellular processes within cells that are competent or incompetent for autophagy, we generated a novel experimental cell line in which autophagy can be restored by ATG5 protein stabilization in an otherwise Atg5-deficient background. We confirmed that IAV induced autophagosome formation and p62 accumulation in infected cells and demonstrated that perturbation of autophagy did not impact viral infection or replication in ATG5-stablized cells. Notably, the induction of interferon-stimulated genes (ISGs) by IAV was diminished when cells were autophagy competent. We further demonstrated that, in the absence of ATG5, IAV-induced interferon-β (IFN-β) expression was increased as compared to levels in autophagy-competent lines, a mechanism that was independent of IAV non-structural protein 1. In sum, we report that induction of autophagy by IAV infection reduces ISG expression in infected cells by limiting IFN-β expression, which may benefit viral replication and spread.

Expression and Regulation of the Retinoic Acid Receptor Beta Gene in Human Mammary Epithelial Cells

DTIC Science & Technology

1996-09-01

Balb/c nu/nu female mice. We have obtained experimental design advice from Dr. Janet Price, University of Texas, M.D. Anderson Cancer Center, Houston...author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation...identified eight genes regulated by retinoic acid in either breast cancer cell lines and/or normal HMECs. We have continued to evaluate the methodology to

A search for the prewetting line. [in binary liquid system at vapor-liquid interface

NASA Technical Reports Server (NTRS)

Schmidt, J. W.; Moldover, M. R.

1986-01-01

This paper describes efforts to locate the prewetting line in a binary liquid system (isopropanol-perfluoromethylcyclohexane) at the vapor-liquid interface. Tight upper bounds were placed on the temperature separation (0.2 K) between the prewetting line and the line of bulk liquid phase separation. The prewetting line in systems at equilibrium was not detected. Experimental signatures indicative of the prewetting line occurred only in nonequilibrium situations. Several theories predict that the adsorption of one of the components (the fluorocarbon, in this case) at the liquid-vapor interface should increase abruptly, at a temperature sightly above the temperature at which the mixture separates into two liquid phases. A regular solution calculation indicates that this prewetting line should have been easily detectable with the instruments used in this experiment. Significant features of the experiment are: (1) low-gradient thermostatting, (2) in situ stirring, (3) precision ellipsometry from the vapor-liquid interface, (4) high resolution differential index of refraction measurements using a novel cell design, and (5) computer control.

Simultaneous detection of MCF-7 and HepG2 cells in blood by ICP-MS with gold nanoparticles and quantum dots as elemental tags.

PubMed

Li, Xiaoting; Chen, Beibei; He, Man; Wang, Han; Xiao, Guangyang; Yang, Bin; Hu, Bin

2017-04-15

In this work, we demonstrate a novel method based on inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticles (Au NPs) and quantum dots (QDs) labeling for the simultaneous counting of two circulating tumor cell lines (MCF-7 and HepG2 cells) in human blood. MCF-7 and HepG2 cells were captured by magnetic beads coupled with anti-EpCAM and then specifically labeled by CdSe QDs-anti-ASGPR and Au NPs-anti-MUC1, respectively, which were used as signal probes for ICP-MS measurement. Under the optimal experimental conditions, the limits of detection of 50 MCF-7, 89 HepG2 cells and the linear ranges of 200-40000 MCF-7, 300-30000 HepG2 cells were obtained, and the relative standard deviations for seven replicate detections of 800 MCF-7 and HepG2 cells were 4.6% and 5.7%, respectively. This method has the advantages of high sensitivity, low sample consumption, wide linear range and can be extended to the simultaneous detection of multiple CTC lines in human peripheral blood. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo and in vitro infection dynamics of honey bee viruses.

PubMed

Carrillo-Tripp, Jimena; Dolezal, Adam G; Goblirsch, Michael J; Miller, W Allen; Toth, Amy L; Bonning, Bryony C

2016-02-29

The honey bee (Apis mellifera) is commonly infected by multiple viruses. We developed an experimental system for the study of such mixed viral infections in newly emerged honey bees and in the cell line AmE-711, derived from honey bee embryos. When inoculating a mixture of iflavirids [sacbrood bee virus (SBV), deformed wing virus (DWV)] and dicistrovirids [Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV)] in both live bee and cell culture assays, IAPV replicated to higher levels than other viruses despite the fact that SBV was the major component of the inoculum mixture. When a different virus mix composed mainly of the dicistrovirid Kashmir bee virus (KBV) was tested in cell culture, the outcome was a rapid increase in KBV but not IAPV. We also sequenced the complete genome of an isolate of DWV that covertly infects the AmE-711 cell line, and found that this virus does not prevent IAPV and KBV from accumulating to high levels and causing cytopathic effects. These results indicate that different mechanisms of virus-host interaction affect virus dynamics, including complex virus-virus interactions, superinfections, specific virus saturation limits in cells and virus specialization for different cell types.

Bone formation in vitro and in nude mice by human osteosarcoma cells.

PubMed

Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E

1995-01-01

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.

Characterization of drug-induced transcriptional modules: towards drug repositioning and functional understanding

PubMed Central

Iskar, Murat; Zeller, Georg; Blattmann, Peter; Campillos, Monica; Kuhn, Michael; Kaminska, Katarzyna H; Runz, Heiko; Gavin, Anne-Claude; Pepperkok, Rainer; van Noort, Vera; Bork, Peer

2013-01-01

In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of α-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology. PMID:23632384

A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

PubMed

Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi

2014-05-01

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR. Copyright © 2014 Elsevier Inc. All rights reserved.

Hepa1-6-FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability.

PubMed

Li, Yasha; Liu, Mengnan; Cui, Jiejie; Yang, Ke; Zhao, Li; Gong, Mengjia; Wang, Yi; He, Yun; He, Tongchuan; Bi, Yang

2018-05-01

Reliable animal models are required for the in vivo study of the molecular mechanisms and effects of chemotherapeutic drugs in hepatocarcinoma. In vivo tracing techniques based on firefly luciferase (FLuc) may optimize the non-invasive monitoring of experimental animals. The present study established a murine Hepa1-6-FLuc cell line that stably expressed a retrovirus-delivered FLuc protein gene. The cell morphology, proliferation, migration and invasion ability of Hepa1-6-FLuc cells were the same as that of the Hepa1-6 cells, and thus is suitable to replace Hepa1-6 cells in the construction of hepatocarcinoma animal models. No differences in subcutaneous tumor mass and its pathomorphology from implanted Hepa1-6-FLuc cells were observed compared with Hepa1-6 control tumors. Bioluminescence imaging indicated that the Luc signal of the Hepa1-6-FLuc cells was consistently strengthened with increases in tumor mass; however, the Luc signal of Hepa1-6-AdFLuc became weaker and eventually disappeared during tumor development. Therefore, compared with the transient expression by adenovirus, stable expression of the FLuc gene in Hepa1-6 cells may better reflect cell proliferation and survival in vivo , and provide a reliable source for the establishment of hepatocarcinoma models.

In Vitro Screening for Cytotoxic Activity of Herbal Extracts

PubMed Central

Lombardi, Valter R. M.; Cacabelos, Ramón

2017-01-01

Experimental studies have shown that a variety of chemopreventive plant components affect tumor initiation, promotion, and progression and the main difference, between botanical medicines and synthetic drugs, resides in the presence of complex metabolite mixtures shown by botanical medicine which in turn exert their action on different levels and via different mechanisms. In the present study, we performed an in vitro screening of ethanol extracts from commercial plants in order to investigate potential antitumor activity against human tumor cell lines. Experimental results obtained through a variety of methods and techniques indicated that extracts of I. verum, G. glabra, R. Frangula, and L. usitatissimum present significant reduction in in vitro tumor cell proliferation, suggesting these extracts as possible chemotherapeutical adjuvants for different cancer treatments. PMID:28386288

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

PubMed

Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

1992-01-01

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

Predicting selective drug targets in cancer through metabolic networks

PubMed Central

Folger, Ori; Jerby, Livnat; Frezza, Christian; Gottlieb, Eyal; Ruppin, Eytan; Shlomi, Tomer

2011-01-01

The interest in studying metabolic alterations in cancer and their potential role as novel targets for therapy has been rejuvenated in recent years. Here, we report the development of the first genome-scale network model of cancer metabolism, validated by correctly identifying genes essential for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40% are targeted by known, approved or experimental anticancer drugs, and the rest are new. It further predicts combinations of synthetic lethal drug targets, whose synergy is validated using available drug efficacy and gene expression measurements across the NCI-60 cancer cell line collection. Finally, potential selective treatments for specific cancers that depend on cancer type-specific downregulation of gene expression and somatic mutations are compiled. PMID:21694718

Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.

PubMed

Xiao, Mei; An, LiLong; Yang, XueYi; Ge, Xin; Qiao, Hai; Zhao, Ting; Ma, XiaoFei; Fan, JingZhuang; Zhu, MengYang; Dou, ZhongYing

2008-09-01

The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant transplantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1 x 10(9) mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with beta-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet-like clusters, as identified by dithizone staining, which expressed the transcription factor of the insulin and secreted the insulin and C-peptide. Furthermore, the transplantation of mhPSCs-induced pancreatic islets into the subcapsular region of the kidney in streptozotocin-induced diabetic rats could reduce blood glucose levels and prolong the life time.

Stochastic modeling and experimental analysis of phenotypic switching and survival of cancer cells under stress

NASA Astrophysics Data System (ADS)

Zamani Dahaj, Seyed Alireza; Kumar, Niraj; Sundaram, Bala; Celli, Jonathan; Kulkarni, Rahul

The phenotypic heterogeneity of cancer cells is critical to their survival under stress. A significant contribution to heterogeneity of cancer calls derives from the epithelial-mesenchymal transition (EMT), a conserved cellular program that is crucial for embryonic development. Several studies have investigated the role of EMT in growth of early stage tumors into invasive malignancies. Also, EMT has been closely associated with the acquisition of chemoresistance properties in cancer cells. Motivated by these studies, we analyze multi-phenotype stochastic models of the evolution of cancers cell populations under stress. We derive analytical results for time-dependent probability distributions that provide insights into the competing rates underlying phenotypic switching (e.g. during EMT) and the corresponding survival of cancer cells. Experimentally, we evaluate these model-based predictions by imaging human pancreatic cancer cell lines grown with and without cytotoxic agents and measure growth kinetics, survival, morphological changes and (terminal evaluation of) biomarkers with associated epithelial and mesenchymal phenotypes. The results derived suggest approaches for distinguishing between adaptation and selection scenarios for survival in the presence of external stresses.

[Experimental study on aging effect of Angelica sinensis polysaccharides combined with cytarabine on human leukemia KG1alpha cell lines].

PubMed

Xu, Chun-Yan; Geng, Shan; Liu, Jun; Zhu, Jia-Hong; Zhang, Xian-Ping; Jiang, Rong; Wang, Ya-Ping

2014-04-01

The latest findings of our laboratory showed that Angelica sinensis polysaccharide (ASP) showed a definite effect in regulating the aging of hematopoietic stem cells. Leukemia is a type of malignant hematopoietic tumor in hematopoietic stem cells. There have been no relevant reports about ASP's effect in regulating the aging of leukemia cells. In this study, human acute myeloid leukemia (AML) KG1alpha cell lines in logarithmic growth phase were taken as the study object, and were divided into the ASP group, the cytarabine (Ara-C) group, the ASP + Ara-C group and the control group. The groups were respectively treated with different concentration of ASP, Ara-C and ASP + Ara-C for different periods, with the aim to study the effect of ASP combined with Ara-C in regulating the aging of human acute myeloid leukemia KG1alpha cell lines and its relevant mechanism. The results showed that ASP, Ara-C and ASP + Ara-C could obviously inhibit KG1alpha cell proliferation in vitro, block the cells in G0/G1 phase. The cells showed the aging morphological feature. The percentage of positive stained aging cells was dramatically increased, and could significantly up-regulate the expression of aging-related proteins P16 and RB, which were more obvious in the ASP + Ara-C group. In conclusion, the aging mechanism of KG1alpha cell induced by ASP and Ara-C may be related to the regulation of the expression of aging-related proteins, suggesting that the combined administration of ASP and anticancer drugs plays a better role in the treatment of leukemia .

Effects of an inhibitor of the γ-secretase complex on proliferation and apoptotic parameters in a FOXL2-mutated granulosa tumor cell line (KGN).

PubMed

Irusta, Griselda; Pazos, Maria Camila; Maidana, Camila Pazos; Abramovich, Dalhia; De Zúñiga, Ignacio; Parborell, Fernanda; Tesone, Marta

2013-07-01

Ovarian granulosa cell tumors (GCTs) represent 3%-5% of all ovarian malignancies. Treatments have limited proven efficacy and biologically targeted treatment is lacking. The aim of this study was to investigate the role of Notch signaling in the proliferation, steroidogenesis, apoptosis, and phosphatidylinositol 3-kinase (PI3K)/AKT pathway in a FOXL2-mutated granulosa tumor cell line (KGN) representative of the adult form of GCTs. When Notch signaling is initiated, the receptors expose a cleavage site in the extracellular domain to the metalloproteinase TACE and, following this cleavage, Notch undergoes another cleavage mediated by the presenilin-gamma-secretase complex. To achieve our goal, DAPT, an inhibitor of the gamma-secretase complex, was used to investigate the role of the Notch system in parameters associated with cell growth and death, using a human granulosa cell tumor line (KGN) as an experimental model. We observed that JAGGED1, DLL4, NOTCH1, and NOTCH4 were highly expressed in KGN cells as compared to granulosa-lutein cells obtained from assisted reproductive techniques patients. The proliferation and viability of KGN cells, as well as progesterone and estradiol production, decreased in the presence of 20 μM DAPT. Apoptotic parameters like PARP and caspase 8 cleavages, BAX, and BCLXs increased in KGN cells cultured with DAPT, whereas others such as BCL2, BCLXl, FAS, and FAS ligand did not change. AKT phosphorylation decreased and PTEN protein increased when Notch signaling was inhibited in KGN cells. We conclude that the Notch system acts as a survival pathway in KGN cells, and might be interacting with the PI3K/AKT pathway.

Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

PubMed

Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

2017-12-01

Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

Positive regulation of spondin 2 by thyroid hormone is associated with cell migration and invasion.

PubMed

Liao, Chen-Hsin; Yeh, Shih-Chi; Huang, Ya-Hui; Chen, Ruey-Nan; Tsai, Ming-Ming; Chen, Wei-Jan; Chi, Hsiang-Cheng; Tai, Pei-Ju; Liao, Chia-Jung; Wu, Sheng-Ming; Cheng, Wan-Li; Pai, Li-Mei; Lin, Kwang-Huei

2010-03-01

The thyroid hormone 3,3',5-triiodo-L-thyronine (T(3)) regulates growth, development, and differentiation processes in animals. These activities are mediated by the nuclear thyroid hormone receptors (TRs). Microarray analyses were performed previously to study the mechanism of regulation triggered by T(3) treatment in hepatoma cell lines. The results showed that spondin 2 was regulated positively by T(3). However, the underlying mechanism and the physiological role of T(3) in the regulation of spondin 2 are not clear. To verify the microarray results, spondin 2 was further investigated using semi-quantitative reverse transcription-PCR and western blotting. After 48 h of T(3) treatment in the HepG2-TR alpha 1#1 cell line, spondin 2 mRNA and protein levels increased by 3.9- to 5.7-fold. Similar results were observed in thyroidectomized rats. To localize the regulatory region in spondin 2, we performed serial deletions of the promoter and chromatin immunoprecipitation assays. The T(3) response element on the spondin 2 promoter was localized in the -1104/-1034 or -984/-925 regions. To explore the effect of spondin 2 on cellular function, spondin 2 knockdown cell lines were established from Huh7 cells. Knockdown cells had higher migration ability and invasiveness compared with control cells. Conversely, spondin 2 overexpression in J7 cells led to lower migration ability and invasiveness compared with control cells. Furthermore, this study demonstrated that spondin 2 overexpression in some types of hepatocellular carcinomas is TR dependent. Together, these experimental findings suggest that spondin 2, which is regulated by T(3), has an important role in cell invasion, cell migration, and tumor progression.

Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS

PubMed Central

2014-01-01

Background Clear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines. Methods Hewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo. Results Hewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling. Conclusions CCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism. PMID:24946937

Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS.

PubMed

Outani, Hidetatsu; Tanaka, Takaaki; Wakamatsu, Toru; Imura, Yoshinori; Hamada, Kenichiro; Araki, Nobuhito; Itoh, Kazuyuki; Yoshikawa, Hideki; Naka, Norifumi

2014-06-19

Clear cell sarcoma (CCS) is a therapeutically unresolved, aggressive, soft tissue sarcoma (STS) that predominantly affects young adults. This sarcoma is defined by t(12;22)(q13;q12) translocation, which leads to the fusion of Ewing sarcoma gene (EWS) to activating transcription factor 1 (ATF1) gene, producing a chimeric EWS-ATF1 fusion gene. We established a novel CCS cell line called Hewga-CCS and developed an orthotopic tumor xenograft model to enable comprehensive bench-side investigation for intensive basic and preclinical research in CCS with a paucity of experimental cell lines. Hewga-CCS was derived from skin metastatic lesions of a CCS developed in a 34-year-old female. The karyotype and chimeric transcript were analyzed. Xenografts were established and characterized by morphology and immunohistochemical reactivity. Subsequently, the antitumor effects of pazopanib, a recently approved, novel, multitargeted, tyrosine kinase inhibitor (TKI) used for the treatment of advanced soft tissue sarcoma, on Hewga-CCS were assessed in vitro and in vivo. Hewga-CCS harbored the type 2 EWS-ATF1 transcript. Xenografts morphologically mimicked the primary tumor and expressed S-100 protein and antigens associated with melanin synthesis (Melan-A, HMB45). Pazopanib suppressed the growth of Hewga-CCS both in vivo and in vitro. A phospho-receptor tyrosine kinase array revealed phosphorylation of c-MET, but not of VEGFR, in Hewga-CCS. Subsequent experiments showed that pazopanib exerted antitumor effects through the inhibition of HGF/c-MET signaling. CCS is a rare, devastating disease, and our established CCS cell line and xenograft model may be a useful tool for further in-depth investigation and understanding of the drug-sensitivity mechanism.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells

PubMed Central

Alonezi, Sanad; Tusiimire, Jonans; Wallace, Jennifer; Dufton, Mark J.; Parkinson, John A.; Young, Louise C.; Clements, Carol J.; Park, Jin-Kyu; Jeon, Jong-Woon; Ferro, Valerie A.; Watson, David G.

2017-01-01

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 μg/mL melittin + 2 μg/mL cisplatin) and A2780CR (combination treatment 2 μg/mL melittin + 10 μg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R2) and goodness of prediction (Q2), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone. PMID:28420117

[Tumor cells transfer between the patient and laboratory animal as a basic methodological approach to the study of cancerogenesis and identification of biomarkers].

PubMed

Klos, D; Stašek, M; Loveček, M; Skalický, P; Vrba, R; Aujeský, R; Havlík, R; Neoral, Č; Varanashi, L; Hajdúch, M; Vrbková, J; Džubák, P

The investigation of prognostic and predictive factors for early diagnosis of tumors, their surveillance and monitoring of the impact of therapeutic modalities using hybrid laboratory models in vitro/in vivo is an experimental approach with a significant potential. It is preconditioned by the preparation of in vivo tumor models, which may face a number of potential technical difficulties. The assessment of technical success of grafting and xenotransplantation based on the type of the tumor or cell line is important for the preparation of these models and their further use for proteomic and genomic analyses. Surgically harvested gastrointestinal tract tumor tissue was processed or stable cancer cell lines were cultivated; the viability was assessed, and subsequently the cells were inoculated subcutaneously to SCID mice with an individual duration of tumor growth, followed by its extraction. We analysed 140 specimens of tumor tissue including 17 specimens of esophageal cancer (viability 13/successful inoculations 0), 13 tumors of the cardia (11/0), 39 gastric tumors (24/4), 47 pancreatic tumors (34/1) and 24 specimens of colorectal cancer (22/9). 3 specimens were excluded due to histological absence of the tumor (complete remission after neoadjuvant therapy in 2 cases of esophageal carcinoma, 1 case of chronic pancreatitis). We observed successful inoculation in 17 of 28 tumor cell lines. The probability of successful grafting to the mice model in tumors of the esophagus, stomach and pancreas is significantly lower in comparison with colorectal carcinoma and cell lines generated tumors. The success rate is enhanced upon preservation of viability of the harvested tumor tissue, which depends on the sequence of clinical and laboratory algorithms with a high level of cooperation.Key words: proteomic analysis - xenotransplantation - prognostic and predictive factors - gastrointestinal tract tumors.

Efficacy of ribavirin against malignant glioma cell lines: Follow-up study

PubMed Central

Ochiai, Yushi; Sano, Emiko; Okamoto, Yutaka; Yoshimura, Sodai; Makita, Kotaro; Yamamuro, Shun; Ohta, Takashi; Ogino, Akiyoshi; Tadakuma, Hisashi; Ueda, Takuya; Nakayama, Tomohiro; Hara, Hiroyuki; Yoshino, Atsuo; Katayama, Yoichi

2018-01-01

Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 µM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time-lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin. PMID:29251333

Translational Genomics Research Institute: Quantified Cancer Cell Line Encyclopedia (CCLE) RNA-seq Data | Office of Cancer Genomics

Cancer.gov

Many applications analyze quantified transcript-level abundances to make inferences.  Having completed this computation across the large sample set, the CTD2 Center at the Translational Genomics Research Institute presents the quantified data in a straightforward, consolidated form for these types of analyses.   Experimental Approaches  

Octyl Methoxycinnamate Modulates Gene Expression and Prevents Cyclobutane Pyrimidine Dimer Formation but not Oxidative DNA Damage in UV-Exposed Human Cell Lines

PubMed Central

Duale, Nur; Olsen, Ann-Karin; Christensen, Terje; Butt, Shamas T.; Brunborg, Gunnar

2010-01-01

Octyl methoxycinnamate (OMC) is one of the most widely used sunscreen ingredients. To analyze biological effects of OMC, an in vitro approach was used implying ultraviolet (UV) exposure of two human cell lines, a primary skin fibroblast (GM00498) and a breast cancer (MCF-7) cell lines. End points include cell viability assessment, assay of cyclobutane pyrimidine dimers (CPDs) and oxidated DNA lesions using alkaline elution and lesion-specific enzymes, and gene expression analysis of a panel of 17 DNA damage–responsive genes. We observed that OMC provided protection against CPDs, and the degree of protection correlated with the OMC-mediated reduction in UV dose. No such protection was found with respect to oxidative DNA lesions. Upon UV exposure in the presence of OMC, the gene expression studies showed significant differential changes in some of the genes studied and the expression of p53 protein was also changed. For some genes, the change in expression seemed to be delayed in time by OMC. The experimental approach applied in this study, using a panel of 17 genes in an in vitro cellular system together with genotoxicity assays, may be useful in the initial screening of active ingredients in sunscreens. PMID:20071424

MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma

PubMed Central

Lau, Diana T.; Flemming, Claudia L.; Gherardi, Samuele; Perini, Giovanni; Oberthuer, André; Fischer, Matthias; Juraeva, Dilafruz; Brors, Benedikt; Xue, Chengyuan; Norris, Murray D.; Marshall, Glenn M.; Haber, Michelle

2015-01-01

MYCN amplification occurs in 20% of neuroblastomas and is strongly related to poor clinical outcome. We have identified folate-mediated one-carbon metabolism as highly upregulated in neuroblastoma tumors with MYCN amplification and have validated this finding experimentally by showing that MYCN amplified neuroblastoma cell lines have a higher requirement for folate and are significantly more sensitive to the antifolate methotrexate than cell lines without MYCN amplification. We have demonstrated that methotrexate uptake in neuroblastoma cells is mediated principally by the reduced folate carrier (RFC; SLC19A1), that SLC19A1 and MYCN expression are highly correlated in both patient tumors and cell lines, and that SLC19A1 is a direct transcriptional target of N-Myc. Finally, we assessed the relationship between SLC19A1 expression and patient survival in two independent primary tumor cohorts and found that SLC19A1 expression was associated with increased risk of relapse or death, and that SLC19A1 expression retained prognostic significance independent of age, disease stage and MYCN amplification. This study adds upregulation of folate-mediated one-carbon metabolism to the known consequences of MYCN amplification, and suggests that this pathway might be targeted in poor outcome tumors with MYCN amplification and high SLC19A1 expression. PMID:25860940

Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation.

PubMed

Shi, Puyu; Oh, You-Take; Deng, Liang; Zhang, Guojing; Qian, Guoqing; Zhang, Shuo; Ren, Hui; Wu, Grant; Legendre, Benjamin; Anderson, Emily; Ramalingam, Suresh S; Owonikoko, Taofeek K; Chen, Mingwei; Sun, Shi-Yong

2017-11-01

Purpose: The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental Design: AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytometry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenograft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results: AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo Conclusions: Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291. Clin Cancer Res; 23(21); 6567-79. ©2017 AACR . ©2017 American Association for Cancer Research.

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells

PubMed Central

Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N.; Glenn, Sean T.; Liu, Song; Trump, Donald L.; Johnson, Candace S.

2014-01-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. MiRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253 J and 253J-BV cells express endogenous vitamin D receptor (VDR) which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253 J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. PMID:25263658

BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

PubMed Central

Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

2016-01-01

BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

In vivo contribution of nestin- and GLAST-lineage cells to adult hippocampal neurogenesis

PubMed Central

DeCarolis, Nathan A.; Mechanic, Maxwell; Petrik, David; Carlton, Adam; Ables, Jessica L.; Malhotra, Shveta; Bachoo, Robert; Götz, Magdalena; Lagace, Diane C.; Eisch, Amelia J.

2013-01-01

Radial glia-like cells (RGCs) are the hypothesized source of adult hippocampal neurogenesis. However, the current model of hippocampal neurogenesis does not fully incorporate the in vivo heterogeneity of RGCs. In order to better understand the contribution of different RGC subtypes to adult hippocampal neurogenesis, we employed widely-used transgenic lines (Nestin-CreERT2 and GLAST∷CreERT2 mice) to explore how RGCs contribute to neurogenesis under basal conditions and after stimulation and depletion of neural progenitor cells. We first used these inducible fate-tracking transgenic lines to define the similarities and differences in the contribution of nestin- and GLAST-lineage cells to basal long-term hippocampal neurogenesis. We then explored the ability of nestin- and GLAST-lineage RGCs to contribute to neurogenesis, after experimental manipulations that either ablate neurogenesis (i.c.v. application of the anti-mitotic AraC, cytosine-β-D-arabinofuranoside) or stimulate neurogenesis (wheel running). Interestingly, in both ablation and stimulation experiments, labeled RGCs in GLAST∷CreERT2 mice appear to contribute to neurogenesis, whereas RGCs in Nestin-CreERT2 mice do not. Finally, using NestinGFP reporter mice, we expanded on previous research by showing that not all RGCs in the adult dentate gyrus subgranular zone express nestin, and therefore RGCs are antigenically heterogeneous. These findings are important for the field, as they allow appropriately conservative interpretation of existing and future data that emerge from these inducible transgenic lines. These findings also raise important questions about the differences between transgenic driver lines, the heterogeneity of RGCs, and the potential differences in progenitor cell behavior between transgenic lines. As these findings highlight the possible differences in the contribution of nestin and GLAST lineage cells to long-term neurogenesis in vivo, they indicate that the current models of hippocampal neurogenesis should be modified to include RGC lineage heterogeneity. PMID:23554226

In vitro and in vivo antitumor effects of the essential oil from the leaves of Guatteria friesiana.

PubMed

Britto, Anny C S; de Oliveira, Allan C A; Henriques, Raissa M; Cardoso, Gabriella M B; Bomfim, Diogo S; Carvalho, Adriana A; Moraes, Manoel O; Pessoa, Claudia; Pinheiro, Maria L B; Costa, Emmanoel V; Bezerra, Daniel P

2012-03-01

Guatteria friesiana (W. A. Rodrigues) Erkens & Maas (synonym Guatteriopsis friesiana W. A. Rodrigues), popularly known as "envireira", is a medicinal plant found in the Brazilian and Colombian Amazon basin that is used in traditional medicine for various purposes. Recent studies on this species have demonstrated antimicrobial activity. In this study, the antitumor activity of the essential oil from the leaves of G. friesiana (EOGF) and its main components ( α-, β-, and γ-eudesmol) were determined using experimental models. In the in vitro study, EOGF and its components α-, β-, and γ-eudesmol displayed cytotoxicity against tumor cell lines, showing IC₅₀ values in the range of 1.7 to 9.4 µg/mL in the HCT-8 and HL-60 cell lines for EOGF, 5.7 to 19.4 µg/mL in the HL-60 and MDA-MB-435 cell lines for α-eudesmol, 24.1 to > 25 µg/mL in the SF-295 and MDA-MB-435 cell lines for β-eudesmol, and 7.1 to 20.6 µg/mL in the SF-295 and MDA-MB-435 cell lines for γ-eudesmol, respectively. In the in vivo study, the antitumor effect of EOGF was evaluated in mice inoculated with sarcoma 180 tumor cells. Tumor growth inhibition rates were 43.4-54.2 % and 6.6-42.8 % for the EOGF treatment by intraperitoneal (50 and 100 mg/kg/day) and oral (100 and 200 mg/kg/day) administration, respectively. The treatment with EOGF did not significantly affect body mass, macroscopy of the organs, or blood leukocyte counts. Based on these results, we can conclude that EOGF possesses significant antitumor activity and has only low systemic toxicity. These effects could be assigned to its components α-, β-, and γ-eudesmol. © Georg Thieme Verlag KG Stuttgart · New York.

Silicon solar cell efficiency improvement: Status and outlook

NASA Technical Reports Server (NTRS)

Wolf, M.

1985-01-01

Efficiency and operating life is an economic attribute in silicon solar cells application. The efficiency improvements made during the 30 year existence of the silicon solar cells, from about 6% efficiency at the beginning to 19% in the most recent experimental cells is illustrated. In the more stationary periods, the effort was oriented towards improving radiation resistance and yields on the production lines, while, in other periods, the emphasis was on reaching new levels of efficiency through better cell design and improved material processing. First results were forthcoming from the recent efforts. Considerably more efficiency advancement in silicon solar cells is expected, and the anticipated attainment of efficiencies significantly above 20% is discussed. Major advances in material processing and in the resulting material perfection are required.

Effects of continuous wave and fractionated diode laser on human fibroblast cancer and dermal normal cells by zinc phthalocyanine in photodynamic therapy: A comparative study.

PubMed

Navaeipour, Farzaneh; Afsharan, Hadi; Tajalli, Habib; Mollabashi, Mahmood; Ranjbari, Farideh; Montaseri, Azadeh; Rashidi, Mohammad-Reza

2016-08-01

In this experimental study, cancer and normal cells behavior during an in vitro photodynamic therapy (PDT) under exposure of continuous wave (CW) and fractionated mode of laser with different irradiation power and time intervals was compared and investigated. At the first, human fibroblast cancer cell line (SW 872) and human dermal normal (HFFF2) cell line were incubated with different concentrations of zinc phthalocyanine (ZnPc), as a PDT drug. The cells, then, were irradiated with a 675nm diode laser and the cell viability was evaluated using MTT assay. Under optimized conditions, the viability of the cancer cells was eventually reduced to 3.23% and 13.17%, and that of normal cells was decreased to 20.83% and 36.23% using CW and fractionated diode lasers, respectively. In general, the ratio of ZnPc LD50 values for the normal cells to the cancer cells with CW laser was much higher than that of the fractionated laser. Subsequently, cancer cells in comparison with normal ones were found to be more sensitive toward the photodynamic damage induced by ZnPc. In addition, treatment with CW laser was found to be more effective against the cancer cells with a lower toxicity to the normal cells compared with the fractionated diode laser. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of Invasion Dynamics of Matrix-Embedded Cells in a Multisample Format.

PubMed

Van Troys, Marleen; Masuzzo, Paola; Huyck, Lynn; Bakkali, Karima; Waterschoot, Davy; Martens, Lennart; Ampe, Christophe

2018-01-01

In vitro tests of cancer cell invasion are the "first line" tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.

Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis.

PubMed

Medjkane, Souhila; Perez-Sanchez, Cristina; Gaggioli, Cedric; Sahai, Erik; Treisman, Richard

2009-03-01

Rho GTPases control cytoskeletal dynamics through cytoplasmic effectors and regulate transcriptional activation through myocardin-related transcription factors (MRTFs), which are co-activators for serum response factor (SRF). We used RNA interference to investigate the contribution of the MRTF-SRF pathway to cytoskeletal dynamics in MDA-MB-231 breast carcinoma and B16F2 melanoma cells, in which basal MRTF-SRF activity is Rho-dependent. Depletion of MRTFs or SRF reduced cell adhesion, spreading, invasion and motility in culture, without affecting proliferation or inducing apoptosis. MRTF-depleted tumour cell xenografts showed reduced cell motility but proliferated normally. Tumour cells depleted of MRTF or SRF failed to colonize the lung from the bloodstream, being unable to persist after their arrival in the lung. Only a few genes show MRTF-dependent expression in both cell lines. Two of these, MYH9 (NMHCIIa) and MYL9 (MLC2), are also required for invasion and lung colonization. Conversely, expression of activated MAL/MRTF-A increases lung colonization by poorly metastatic B16F0 cells. Actin-based cell behaviour and experimental metastasis thus require Rho-dependent nuclear signalling through the MRTF-SRF network.

Influenza A Virus Polymerase Is a Site for Adaptive Changes during Experimental Evolution in Bat Cells

PubMed Central

Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.

2014-01-01

ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity. PMID:25142579

Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias.

PubMed

Parton, Angela; Forest, David; Kobayashi, Hiroshi; Dowell, Lori; Bayne, Christopher; Barnes, David

2007-02-01

Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.

The Distribution of Chromosomal Aberrations in Human Cells Predicted by a Generalized Time-Dependent Model of Radiation-Induced Formation of Aberrations

NASA Technical Reports Server (NTRS)

Ponomarev, Artem L.; George, K.; Cucinotta, F. A.

2011-01-01

New experimental data show how chromosomal aberrations for low- and high-LET radiation are dependent on DSB repair deficiencies in wild-type, AT and NBS cells. We simulated the development of chromosomal aberrations in these cells lines in a stochastic track-structure-dependent model, in which different cells have different kinetics of DSB repair. We updated a previously formulated model of chromosomal aberrations, which was based on a stochastic Monte Carlo approach, to consider the time-dependence of DSB rejoining. The previous version of the model had an assumption that all DSBs would rejoin, and therefore we called it a time-independent model. The chromosomal-aberrations model takes into account the DNA and track structure for low- and high-LET radiations, and provides an explanation and prediction of the statistics of rare and more complex aberrations. We compared the program-simulated kinetics of DSB rejoining to the experimentally-derived bimodal exponential curves of the DSB kinetics. We scored the formation of translocations, dicentrics, acentric and centric rings, deletions, and inversions. The fraction of DSBs participating in aberrations was studied in relation to the rejoining time. Comparisons of simulated dose dependence for simple aberrations to the experimental dose-dependence for HF19, AT and NBS cells will be made.

Epithelial Membrane Protein-2 is a Novel Therapeutic Target in Ovarian Cancer

PubMed Central

Fu, Maoyong; Maresh, Erin L.; Soslow, Robert A.; Alavi, Mohammad; Mah, Vei; Zhou, Qin; Iasonos, Alexia; Goodglick, Lee; Gordon, Lynn K.; Braun, Jonathan; Wadehra, Madhuri

2010-01-01

Purpose The tetraspan protein epithelial membrane protein-2 (EMP2) has been shown to regulate the surface display and signaling from select integrin pairs, and it was recently identified as a prognostic biomarker in human endometrial cancer. In this study, we assessed the role of EMP2 in human ovarian cancer. Experimental Design We examined the expression of EMP2 within a population of women with ovarian cancer using tissue microarray assay technology. We evaluated the efficacy of EMP2-directed antibody therapy using a fully human recombinant bivalent antibody fragment (diabody) in vitro and ovarian cancer xenograft models in vivo. Results EMP2 was found to be highly expressed in over 70% of serous and endometrioid ovarian tumors compared to non-malignant ovarian epithelium using a human ovarian cancer tissue microarray. Using anti-EMP2 diabody, we evaluated the in vitro response of 9 human ovarian cancer cell lines with detectable EMP2 expression. Treatment of human ovarian cancer cell lines with anti-EMP2 diabodies induced cell death and retarded cell growth, and these response rates correlated with cellular EMP2 expression. We next assessed the effects of anti-EMP2 diabodies in mice bearing xenografts from the ovarian endometrioid carcinoma cell line OVCAR5. Anti-EMP2 diabodies significantly suppressed tumor growth and induced cell death in OVCAR5 xenografts. Conclusions These findings indicate that EMP2 is expressed in the majority of ovarian tumors and it may be a feasible target in vivo. PMID:20670949

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

PubMed

Guruceaga, Elizabeth; Garin-Muga, Alba; Prieto, Gorka; Bejarano, Bartolomé; Marcilla, Miguel; Marín-Vicente, Consuelo; Perez-Riverol, Yasset; Casal, J Ignacio; Vizcaíno, Juan Antonio; Corrales, Fernando J; Segura, Victor

2017-12-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach

PubMed Central

2017-01-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations. PMID:28960077

[Experimental study on carcinogenesis by human papillomavirus type 8 E7 gene].

PubMed

Nishikawa, T

1994-05-01

Human papillomavirus (HPV) 5 and HPV8 are often detected in skin cancers developed in patients suffering from epidermodysplasia verruciformis, as well as in skin cancers developed in immunosuppressed patients. In the present study, in order to examine the transforming activity of the HPV8E7 gene, the HPV8E7 and HPV8E6/E7 genes were cloned into the expression vector (pcD2-Y), under the SV40 enhancer/promoter to construct pcD2-8E7 and pcD2-8E6/E7, respectively. The E7 and E6/E7 genes of genital high-risk HPV16 were also cloned into pcD2-Y to construct pcD2-16E7 and pcD2-16E6/E7, respectively. They were tested for their ability to collaboratively transform primary rat embryo fibroblasts (REFs) with activated H-ras gene. Transfection experiments of REFs having an activated H-ras gene revealed that pcD2-8E7, as well as pcD2-16E7 and pcD2-16E6/E7, induced transformation of cells in G418-resistant colonies at efficiencies of 11.9%, 43.0% and 53.0%, respectively. Transformed cell lines induced by activated H-ras gene and pcD2-8E7 or pcD2-16E7 were named 8RE and 16RE cell lines, respectively. Tumor induction in syngeneic newborn rats by injected the 8RE cells was higher than that of the 16RE cells. In cytological and histological examination, the 8RE cell lines and their induced tumors were different from the 16RE cell lines and their induced tumors. The 8RE cell lines showed the characteristic transformation with efficient growth ability on plastic and colony formation in 0.3% soft agar. These results support the hypothesis that the HPV8E7 gene plays an important role in the carcinogenesis of skin cancers.

GPER Mediates Non-Genomic Effects of Estrogen.

PubMed

Pupo, Marco; Maggiolini, Marcello; Musti, Anna Maria

2016-01-01

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

LABORATORY MEASUREMENTS OF WHITE DWARF PHOTOSPHERIC SPECTRAL LINES: Hβ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Falcon, Ross E.; Gomez, T. A.; Montgomery, M. H.

2015-06-20

We spectroscopically measure multiple hydrogen Balmer line profiles from laboratory plasmas to investigate the theoretical line profiles used in white dwarf (WD) atmosphere models. X-ray radiation produced at the Z Pulsed Power Facility at Sandia National Laboratories initiates plasma formation in a hydrogen-filled gas cell, replicating WD photospheric conditions. Here we present time-resolved measurements of Hβ and fit this line using different theoretical line profiles to diagnose electron density, n{sub e}, and n = 2 level population, n{sub 2}. Aided by synthetic tests, we characterize the validity of our diagnostic method for this experimental platform. During a single experiment, wemore » infer a continuous range of electron densities increasing from n{sub e} ∼ 4 to ∼30 × 10{sup 16} cm{sup −3} throughout a 120-ns evolution of our plasma. Also, we observe n{sub 2} to be initially elevated with respect to local thermodynamic equilibrium (LTE); it then equilibrates within ∼55 ns to become consistent with LTE. This supports our electron-temperature determination of T{sub e} ∼ 1.3 eV (∼15,000 K) after this time. At n{sub e} ≳ 10{sup 17} cm{sup −3}, we find that computer-simulation-based line-profile calculations provide better fits (lower reduced χ{sup 2}) than the line profiles currently used in the WD astronomy community. The inferred conditions, however, are in good quantitative agreement. This work establishes an experimental foundation for the future investigation of relative shapes and strengths between different hydrogen Balmer lines.« less

Verification of ALDH Activity as a Biomarker in Colon Cancer Stem Cells-Derived HT-29 Cell Line.

PubMed

Khorrami, Samaneh; Zavaran Hosseini, Ahmad; Mowla, Seyed Javad; Malekzadeh, Reza

2015-10-01

Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ''stemness'' genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn't seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

Counter-Flow Cooling Tower Test Cell

NASA Astrophysics Data System (ADS)

Dvořák, Lukáš; Nožička, Jiří

2014-03-01

The article contains a design of a functional experimental model of a cross-flow mechanical draft cooling tower and the results and outcomes of measurements. This device is primarily used for measuring performance characteristics of cooling fills, but with a simple rebuild, it can be used for measuring other thermodynamic processes that take part in so-called wet cooling. The main advantages of the particular test cell lie in the accuracy, size, and the possibility of changing the water distribution level. This feature is very useful for measurements of fills of different heights without the influence of the spray and rain zone. The functionality of this test cell has been verified experimentally during assembly, and data from the measurement of common film cooling fills have been compared against the results taken from another experimental line. For the purpose of evaluating the data gathered, computational scripts were created in the MATLAB numerical computing environment. The first script is for exact calculation of the thermal balance of the model, and the second is for determining Merkel's number via Chebyshev's method.

Regenerative toxicology: the role of stem cells in the development of chronic toxicities.

PubMed

Canovas-Jorda, David; Louisse, Jochem; Pistollato, Francesca; Zagoura, Dimitra; Bremer, Susanne

2014-01-01

Human stem cell lines and their derivatives, as alternatives to the use of animal cells or cancer cell lines, have been widely discussed as cellular models in predictive toxicology. However, the role of stem cells in the development of long-term toxicities and carcinogenesis has not received great attention so far, despite growing evidence indicating the relationship of stem cell damage to adverse effects later in life. However, testing this in vitro is a scientific/technical challenge in particular due to the complex interplay of factors existing under physiological conditions. Current major research programs in stem cell toxicity are not aiming to demonstrate that stem cells can be targeted by toxicants. Therefore, this knowledge gap needs to be addressed in additional research activities developing technical solutions and defining appropriate experimental designs. The current review describes selected examples of the role of stem cells in the development of long-term toxicities in the brain, heart or liver and in the development of cancer. The presented examples illustrate the need to analyze the contribution of stem cells to chronic toxicity in order to make a final conclusion whether stem cell toxicities are an underestimated risk in mechanism-based safety assessments. This requires the development of predictive in vitro models allowing the assessment of adverse effects to stem cells on chronic toxicity and carcinogenicity.

EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity.

PubMed

Olivier, Stéphane; Jacoby, Marine; Brillon, Cédric; Bouletreau, Sylvana; Mollet, Thomas; Nerriere, Olivier; Angel, Audrey; Danet, Sévérine; Souttou, Boussad; Guehenneux, Fabienne; Gauthier, Laurent; Berthomé, Mathilde; Vié, Henri; Beltraminelli, Nicola; Mehtali, Majid

2010-01-01

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive grown characteristics such as a very short population doubling time of 12 to 14 hours, a capacity to reach very high cell density (> 30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

PubMed Central

2011-01-01

Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests. PMID:21824406

Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

PubMed

Carroll, Kelli J; North, Trista E

2014-08-01

Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Chemopreventive potential of β-Sitosterol in experimental colon cancer model - an In vitro and In vivo study

PubMed Central

2010-01-01

Background Asclepias curassavica Linn. is a traditional medicinal plant used by tribal people in the western ghats, India, to treat piles, gonorrhoea, roundworm infestation and abdominal tumours. We have determined the protective effect of β-sitosterol isolated from A. curassavica in colon cancer, using in vitro and in vivo models. Methods The active molecule was isolated, based upon bioassay guided fractionation, and identified as β-sitosterol on spectral evidence. The ability to induce apoptosis was determined by its in vitro antiradical activity, cytotoxic studies using human colon adenocarcinoma and normal monkey kidney cell lines, and the expression of β-catenin and proliferating cell nuclear antigen (PCNA) in human colon cancer cell lines (COLO 320 DM). The chemopreventive potential of β-sitosterol in colon carcinogenesis was assessed by injecting 1,2-dimethylhydrazine (DMH, 20 mg/kg b.w.) into male Wistar rats and supplementing this with β-sitosterol throughout the experimental period of 16 weeks at 5, 10, and 20 mg/kg b.w. Results β-sitosterol induced significant dose-dependent growth inhibition of COLO 320 DM cells (IC50 266.2 μM), induced apoptosis by scavenging reactive oxygen species, and suppressed the expression of β-catenin and PCNA antigens in human colon cancer cells. β-sitosterol supplementation reduced the number of aberrant crypt and crypt multiplicity in DMH-initiated rats in a dose-dependent manner with no toxic effects. Conclusion We found doses of 10-20 mg/kg b.w. β-sitosterol to be effective for future in vivo studies. β-sitosterol had chemopreventive potential by virtue of its radical quenching ability in vitro, with minimal toxicity to normal cells. It also attenuated β-catenin and PCNA expression, making it a potential anticancer drug for colon carcinogenesis. PMID:20525330

Evaporation of binary mixtures in microgravity

NASA Technical Reports Server (NTRS)

Girgis, Morris; Matta, Nabil; Kolli, Kiran; Brown, Leon; Chubb, Kevin

1995-01-01

The motivation of this research is to obtain a better understanding of phase-change heat transfer within single and binary liquid meniscii, both in 1-g and 0-g environments. During phase 1 and part of phase 2, in a glass test cell with an inclined heated plate, 1-6 experiments on pentane with additions of decane up to 3% were conducted to determine the optimum concentration that will exhibit the maximum heat transfer and stability. During phase 2 emphasis was given to explore fundamental research issues and to ultimately develop a reliable capillary pumped loop (CPL) device for low gravity. In related experimental work, it was found that thermocapillary stresses near the contract line could result in a degraded wettability which ultimately could explain the observed failure of CPL devices in zero-gravity environment. Therefore, the current experimental effort investigates the effect of adding binary constituents in improving the thermocapillary characteristics near the contact line within the loop configuration. Achievements during second phase include: (1) Further enhancement of Central State University's Microgravity Laboratory by adding or improving upon capabilities of photography, video imaging, fluid visualization, and general experimental testing capabilities; (2) Experimental results for the inclined plate cell; (3) Modeling effort with a detailed scaling analysis; (4) Additional testing with a tube loop configuration to extend experimental work by Dickens, et al.; (5) Fabrication of a capillary loop to be tested using binary fluid (pentane/decane). The device that has been recently completed will be set up horizontally so that the effect of gravity on the performance is negligible. Testing will cover a wide range of parameters such as decane/pentane concentration, heat input value, heat input location (below or above meniscus), and loop temperature.

Fourier transform spectroscopy of the nu3 band of the N3 radical

NASA Technical Reports Server (NTRS)

Brazier, C. R.; Bernath, P. F.; Burkholder, James B.; Howard, Carleton J.

1988-01-01

The nu3 transitions of N3 radicals produced by HN3-Cl reactions in a multipass cell (effective path length 100 m) are investigated experimentally using a Fourier-transform spectrometer with maximum resolution 0.004/cm. A total of 176 rotation-vibration lines are listed in a table and used, in combination with published data on 240 optical lines (Douglas and Jones, 1965), to determine the nu3 molecular constants. The lower-than-expected value of the nu3 fundamental frequency (1644.6784/cm) is attributed to the vibronic interaction discussed by Kawaguchi et al. (1981).

Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling

PubMed Central

Dasgupta, Santanu; Menezes, Mitchell E.; Das, Swadesh K.; Emdad, Luni; Janjic, Aleksandar; Bhatia, Shilpa; Mukhopadhyay, Nitai D; Shao, Chunbo; Sarkar, Devanand; Fisher, Paul B.

2013-01-01

Purpose Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Experimental Design Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors. Results Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident. Conclusions Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC. PMID:23873690

Comparison of cellular lethality in DNA repair-proficient or -deficient cell lines resulting from exposure to 70 MeV/n protons or 290 MeV/n carbon ions.

PubMed

Genet, Stefan C; Maeda, Junko; Fujisawa, Hiroshi; Yurkon, Charles R; Fujii, Yoshihiro; Romero, Ashley M; Genik, Paula C; Fujimori, Akira; Kitamura, Hisashi; Kato, Takamitsu A

2012-11-01

Charged particle therapy utilizing protons or carbon ions has been rapidly intensifying over recent years. The present study was designed to jointly investigate these two charged particle treatment modalities with respect to modeled anatomical depth-dependent dose and linear energy transfer (LET) deliveries to cells with either normal or compromised DNA repair phenotypes. We compared cellular lethality in response to dose, LET and Bragg peak location for accelerated protons and carbon ions at 70 and 290 MeV/n, respectively. A novel experimental live cell irradiation OptiCell™ in vitro culture system using three different Chinese hamster ovary (CHO) cells as a mammalian model was conducted. A wild-type DNA repair-competent CHO cell line (CHO 10B2) was compared to two other CHO cell lines (51D1 and xrs5), each genetically deficient with respect to one of the two major DNA repair pathways (homologous recombination and non-homologous end joining pathways, respectively) following genotoxic insults. We found that wild-type and homologous recombination-deficient (Rad51D) cellular lethality was dependent on both the dose and LET of the carbon ions, whereas it was only dependent on dose for protons. The non-homologous end joining deficient cell line (Ku80 mutant) showed nearly identical dose-response profiles for both carbon ions and protons. Our results show that the increasingly used modality of carbon ions as charged particle therapy is advantageous to protons in a radiotherapeutic context, primarily for tumor cells proficient in non-homologous end joining DNA repair where cellular lethality is dependent not only on the dose as in the case of more common photon therapeutic modalities, but more importantly on the carbon ion LETs. Genetic characterization of patient tumors would be key to individualize and optimize the selection of radiation modality, clinical outcome and treatment cost.

Photoluminescent graphene quantum dots for in vivo imaging of apoptotic cells

NASA Astrophysics Data System (ADS)

Roy, Prathik; Periasamy, Arun Prakash; Lin, Chiu-Ya; Her, Guor-Mour; Chiu, Wei-Jane; Li, Chi-Lin; Shu, Chia-Lun; Huang, Chih-Ching; Liang, Chi-Te; Chang, Huan-Tsung

2015-01-01

Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications.Apoptosis (programmed cell death) is linked to many incurable neurodegenerative, cardiovascular and cancer causing diseases. Numerous methods have been developed for imaging apoptotic cells in vitro; however, there are few methods available for imaging apoptotic cells in live animals (in vivo). Here we report a novel method utilizing the unique photoluminescence properties of plant leaf-derived graphene quantum dots (GQDs) modified with annexin V antibody (AbA5) to form (AbA5)-modified GQDs (AbA5-GQDs) enabling us to label apoptotic cells in live zebrafish (Danio rerio). The key is that zebrafish shows bright red photoluminescence in the presence of apoptotic cells. The toxicity of the GQDs has also been investigated with the GQDs exhibiting high biocompatibility as they were excreted from the zebrafish's body without affecting its growth significantly at a concentration lower than 2 mg mL-1 over a period of 4 to 72 hour post fertilization. The GQDs have further been used to image human breast adenocarcinoma cell line (MCF-7 cells), human cervical cancer cell line (HeLa cells), and normal human mammary epithelial cell line (MCF-10A). These results are indispensable to further the advance of graphene-based nanomaterials for biomedical applications. Electronic supplementary information (ESI) available: Experimental discussion on synthesis, characterization, cellular imaging, cytotoxicity of GQDs in addition to its effect on zebrafish embryos, preparation of annexin V (A5)-modified GQDs (AbA5-GQDs), staining procedures and imaging are given. Figures for XRD, UV-vis absorption, photoluminescence of GQDs, mortality of zebrafish, time course recording of morphology of zebrafish embryos and morphology of adult zebrafish exposed to GQDs are illustrated. See DOI: 10.1039/c4nr07005d

Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

PubMed

Schrader, Thorsten; Münter, Klaus; Kleine-Ostmann, Thomas; Schmid, Ernst

2008-12-01

The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-β1 and IL-6

PubMed Central

Ruff, Kristin R; Puetter, Adriane; Levy, Laura S

2007-01-01

Background AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation. Methods Growth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3. Results Results confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1. Conclusion These studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3. PMID:17324269

Glycogen synthase kinase-3 inhibition sensitizes human induced pluripotent stem cells to thiol-containing antioxidants induced apoptosis.

PubMed

Tu, Chengyi; Xu, Robert; Koleti, Meghana; Zoldan, Janet

2017-08-01

Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome. Copyright © 2017. Published by Elsevier B.V.

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells.

PubMed

Barnes, D W

2012-04-01

Two of the most commonly used elasmobranch experimental model species are the spiny dogfish Squalus acanthias and the little skate Leucoraja erinacea. Comparative biology and genomics with these species have provided useful information in physiology, pharmacology, toxicology, immunology, evolutionary developmental biology and genetics. A wealth of information has been obtained using in vitro approaches to study isolated cells and tissues from these organisms under circumstances in which the extracellular environment can be controlled. In addition to classical work with primary cell cultures, continuously proliferating cell lines have been derived recently, representing the first cell lines from cartilaginous fishes. These lines have proved to be valuable tools with which to explore functional genomic and biological questions and to test hypotheses at the molecular level. In genomic experiments, complementary (c)DNA libraries have been constructed, and c. 8000 unique transcripts identified, with over 3000 representing previously unknown gene sequences. A sub-set of messenger (m)RNAs has been detected for which the 3' untranslated regions show elements that are remarkably well conserved evolutionarily, representing novel, potentially regulatory gene sequences. The cell culture systems provide physiologically valid tools to study functional roles of these sequences and other aspects of elasmobranch molecular cell biology and physiology. Information derived from the use of in vitro cell cultures is valuable in revealing gene diversity and information for genomic sequence assembly, as well as for identification of new genes and molecular markers, construction of gene-array probes and acquisition of full-length cDNA sequences. © 2012 The Author. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

Biomarkers in Tumorigenesis Using Cancer Cell Lines: A Systematic Review

PubMed Central

K, Lizbeth Raju; Augustine, Dominic; Rao, Roopa S; SV, Sowmya; Haragannavar, Vanishri C; Nambiar, Shwetha; Prasad, Kavitha; Awan, Kamran Habib; Patil, Shankargouda

2017-01-01

Cancer is a leading cause of death worldwide. Despite many research advancements in the field, the genetic changes regulating the transformation of normal oral cells into malignant cells have not been fully elucidated. Several studies have evaluated carcinogenesis at the molecular level. Cancer cell lines are commonly used in biomedical research because they provide an unlimited source of cells and represent various stages of initiation and progression of carcinogenesis in vitro. Aims: The objective of the study was to review original research articles using cancer cell lines as a tool to understand carcinogenesis and to identify the genes involved in tumor development. Additionally, we also examined the application of the genes as predictive biomarkers. Methods and Materials: Several databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 1985 to December 2016 using various combinations of the following key words: “mouth neoplasm”, “cell lines”, and “tumorigenesis”. Original experimental studies published in English were included. We excluded letters to the editor, historic reviews, and unpublished data from the analysis. Results: There were 17 studies (in vitro) included in the analysis. There were 14 genes and 4 miRNAs involved in malignant transformation of oral keratinocytes into cancer cells. The most commonly studied genes were p53, cyclin D1, and hTERT. Conclusion: Additional reviews and studies are needed to identify a panel of genes specific to various potentially malignant disorders and to aid in the early detection of oral squamous cell carcinoma (OSCC) because tumorigenesis involves the mutation of multiple genes. Furthermore, improving advanced cost-effective diagnostic methods may benefit the public health sector. PMID:28950674

Antagonism of SET using OP449 enhances the efficacy of tyrosine kinase inhibitors and overcome drug resistance in myeloid leukemia

PubMed Central

Agarwal, Anupriya; MacKenzie, Ryan J.; Pippa, Raffaella; Eide, Christopher A.; Oddo, Jessica; Tyner, Jeffrey W.; Sears, Rosalie; Vitek, Michael P.; Odero, María D.; Christensen, Dale; Druker, Brian J.

2014-01-01

Purpose The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A. Experimental Design In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis and colonogenic assays. Efficacy of target inhibition by OP449 is evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model. Results We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells. Conclusions We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. PMID:24436473

In vitro fluoride release from a different kind of conventional and resin modified glass-ionomer cements

PubMed Central

Selimović-Dragaš, Mediha; Hasić-Branković, Lajla; Korać, Fehim; Đapo, Nermin; Huseinbegović, Amina; Kobašlija, Sedin; Lekić, Meliha; Hatibović-Kofman, Šahza

2013-01-01

Fluoride release is important characteristic of glass-ionomer cements. Quantity of fluoride ions released from the glass-ionomer cements has major importance in definition of their biological activity. The objectives of this study were to define the quantity of fluoride ions released from the experimental glass-ionomer cements and to define the effect of fluoride ions released from the experimental glass-ionomer cements on their cytotoxicity. Concentrations of the fluoride ions released in the evaluated glass-ionomer cements were measured indirectly, by the fluoride-selective WTW, F500 electrode potential, combined with reference R503/D electrode. Statistical analyses of F-ion concentrations released by all glass-ionomers evaluated at two time points, after 8 and after 24 hours, show statistically higher fluoride releases from RMGICs: Vitrebond, Fuji II LC and Fuji Plus, when compared to conventional glass-ionomer cements: Fuji Triage, Fuji IX GP Fast and Ketac Silver, both after 8 and after 24 hours. Correlation coefficient between concentrations of fluoride ion released by evaluated glass-ionomer cements and cytotoxic response of UMR-106 osteoblast cell-line are relatively high, but do not reach levels of biological significance. Correlation between concentrations of fluoride ion released and cytotoxic response of NIH3T3 mouse fibroblast cell line after 8 hours is high, positive and statistically significant for conventional GICs, Fuji Triage and Fuji IX GP Fast, and RMGIC, Fuji II LC. Statistically significant Correlation coefficient between concentrations of fluoride ion released and cytotoxic response of NIH3T3 cell line after 24 hours is defined for RMGIC Fuji II LC only. PMID:23988173

Hemochromatosis Enhances Tumor Progression via Upregulation of Intracellular Iron in Head and Neck Cancer

PubMed Central

Lenarduzzi, Michelle; Hui, Angela B. Y.; Yue, Shijun; Ito, Emma; Shi, Wei; Williams, Justin; Bruce, Jeff; Sakemura-Nakatsugawa, Noriko; Xu, Wei; Schimmer, Aaron; Liu, Fei-Fei

2013-01-01

Introduction Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC. Experimental Design Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry. Results In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression. Conclusions Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management. PMID:23991213

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity.

PubMed

Edahiro, Keitaro; Iimori, Makoto; Kobunai, Takashi; Morikawa-Ichinose, Tomomi; Miura, Daisuke; Kataoka, Yuki; Niimi, Shinichiro; Wakasa, Takeshi; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

2018-06-04

Acquired resistance to therapeutic drugs is a serious problem for cancer patients receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analog-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1-/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The present data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. 5-fluorouracil-based chemotherapy would be effective even in tumors that become refractory to trifluridine during combined trifluridine/tipiracil treatment. Copyright ©2018, American Association for Cancer Research.

Theoretical and experimental investigation of 'grating' type photovoltaic cells

NASA Technical Reports Server (NTRS)

Loferski, J. J.; Crisman, E. E.; Armitage, W.; Chen, L. Y.

1974-01-01

The fabrication procedure and properties of 'grating' cells made by forming a fine grating pattern of aluminum alloyed into n-silicon wafers are described. The finest grating lines achieved in the cells described were 5 microns; the smallest spacing was about 15 microns. The best temperature for alloying was found to be about 600 C, a bit above the Si-Al eutectic temperature (576 C). The short-circuit current obtained from the best of these cells exposed to 100 mW/sq cm of (simulated air mass zero) illumination was at least equal to that obtained from conventional diffused cells, but their open-circuit voltage was lower. Their quantum yield was strongly blue-shifted; it was flat from 4000 to 8500 A.

Regulated and Unregulated Clinical Trials of Stem Cell Therapies for Stroke

PubMed Central

Liska, Michael G.; Crowley, Marci G.; Borlongan, Cesar V.

2017-01-01

Several lines of laboratory investigations reporting solid safety profiles and robust efficacy readouts of stem cells in clinically relevant animal models have advanced stem cell transplantation as an experimental therapy for stroke. Unfortunately, translating laboratory findings into effective clinical trials entails rigorous regulatory examinations, which posed a major challenge in the application of stem cells to patients. As a consequence of this slow pace of clinical entry, and a media-propagated hype narrating stem cells as a “magic bullet”, a dangerous market has been created for unregulated stem cell clinics. These clinics are often guilty of misleading patients and delivering low-quality, even harmful, treatments. Additionally, these medical tourism-purported clinical procedures, which have been performed even in the US, are likely to negatively impact on the true science and clinical value of stem cells. For the full potential of stem cell therapies to be realized, these pressing public misconceptions and regulatory clinical concerns must be addressed. Here, we provide the scientific evidence supporting the safe and effective conduct of stem cells. Arguably, relying on such evidence-based science to dictate the translation of stem cells from the laboratory to the clinic should allow an objective assessment of the risks and the rewards, and the delineation of the hype from hope of this experimental stroke therapy. PMID:28127687

Identification of a new gene regulatory circuit involving B cell receptor activated signaling using a combined analysis of experimental, clinical and global gene expression data

PubMed Central

Schrader, Alexandra; Meyer, Katharina; Walther, Neele; Stolz, Ailine; Feist, Maren; Hand, Elisabeth; von Bonin, Frederike; Evers, Maurits; Kohler, Christian; Shirneshan, Katayoon; Vockerodt, Martina; Klapper, Wolfram; Szczepanowski, Monika; Murray, Paul G.; Bastians, Holger; Trümper, Lorenz; Spang, Rainer; Kube, Dieter

2016-01-01

To discover new regulatory pathways in B lymphoma cells, we performed a combined analysis of experimental, clinical and global gene expression data. We identified a specific cluster of genes that was coherently expressed in primary lymphoma samples and suppressed by activation of the B cell receptor (BCR) through αIgM treatment of lymphoma cells in vitro. This gene cluster, which we called BCR.1, includes numerous cell cycle regulators. A reduced expression of BCR.1 genes after BCR activation was observed in different cell lines and also in CD10+ germinal center B cells. We found that BCR activation led to a delayed entry to and progression of mitosis and defects in metaphase. Cytogenetic changes were detected upon long-term αIgM treatment. Furthermore, an inverse correlation of BCR.1 genes with c-Myc co-regulated genes in distinct groups of lymphoma patients was observed. Finally, we showed that the BCR.1 index discriminates activated B cell-like and germinal centre B cell-like diffuse large B cell lymphoma supporting the functional relevance of this new regulatory circuit and the power of guided clustering for biomarker discovery. PMID:27166259

Transport channel of secondary ion beam of experimental setup for selective laser ionization with gas cell GALS

NASA Astrophysics Data System (ADS)

Gulbekyan, G. G.; Zemlyanoy, S. G.; Bashevoy, V. V.; Ivanenko, I. A.; Kazarinov, N. Yu; Kazacha, V. I.; Osipov, N. F.

2017-07-01

GALS is the experimental setup intended for production and research of isobaric and isotopically pure heavy neutron-rich nuclei. The beam line consists of two parts. The initial part is used for transport of the primary 136Xe ion beam with the energy of 4.5-9.0 MeV/amu from the FLNR cyclotron U-400M to the Pb target for production of the studying ion beams. These beams have the following design parameters: the charge Z = +1, the mass A = 180-270 and the kinetic energy W = 40 keV. The second part placed after the target consists of the SPIG (QPIG) system, the accelerating gap, the electrostatic Einzel lens, 90-degree spectrometric magnet (calculated value of the mass-resolution is equal to 1400) and the beam line for the transportation of the ions from the magnet focal plane to a particle detector. The results of simulation of the particle dynamics and the basic parameters of all elements of the beam line are presented.

Dissecting and Targeting Latent Metastasis

DTIC Science & Technology

2013-09-01

particular. Developing and using experimental models of LMBC to elucidate these survival mechanisms will provide a basis for therapeutically targeting...stochastic, low frequency generation of lethal macrometastatic lesion. Progress Item 1.1. Using this approach, we have successfully isolated latent...cells lines are being used to illuminate the molecular basis for the latent metastasis state. Progress Item 1.2. In parallel with the isolation of

In vitro effect of phototherapy with low-intensity laser on HSV-1 and epithelial cells

NASA Astrophysics Data System (ADS)

Eduardo, Fernanda P.; Mehnert, Dolores U.; Monezi, Telma A.; Zezell, Denise M.; Schubert, Mark M.; Eduardo, Carlos P.; Marques, Márcia M.

2007-02-01

The effects of phototherapy on herpes lesions have been clinically demonstrated by either preventing the lesion formation or speeding their repair. The aim of this in vitro study was analyze the effect of phototherapy on epithelial cells and HSV-1 in culture. Cultures of HSV-1 and epithelial cells (Vero cell line) were used. The irradiations were done using a GaAlAs laser (660 e 780 nm, 4.0 mm2). One, two and three irradiations with 6 h-intervals were done. The experimental groups were: Control: non-irradiated; 660 nm and 3 J/cm2 (2.8 sec); 660 nm and 5 J/cm2 (3.8 sec); 780 nm and 3 J/cm2 (1.9 sec), and 780 nm and 5 J/cm2 (2.5 sec). The HSV-1 cytopatic effect and the cell viability of irradiated cultures and controls were analyzed in four different conditions: irradiation of non-infected epithelial cells; epithelial cells irradiated prior infection; virus irradiated prior infection; irradiation of HSV infected cells. The mitochondrial activity and cytopathic effects were assessed. The number of irradiations influenced the cell growth positively and proportionally, except for the 660 nm/ 3 J/cm2 group. Any variation in cytopathic effects was observed amongst the experimental groups. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Under the experimental conditions of this study we concluded that phototherapy is capable of enhancing epithelial cell growth and prolonging cell viability of HSV-1 infected cells. Positive benefits of phototherapy could be resultant from prolongation of infected cells viability, corroborating with host defenses.

Advances in cholangiocyte immunobiology

PubMed Central

Syal, Gaurav; Fausther, Michel

2012-01-01

Cholangiocytes, or bile duct epithelia, were once thought to be the simple lining of the conduit system comprising the intra- and extrahepatic bile ducts. Growing experimental evidence demonstrated that cholangiocytes are in fact the first line of defense of the biliary system against foreign substances. Experimental advances in recent years have unveiled previously unknown roles of cholangiocytes in both innate and adaptive immune responses. Cholangiocytes can release inflammatory modulators in a regulated fashion. Moreover, they express specialized pattern-recognizing molecules that identify microbial components and activate intracellular signaling cascades leading to a variety of downstream responses. The cytokines secreted by cholangiocytes, in conjunction with the adhesion molecules expressed on their surface, play a role in recruitment, localization, and modulation of immune responses in the liver and biliary tract. Cholangiocyte survival and function is further modulated by cytokines and inflammatory mediators secreted by immune cells and cholangiocytes themselves. Because cholangiocytes act as professional APCs via expression of major histocompatibility complex antigens and secrete antimicrobial peptides in bile, their role in response to biliary infection is critical. Finally, because cholangiocytes release mediators critical to myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells, cholangiocytes may be essential in the pathogenesis of biliary cirrhosis. PMID:22961800

Advances in cholangiocyte immunobiology.

PubMed

Syal, Gaurav; Fausther, Michel; Dranoff, Jonathan A

2012-11-15

Cholangiocytes, or bile duct epithelia, were once thought to be the simple lining of the conduit system comprising the intra- and extrahepatic bile ducts. Growing experimental evidence demonstrated that cholangiocytes are in fact the first line of defense of the biliary system against foreign substances. Experimental advances in recent years have unveiled previously unknown roles of cholangiocytes in both innate and adaptive immune responses. Cholangiocytes can release inflammatory modulators in a regulated fashion. Moreover, they express specialized pattern-recognizing molecules that identify microbial components and activate intracellular signaling cascades leading to a variety of downstream responses. The cytokines secreted by cholangiocytes, in conjunction with the adhesion molecules expressed on their surface, play a role in recruitment, localization, and modulation of immune responses in the liver and biliary tract. Cholangiocyte survival and function is further modulated by cytokines and inflammatory mediators secreted by immune cells and cholangiocytes themselves. Because cholangiocytes act as professional APCs via expression of major histocompatibility complex antigens and secrete antimicrobial peptides in bile, their role in response to biliary infection is critical. Finally, because cholangiocytes release mediators critical to myofibroblastic differentiation of portal fibroblasts and hepatic stellate cells, cholangiocytes may be essential in the pathogenesis of biliary cirrhosis.

X-ray fast tomography and its applications in dynamical phenomena studies in geosciences at Advanced Photon Source

NASA Astrophysics Data System (ADS)

Xiao, Xianghui; Fusseis, Florian; De Carlo, Francesco

2012-10-01

State-of-art synchrotron radiation based micro-computed tomography provides high spatial and temporal resolution. This matches the needs of many research problems in geosciences. In this letter we report the current capabilities in microtomography at sector 2BM at the Advanced Photon Source (APS) of Argonne National Laboratory. The beamline is well suited to routinely acquire three-dimensional data of excellent quality with sub-micron resolution. Fast cameras in combination with a polychromatic beam allow time-lapse experiments with temporal resolutions of down to 200 ms. Data processing utilizes quantitative phase retrieval to optimize contrast in phase contrast tomographic data. The combination of these capabilities with purpose-designed experimental cells allows for a wide range of dynamic studies on geoscientific topics, two of which are summarized here. In the near future, new experimental cells capable of simulating conditions in most geological reservoirs will be available for general use. Ultimately, these advances will be matched by a new wide-field imaging beam line, which will be constructed as part of the APS upgrade. It is expected that even faster tomography with larger field of view can be conducted at this beam line, creating new opportunities for geoscientific studies.

Cole-Cole, linear and multivariate modeling of capacitance data for on-line monitoring of biomass.

PubMed

Dabros, Michal; Dennewald, Danielle; Currie, David J; Lee, Mark H; Todd, Robert W; Marison, Ian W; von Stockar, Urs

2009-02-01

This work evaluates three techniques of calibrating capacitance (dielectric) spectrometers used for on-line monitoring of biomass: modeling of cell properties using the theoretical Cole-Cole equation, linear regression of dual-frequency capacitance measurements on biomass concentration, and multivariate (PLS) modeling of scanning dielectric spectra. The performance and robustness of each technique is assessed during a sequence of validation batches in two experimental settings of differing signal noise. In more noisy conditions, the Cole-Cole model had significantly higher biomass concentration prediction errors than the linear and multivariate models. The PLS model was the most robust in handling signal noise. In less noisy conditions, the three models performed similarly. Estimates of the mean cell size were done additionally using the Cole-Cole and PLS models, the latter technique giving more satisfactory results.

New phenanthrene and 9, 10-dihydrophenanthrene derivatives from the stems of Dendrobium officinale with their cytotoxic activities.

PubMed

Zhao, Gui-Yun; Deng, Bo-Wen; Zhang, Chong-Yu; Cui, Yi-Da; Bi, Jia-Yi; Zhang, Guo-Gang

2018-01-01

Two new phenanthrene and 9, 10-dihydrophenanthrene derivatives (1-2) with six known congeners (3-8) were isolated from the extraction of stems of Dendrobium officinale. Compounds 1 and 2 were based on carbon skeleton in which phenanthrene and 9, 10-dihydrophenanthrene moiety were linked with a phenylpropane unit through a dioxane bridge, respectively. Their structures were determined by comprehensive NMR spectroscopic data, the absolute configuration of new compounds were determined by comparing their experimental and calculated ECD for the first time. All the compounds were investigated contains two cancer cell lines (HI-60, THP-1). All the isolates showed cytotoxicity, especially compound 4 showed markedly cytotoxic activities against HI-60 and THP-1 cell lines with IC 50 values of 11.96 and 8.92 μM.

Development of a Novel CD4+ TCR Transgenic Line That Reveals a Dominant Role for CD8+ Dendritic Cells and CD40 Signaling in the Generation of Helper and CTL Responses to Blood-Stage Malaria.

PubMed

Fernandez-Ruiz, Daniel; Lau, Lei Shong; Ghazanfari, Nazanin; Jones, Claerwen M; Ng, Wei Yi; Davey, Gayle M; Berthold, Dorothee; Holz, Lauren; Kato, Yu; Enders, Matthias H; Bayarsaikhan, Ganchimeg; Hendriks, Sanne H; Lansink, Lianne I M; Engel, Jessica A; Soon, Megan S F; James, Kylie R; Cozijnsen, Anton; Mollard, Vanessa; Uboldi, Alessandro D; Tonkin, Christopher J; de Koning-Ward, Tania F; Gilson, Paul R; Kaisho, Tsuneyasu; Haque, Ashraful; Crabb, Brendan S; Carbone, Francis R; McFadden, Geoffrey I; Heath, William R

2017-12-15

We describe an MHC class II (I-A b )-restricted TCR transgenic mouse line that produces CD4 + T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4 + T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human ( Plasmodium falciparum ) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8 + T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4 + T cells and the previously described PbT-I CD8 + T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8 + DC (a subset of XCR1 + DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4 + T cell responses. Depletion of CD8 + DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4 + T cell immunity during malaria and provides evidence that CD4 + T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8 + DC. Copyright © 2017 by The American Association of Immunologists, Inc.

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology

PubMed Central

Dong, Jia; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K.N.; Knobeloch, Daniel; Gerlach, Jörg C.; Zeilinger, Katrin

2008-01-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes. PMID:19003182

Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

PubMed

Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

2008-07-01

Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia.

PubMed

Rathe, Susan K; Moriarity, Branden S; Stoltenberg, Christopher B; Kurata, Morito; Aumann, Natalie K; Rahrmann, Eric P; Bailey, Natashay J; Melrose, Ellen G; Beckmann, Dominic A; Liska, Chase R; Largaespada, David A

2014-08-13

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in Dck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC₅₀ of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Experimental research on Ku-band magnetically insulated transmission line oscillator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Tao; Zhang, Jiande; He, Juntao

2015-10-15

An improved Ku-band magnetically insulated transmission line oscillator is proposed and investigated experimentally. In the particle-in-cell simulation, the Ku-band MILO generates the microwave with a power of 1.62 GW and a frequency of 13 GHz at the input voltage of 474 kV. The device is fabricated based on the simulation results, and an experiment system is designed. In the preliminary experiments, output microwave with frequency of 13.02 GHz, power of 150 MW, and pulse width of 17 ns is generated, under the diode voltage of 450 kV. Analysis on the experiment results shows that plasma produced due to the large current hitting to the outside of themore » collection tank is the essential cause for the low amplitude of the microwave power and short pulse width.« less

Synthesis, spectroscopic, physicochemical and structural characterization of tetrandrine-based macrocycles functionalized with acridine and anthracene groups: DNA binding and anti-proliferative activity.

PubMed

Calvillo-Páez, Viviana; Sotelo-Mundo, Rogerio R; Leyva-Peralta, Mario; Gálvez-Ruiz, Juan Carlos; Corona-Martínez, David; Moreno-Corral, Ramón; Escobar-Picos, Raymundo; Höpfl, Herbert; Juárez-Sánchez, Octavio; Lara, Karen Ochoa

2018-04-25

In this work, we report on the synthesis of two new mono-alkylated tetrandrine derivatives with acridine and anthracene units, MAcT and MAnT. The compounds were fully characterized by physicochemical techniques and single-crystal X-ray diffraction analysis. In addition, both derivatives were studied as nucleotide receptors and double-stranded DNA binders in aqueous phosphate buffer at pH = 7.2 using UV-vis and fluorescence spectroscopy. According to the molecular recognition studies, MAcT and MAnT exhibit high affinity (K ∼ 10 5  M -1 ) and selectivity for ds-DNA, presumably in an intercalation mode. Finally, the anti-proliferative effects of the tetrandrine derivatives on different cancer cell lines were explored, revealing promising activities. Particularly, the mono-anthracene tetrandrine derivative MAnT showed an IC 50 of 2.74 μg/mL on the HeLa cervical cancer cell line, representing a value 3.3 times smaller than that obtained for unsubstituted tetrandrine. Examination of the cytotoxic effects on the HeLa cell line by inverted microscopy suggests that the cell death mechanism consists basically in apoptosis. The molecular modelling of three ds-DNA-MAcT complexes, suggested that the macrocycles may use an intercalation binding mode towards DNA. MAcT is predicted to bind into the major groove of the ds-DNA providing non-covalent interactions such as electrostatic, van der Waals and hydrophobic interactions that lead to selectivity. Overall experimental data supports the mode of action of MAnT and MAcT as cytotoxic compounds against cancer cell lines via a DNA interaction mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

CLO: The cell line ontology

PubMed Central

2014-01-01

Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

Exploiting the co-reliance of tumours upon transport of amino acids and lactate: Gln and Tyr conjugates of MCT1 inhibitors.

PubMed

Nair, Reji N; Mishra, Jitendra K; Li, Fangzheng; Tortosa, Mariola; Yang, Chunying; Doherty, Joanne R; Cameron, Michael; Cleveland, John L; Roush, William R; Bannister, Thomas D

2016-05-01

Glutamine and tyrosine-based amino acid conjugates of monocarboxylate transporter types 1 and 2 inhibitors (MCT1/2) were designed, synthesized and evaluated for their potency in blocking the proliferation of a human B lymphoma cell line that expresses the transporters Asct2, LAT1 and MCT1. Appropriate placement of an amino acid transporter recognition element was shown to augment anti-tumour efficacy vs. Raji cells. Amino acid conjugation also improves the pharmacodynamic properties of experimental MCT1/2 inhibitors.

Supraependymal cells of hypothalamic third ventricle: identification as resident phagocytes of the brain.

PubMed

Bleier, R; Albrecht, R; Cruce, J A

1975-07-25

Cells lying on the ventricular surface of the hypothalamic ependyma of the tegu lizard exhibit the pseudopodial and flaplike processes characteristic of macrophages found elsewhere. Since they ingest latex beads, they may be considered a resident phagocytic system of the brain. The importance of ependyma and ventricular phagocytes as a first line of defense against viral invasion of the brain, as well as their role in the pathogenesis of certain virus-related diseases, is suggested by a number of experimental and clinical observations.

Cloning of ES cells and mice by nuclear transfer.

PubMed

Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2009-01-01

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

High resolution, low cost solar cell contact development

NASA Technical Reports Server (NTRS)

Mardesich, N.

1979-01-01

The experimental work demonstrating the feasibility of the MIDFILM process as a low cost means of applying solar cell collector metallization as reported. Cell efficiencies of above 14% (AMl, 28 C) were achieved with fritted silver metallization. Environmental tests suggest that the metallization is slightly humidity sensitive and degradation is observed on cells with high series resistance. The major yield loss in the fabrication of cells was due to discontinuous grid lines, resulting in high series resitance. Standard lead-tin solder plated interconnections do not appear compatible with the MIDFILM contact. Copper, nickel and molybdemun base powder were investigated as low cost metallization systems. The copper based powder degraded the cell response. The nickel and molybdenum base powders oxidized when sintered in the oxidizing atmosphere necessary to ash the photoresin.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Daisuke; Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp; Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2013-05-17

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present inmore » esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.« less

Effect of quantum dots on the biological behavior of the EJ human bladder urothelial carcinoma cell line.

PubMed

Yuan, Run; Yu, Wei-Min; Cheng, Fan; Zhang, Xiao-Bin; Ruan, Yuan; Cao, Zhi-Xiu; Larré, Stéphane

2015-10-01

Quantum dots (QDs) are a type of fluorescent label with applications in biological molecules, cells and in vivo imaging. The current study investigated the effect of QDs on the toxicity, proliferation, migration and invasion of the EJ human bladder cancer cell line in vitro. The cell counting kit‑8 test was used to measure the survival rate of EJ cells following incubation with varying concentrations of QDs. Additionally, the effect of QDs on tumor cell migration and invasion was evaluated using the Transwell chamber assay, and cell proliferation rate was assessed using a hemocytometer. Data from the current study demonstrated no significant differences in survival rate between the experimental and control groups with the conventionally used concentrations (5, 10 and 20 nM) of QD605 (P>0.05). However, with high concentrations of QD605 (40 and 80 nM), significant differences were observed (P<0.001). The survival rate of EJ cells, however, remained at 92.6%. In addition, no significant differences were observed between the EJ cells labeled with transactivator of transcription (TAT)‑QD605 and the unlabeled EJ cells with regard to proliferation, migration and invasion (P>0.05). Thus, the results of the current study indicate that QDs exhibit a certain degree of influence on the activity of the EJ bladder cancer cell line at high concentrations. However, at the concentrations that QDs are conventionally used, there was little impact on the survival of the EJ cells. In addition, the proliferation, migration and invasion abilities of the EJ cells were not affected by TAT‑QDs. Therefore, the peptide‑conjugated QDs have potential to be applied in the imaging and tracking of live cells in vitro and of animals in vivo. Notably, QDs may provide the foundation for a novel, non‑invasive imaging strategy for the early diagnosis of tumors.

Mechanistic Exploration of Cancer Stem Cell Marker Voltage-Dependent Calcium Channel α2δ1 Subunit-mediated Chemotherapy Resistance in Small-Cell Lung Cancer.

PubMed

Yu, Jiangyong; Wang, Shuhang; Zhao, Wei; Duan, Jianchun; Wang, Zhijie; Chen, Hanxiao; Tian, Yanhua; Wang, Di; Zhao, Jun; An, Tongtong; Bai, Hua; Wu, Meina; Wang, Jie

2018-05-01

Purpose: Chemoresistance in small-cell lung cancer (SCLC) is reportedly attributed to the existence of resistant cancer stem cells (CSC). Studies involving CSC-specific markers and related mechanisms in SCLC remain limited. This study explored the role of the voltage-dependent calcium channel α2δ1 subunit as a CSC marker in chemoresistance of SCLC, and explored the potential mechanisms of α2δ1-mediated chemoresistance and strategies of overcoming the resistance. Experimental Design: α2δ1-positive cells were identified and isolated from SCLC cell lines and patient-derived xenograft (PDX) models, and CSC-like properties were subsequently verified. Transcriptome sequencing and Western blotting were carried out to identify pathways involved in α2δ1-mediated chemoresistance in SCLC. In addition, possible interventions to overcome α2δ1-mediated chemoresistance were examined. Results: Different proportions of α2δ1 + cells were identified in SCLC cell lines and PDX models. α2δ1 + cells exhibited CSC-like properties (self-renewal, tumorigenic, differentiation potential, and high expression of genes related to CSCs and drug resistance). Chemotherapy induced the enrichment of α2δ1 + cells instead of CD133 + cells in PDXs, and an increased proportion of α2δ1 + cells corresponded to increased chemoresistance. Activation and overexpression of ERK in the α2δ1-positive H1048 cell line was identified at the protein level. mAb 1B50-1 was observed to improve the efficacy of chemotherapy and delay relapse as maintenance therapy in PDX models. Conclusions: SCLC cells expressing α2δ1 demonstrated CSC-like properties, and may contribute to chemoresistance. ERK may play a key role in α2δ1-mediated chemoresistance. mAb 1B50-1 may serve as a potential anti-SCLC drug. Clin Cancer Res; 24(9); 2148-58. ©2018 AACR . ©2018 American Association for Cancer Research.

Investigation of synthesized new vanadium(III) complexes of ditolyldithiophosphate ligands by spectroscopic, cyclic voltammetric, DFT, antimicrobial and cytotoxic studies

NASA Astrophysics Data System (ADS)

Kumar, Sandeep; Syed, Atiya; Andotra, Savit; Kaur, Ramanpreet; Vikas; Pandey, Sushil K.

2018-02-01

Vanadium(III) complexes with sulfur donor dithiophosphate ligands corresponding to [{(ArO)2PS2}3V] and [{(ArO)2PS2}2VCl.L] (Ar = o-, m-, p-CH3C6H4 and p-Cl-m-CH3C6H3; L = NC5H5, P(C6H5)3, have been synthesized and characterized by various physico-chemical techniques like elemental analyses, magnetic studies, ESI-Mass, IR, UV and heteronuclear NMR (1H, 13C and 31P) spectral studies. These analyses have contributed to the prediction of structure: by exhibiting significant v(P-S) and v(Pdbnd S) band shifting in comparative IR spectra; shifting of resonance signal in comparative 31P NMR spectra of ligands and complexes and stability of V(III) ion in the complexed state is confirmed by magnetic and UV studies. Therefore, the six coordinated geometry stabilizing the trivalent vanadium atom in the complexes and adducts, respectively has been confirmed. The cyclic voltammetric analyses presented the redox aptitude of the complex under analysis which can be utilized as catalyst in organic synthesis. The geometry of ligands and complexes has been optimized using density functional theory (DFT). The structural parameters, vibrational bands and energy gaps of frontier orbitals (HOMO-LUMO) have also been calculated. The calculated geometric and spectral results reproduced the experimental data with well agreement. The DFT computed frontier molecular orbitals (HOMO-LUMO) and their energies suggest charge transfer occurs within the complexes. Antimicrobial screening of the complexes against two bacterial strains: Gram-positive, Enterrococcus faecalis and Gram-negative, Eischerichia coli and fungus Fusarium oxysporum have shown potential bioactivity. A preliminary cytotoxic analysis has been carried out using the cultivated human cell lines: lung adeno carcinoma cell line A-549, leukemia cell line THP-1, prostate cancer cell line PC3 and colorectal cancer cell line HCT-116.

Extraction, fractionation and re-fractionation of Artemisia nilagirica for anticancer activity and HPLC-ESI-QTOF-MS/MS determination.

PubMed

Sahu, Neha; Meena, Sanjeev; Shukla, Vijaya; Chaturvedi, Priyank; Kumar, Brijesh; Datta, Dipak; Arya, K R

2018-03-01

Medicinal plants used in traditional medicines are affordable, easily accessible, safer, less toxic and considered as a rich or efficient source of bioactive molecules for modern therapeutics. Artemisia nilagirica (AR) has a long history of use in Indian traditional medicine to combat a wide variety of diseases including cancer. Considering the vast potential of traditional healing plants to deliver safer, less toxic and efficient chemotherapeutics, we have examined anticancer activity of ethanolic extract, bioactive fractions and sub-fractions of AR against different human cancer cell lines along with their phytochemical analysis to understand the insights of novel anticancer activities for further preclinical studies. Fresh plant material of AR was procured from the wild, dried and ground. The grinded materials was extracted in ethanol (AR-01) and fractionated into butanol (AR-02), ethyl acetate (AR-03), hexane (AR-04) and water (AR-05). The cytotoxicity was evaluated against three different human cancer cell lines, i.e. colon (DLD-1), lung (A-549), and breast (MCF-7) using Sulforhodamine B (SRB) assay along with non-cancerous VERO cells as control and doxorubicin (DOX) as positive control. As we observed strong cytotoxicity of AR-03 and AR-04 fractions against tested cells and marked cytotoxic effects particularly in colon cancer cell lines, we further re-fractionated, AR-03 into (AR-03A, AR-03B, AR-03C, AR-03D, AR-03E) and AR-04 into (AR-04A, AR-04B, AR-04C) sub-fractions by column chromatography and investigated against the same panel of cell lines in addition to one more colon cancer cell line (HT-29). Phytochemical analysis was performed through HPLC-ESI-QTOF-MS/MS fragmentation. Ethyl acetate (AR-03) and hexane (AR-04) fractions were found to be the most cytotoxic against all the tested cell lines. Further, AR-03E and AR-04A sub-fractions were found more specific cytotoxic selectively against DLD-1 cancer cell lines at 100µg/ml concentration. HPLC-ESI-QTOF-MS/MS determination revealed the presence of 17 compounds in AR-01. Among them, 4 compounds were reported for the first time in this species. However, 3 identified compounds (artemorin, β-santonin and caryophyllene oxide) in AR-03E sub-fraction were commonly present in each bioactive fraction and may be considered as potential and safest cytotoxic agents for anticancer activity. Experimental evidences reported in this paper for anticancer activity validate the traditional wisdom of Artemisia nilagirica as an anticancer herbal drug. To our knowledge, this is our first novel observation of cytotoxicity and selectivity of ethyl acetate and hexane sub-fraction of AR-01 i.e. AR-03E and AR-04A respectively against DLD-1 human cancer cell lines. HPLC-ESI-QTOF-MS/MS determination attributes the identification of cytotoxic compounds which may be used for further preclinical studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Thyroid cell lines in research on goitrogenesis.

PubMed

Gerber, H; Peter, H J; Asmis, L; Studer, H

1991-12-01

Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

[Biocompatibility research of true bone ceramics].

PubMed

Qiao, Wei; Ren, Xiaoqi; Shi, Hao; Li, Jing; Yang, Ting; Ma, Shaoying; Zhao, Yaping; Su, Chengzhong; Li, Baoxing

2017-10-01

To investigate the biocompatibility of true bone ceramic (TBC) and provide experimental basis for clinic application. TBC was prepared from healthy adult bovine cancellous bone by deproteinization and high temperature calcinations. Mouse fibroblast cell line (L929 cells) were cultured with the leaching liquor of TBC in vitro , and the cytotoxicity was evaluated at 2nd, 4th, and 7th days. L929 cells were inoculated into the TBC and cultured for 4 days. The cell adhesion and proliferation on the surface of the TBC were observed by scanning electron microscopy, and evaluated the cell compatibility of TBC. Ten New Zealand white rabbits were divided into 2 groups, and drilled holes at the tibia of both hind limbs. TBC and hydroxyapatite (HA) were implanted into the left side (experimental group) and the right side (control group), respectively. And the biocompatibility of TBC was evaluated by general observation and histological observation at 4 and 26 weeks after implantation. Cytotoxicity test showed that the cytotoxicity level of leaching liquor of TBC was grade 0-1. Cell compatibility experiments showed that the L929 cells adhered well on the surface of TBC and migrated into the pores. The implantation test in vivo showed that experimental group and control group both had mild or moderate inflammatory response at 4 weeks, and new bone formation occurred. At 26 weeks, there was no inflammatory reaction observed in both groups, and new bone formation was observed in varying degrees. TBC have good biocompatibility and can be used to repair bone defect in clinic.

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

PubMed Central

Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

2017-01-01

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA Damaging Agents

PubMed Central

Vasilevskaya, Irina A.; Selvakumaran, Muthu; Hierro, Lucia Cabal; Goldstein, Sara R.; Winkler, Jeffrey D.; O'Dwyer, Peter J.

2015-01-01

Purpose We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. Experimental design In a panel of cell lines we investigated effects of pharmacological and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38 and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Results Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, though synergy is not always hypoxia-specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (non-responsive) lines. In HT29 and SW620 cells CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, where tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. Conclusions These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic. PMID:26023085

Preclinical evaluation of the PARP inhibitor BMN-673 for the treatment of ovarian clear cell cancer

PubMed Central

Samartzis, Eleftherios Pierre; Dedes, Ioannis; Lambros, Maryou B; Natrajan, Rachael; Gauthier, Arnaud; Piscuoglio, Salvatore; Töpfer, Chantal; Vukovic, Vesna; Daley, Frances; Weigelt, Britta; Reis-Filho, Jorge S

2017-01-01

Purpose To determine if models of ovarian clear cell carcinomas (OCCCs) harbouring defects in homologous recombination (HR) DNA repair of double strand breaks (DSBs) are sensitive to cisplatin and/or PARP inhibition. Experimental Design The HR status of 12 OCCC cell lines was determined using RAD51/γH2AX foci formation assays. Sensitivity to cisplatin and the PARP inhibitor BMN-673 was correlated with HR status. BRCA1, BRCA2, MRE11 and PTEN loss of expression was investigated as a potential determinant of BMN-673 sensitivity. A tissue microarray containing 50 consecutive primary OCCC was assessed for PTEN expression using immunohistochemistry. Results A subset of OCCC cells displayed reduced RAD51 foci formation in the presence of DNA DSBs, suggestive of HR defects. HR-defective OCCC cells, with the exception of KOC-7c, had higher sensitivity to cisplatin/ BMN-673 than HR-competent OCCC cell lines (Log10 SF50 –9.4 (SD +/− 0.29) vs –8.1 (SD +/− 0.35), mean difference 1.3, p < 0.01). Of the cell lines studied, two, TOV-21G and KOC-7c, showed loss of PTEN expression. In primary OCCCs, loss of PTEN expression was observed in 10% (5/49) of cases. Conclusions A subset of OCCC cells are sensitive to PARP inhibition in vitro, which can be predicted by HR defects as defined by γH2AX/RAD51 foci formation. These results provide a rationale for the testing of HR deficiency and PARP inhibitors as a targeted therapy in a subset of OCCCs. PMID:28002809

Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

NASA Astrophysics Data System (ADS)

Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

PubMed Central

Sadikot, Takrima; Swink, Megan; Eskew, Jeffery D.; Brown, Douglas; Zhao, Huiping; Kusuma, Bhaskar R.; Rajewski, Roger A.; Blagg, Brian S. J.; Matts, Robert L.; Holzbeierlein, Jeffrey M.

2013-01-01

Abstract The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu. PMID:24127661

Release of copper-amended particles from micronized copper-pressure-treated wood during mechanical abrasion.

PubMed

Civardi, Chiara; Schlagenhauf, Lukas; Kaiser, Jean-Pierre; Hirsch, Cordula; Mucchino, Claudio; Wichser, Adrian; Wick, Peter; Schwarze, Francis W M R

2016-11-28

We investigated the particles released due to abrasion of wood surfaces pressure-treated with micronized copper azole (MCA) wood preservative and we gathered preliminary data on its in vitro cytotoxicity for lung cells. The data were compared with particles released after abrasion of untreated, water (0% MCA)-pressure-treated, chromated copper (CC)-pressure-treated wood, and varnished wood. Size, morphology, and composition of the released particles were analyzed. Our results indicate that the abrasion of MCA-pressure-treated wood does not cause an additional release of nanoparticles from the unreacted copper (Cu) carbonate nanoparticles from of the MCA formulation. However, a small amount of released Cu was detected in the nanosized fraction of wood dust, which could penetrate the deep lungs. The acute cytotoxicity studies were performed on a human lung epithelial cell line and human macrophages derived from a monocytic cell line. These cell types are likely to encounter the released wood particles after inhalation. Our findings indicate that under the experimental conditions chosen, MCA does not pose a specific additional nano-risk, i.e. there is no additional release of nanoparticles and no specific nano-toxicity for lung epithelial cells and macrophages.

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cis activation of the c-myc gene in bovine papilloma virus type 1/human c-myc hybrid plasmids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Modjtahedi, N.; Feunteun, J.; Brison, O.

1988-01-01

The c-myc gene amplification observed in human tumors is likely to represent an activation mechanism aiming at an increased transcription level. In order to evaluate the biological significance of this amplification in the malignant transformation the authors designed an experimental model that could possibly mimic this situation in vitro. They have constructed a series of plasmids which physically link the human c-myc gene to the bovine papilloma virus type 1 genome (BPV1) and therefore should be maintained as amplified episomes upon transformation of rodent cells. Anticipating that the high copy number will bring about the immortalizing capacity of the c-mycmore » gene, the constructions were introduced into primary rat embryo cells. Immortal cell lines were established by transfection of the hybrid plasmids carrying either the complete BPV1 genome or the transforming region of the viral genome. The BPV1 DNA alone or the c-myc gene alone has no activity in this assay. The analysis of the established cell lines demonstrates that the transfected plasmids are present not as free copies as anticipated but rather integrated as tandem repeats. They present data which strongly suggest that the immortalization capacity of the hybrid plasmids reflects the activation of the c-myc gene by the transactivable BPV1 enhancer. Although both the BPV1 early genes and the c-myc gene are actively transcribed, most of the cell lines do not display a transformed phenotype.« less

Metabolic Imaging of Pancreatic Ductal Adenocarcinoma Detects Altered Choline Metabolism

PubMed Central

Penet, Marie-France; Shah, Tariq; Bharti, Santosh; Krishnamachary, Balaji; Artemov, Dmitri; Mironchik, Yelena; Wildes, Flonné; Maitra, Anirban; Bhujwalla, Zaver M.

2014-01-01

Purpose Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets. Experimental Design We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets. Results We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between HPNE and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter-like protein 1 (CTL1) in the PDAC cell lines and tumors. Conclusions Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging-probe independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. PMID:25370468

Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications

PubMed Central

Lange, Sara E.S.; Zheleznyak, Alex; Studer, Matthew; O'Shannessy, Daniel J.; Lapi, Suzanne E.; Van Tine, Brian A.

2016-01-01

Purpose The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab. Results Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs. Experimental Design Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed. Conclusion 89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy. PMID:26909615

Microarray data re-annotation reveals specific lncRNAs and their potential functions in non-small cell lung cancer subtypes.

PubMed

Zhou, Dongbo; Xie, Mingxuan; He, Baimei; Gao, Ying; Yu, Qiao; He, Bixiu; Chen, Qiong

2017-10-01

Non‑small‑cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. The most common subtypes of NSCLC are adenocarcinoma (AC) and squamous cell carcinoma (SCC). However, the pathophysiological mechanisms contributing to AC and SCC are still largely unknown, especially the roles of long non‑coding RNAs (lncRNAs). The present study identified differentially expressed lncRNAs between lung AC and SCC by re‑annotation of NSCLC microarray data analysis profiling. The potential functions of lncRNAs were predicted by using coding‑non‑coding gene co‑expressing network. Reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) was used to investigate lncRNA expression levels in AC cell lines (A549 and L78), SCC cell lines (H226 and H520) and normal cells (NL‑20). Western blotting analysis was used to investigate the protein expression levels in these cell lines. A total of 65 lncRNAs were differentially expressed between AC and SCC including 28 lncRNAs that were downregulated in SCC subtypes compared with those in AC ones, and 37 upregulated lncRNAs in SCC subtypes compared with AC subtypes. Three lncRNAs, sex determining region Y‑box 2 overlapping transcript (SOX2‑OT), NCBP2 antisense RNA 2 (NCBP2‑AS2) and ubiquitin like with PHD and ring finger domains 1 (UHRF1), were predicted to be associated with lung cancer; RT‑qPCR confirmed that SOX2‑OT and NCBP2‑AS2 were associated with lung cancer. Finally, western blot assays demonstrated that there was no difference in β‑catenin and glycogen synthase kinase 3β (GSK‑3β) expression in cancer cells compared with NL‑20, but increased phosphorylated (p‑)β‑catenin and p‑GSK‑3β was detected in lung cancer cell lines compared with NL‑20, particularly in A549 cells. Although these results require further experimental verification, the analysis of lncRNA signatures between AC and SCC has provided insights into the regulatory mechanism of NSCLC development.

Effects of Selective Checkpoint Kinase 1 Inhibition on Cytarabine Cytotoxicity in Acute Myelogenous Leukemia Cells in Vitro

PubMed Central

Schenk, Erin L.; Koh, Brian D.; Flatten, Karen S.; Peterson, Kevin L.; Parry, David; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Karnitz, Larry M.; Kaufmann, Scott H.

2012-01-01

Purpose Previous studies have demonstrated that the replication checkpoint, which involves the kinases ATR and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical AML during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results Immunoblotting demonstrated increased Chk1 phosphorylation, a marker of checkpoint activation, in over half of Chk1-containing AMLs after 48 h of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S phase arrest, but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the anti-proliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions These results not only provide evidence for cytarabine-induced S phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. PMID:22869869

Experimental analysis of a TEM plane transmission line for DNA studies at 900 MHz EM fields

NASA Astrophysics Data System (ADS)

Belloni, F.; Doria, D.; Lorusso, A.; Nassisi, V.; Velardi, L.; Alifano, P.; Monaco, C.; Talà, A.; Tredici, M.; Rainò, A.

2006-07-01

A suitable plane transmission line was developed and its behaviour analysed at 900 MHz radiofrequency fields to study DNA mutability and the repair of micro-organisms. In this work, utilizing such a device, we investigated the behaviour of DNA mutability and repair of Escherichia coli strains. The transmission line was very simple and versatile in changing its characteristic resistance and field intensity by varying its sizes. In the absence of cell samples inside the transmission line, the relative modulation of the electric and/or magnetic field was ±31% with respect to the mean values, allowing the processing of more samples at different exposure fields in a single run. A slight decrease in spontaneous mutability to rifampicin-resistance of the E. coli JC411 strain was demonstrated in mismatch-repair proficient samples exposed to the radio-frequency fields during their growth on solid medium.

Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guseva, Daria; Hannover Medical School, Hannover; Rizvanov, Albert A.

2014-09-05

Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignantmore » transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.« less

Protective activity of Panduratin A against Thioacetamide-induced oxidative damage: demonstration with in vitro experiments using WRL-68 liver cell line

PubMed Central

2013-01-01

Background Chalcone Panduratin A (PA) has been known for its antioxidant property, but its merits against oxidative damage in liver cells has yet to be investigated. Hence, the paper aimed at accomplishing this task with normal embryonic cell line WRL-68. Methods PA was isolated from Boesenbergia rotunda rhizomes and its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and ferric reducing power (FRAP) activities were measured in comparison with that of the standard reference drug Silymarin (SI). Oxidative damage was induced by treating the cells with 0.04 g/ml of toxic thioacetamide for 60 minutes followed by treatment with 1, 10 and 100 μg/ml concentrations of either PA or SI. The severities of oxidative stress in the control and experimental groups of cells were measured by Malondialdehyde (MDA) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. Results PA exhibited an acceptable DPPH scavenging and FRAP activities close to that of Silymarin. Treating the injured cells with PA significantly reduced the MDA level and increased the cell viability, comparable to SI. The activities of SOD, CAT and GPx were significantly elevated in the PA-treated cells in a dose dependent manner and again similar to SI. Conclusion Collectively, data suggested that PA has capacity to protect normal liver cells from oxidative damage, most likely via its antioxidant scavenging ability. PMID:24156366

A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29.

PubMed

Adlerberth, I; Ahrne, S; Johansson, M L; Molin, G; Hanson, L A; Wold, A E

1996-07-01

Two Lactobacillus plantarum strains of human intestinal origin, strains 299 (= DSM 6595) and 299v (= DSM 9843), have proved to be efficient colonizers of the human intestine under experimental conditions. These strains and 17 other L. plantarum strains were tested for the ability to adhere to cells of the human colonic cell line HT-29.L.plantarum 299 and 299v and nine other L. plantarum strains, including all six strains that belong to the same genetic subgroup as L. plantarum 299 and 299v, adhered to HT-29 cells in a manner that could be inhibited by methyl-alpha-D-mannoside. The ability to adhere to HT-29 cells correlated with an ability to agglutinate cells of Saccharomyces cerevisiae and erythrocytes in a mannose-sensitive manner and with adherence to D-mannose-coated agarose beads. L. plantarum 299 and 299v adhered to freshly isolated human colonic and ileal enterocytes, but the binding was not significantly inhibited by methyl-alpha-D-mannoside. Periodate treatment of HT-29 cells abolished mannose-sensitive adherence, confirming that the cell-bound receptor was of carbohydrate nature. Proteinase K treatment of the bacteria also abolished adherence, indicating that the binding involved protein structures on the bacterial cell surface. Thus, a mannose-specific adhesin has been identified in L. plantarum; this adhesin could be involved in the ability to colonize the intestine.

Evaluating cell lines as tumour models by comparison of genomic profiles

PubMed Central

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types. PMID:23839242

Biokinetics and dosimetry of several radiolabelled peptides in cancer cells

NASA Astrophysics Data System (ADS)

Rodríguez-Cortés, J.; Ferro-Flores, G.; de Murphy, C. Arteaga; Pedraza-López, M.; Ramírez-Iglesias, M. A. T.

Radiolabelled peptides have been used as target-specific radiopharmaceuticals. The goal of this research was the in vitro assessment of the uptake, internalization, externalization, and efflux of five radiolabelled peptides in cancer cells to estimate radiation-absorbed doses from experimental biokinetic data. 177Lu-DOTA-octreotate, 188Re-lanreotide, and 99mTc-HYNIC-octreotide were studied in the AR42J cell line. The PC3 and NCIH69 cells were used for 99mTc-HYNIC-bombesin and 177Lu-DOTA-minigastrin, respectively. The cumulated activities in the membrane and cytoplasm were calculated by integration of the experimental time-activity curves and used for dosimetry calculations according to the Medical Internal Radiation Dose (MIRD) cellular methodology. The mean absorbed dose to the cell nucleus were 0.69±0.09, 0.11±0.08, 0.55±0.09, 3.45±0.48, and 3.30±0.65 Gy/Bq for 99mTc-HYNIC-bombesin, 99mTc-HYNIC-octreotide, 177Lu-DOTA-minigastrin, 177Lu-DOTA-octreotate, and 188Re-lanreotide, respectively. If radiopharmaceutical cell kinetics were not used and only uptake data were considered, the calculated doses would be overestimated up to 25 times.

Toxicity of Nanoparticles and an Overview of Current Experimental Models.

PubMed

Bahadar, Haji; Maqbool, Faheem; Niaz, Kamal; Abdollahi, Mohammad

2016-01-01

Nanotechnology is a rapidly growing field having potential applications in many areas. Nanoparticles (NPs) have been studied for cell toxicity, immunotoxicity, and genotoxicity. Tetrazolium-based assays such as MTT, MTS, and WST-1 are used to determine cell viability. Cell inflammatory response induced by NPs is checked by measuring inflammatory biomarkers, such as IL-8, IL-6, and tumor necrosis factor, using ELISA. Lactate dehydrogenase (LDH) assay is used for cell membrane integrity. Different types of cell cultures, including cancer cell lines have been employed as in vitro toxicity models. It has been generally agreed that NPs interfere with either assay materials or with detection systems. So far, toxicity data generated by employing such models are conflicting and inconsistent. Therefore, on the basis of available experimental models, it may be difficult to judge and list some of the more valuable NPs as more toxic to biological systems and vice versa. Considering the potential applications of NPs in many fields and the growing apprehensions of FDA about the toxic potential of nanoproducts, it is the need of the hour to look for new internationally agreed free of bias toxicological models by focusing more on in vivo studies.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

PubMed Central

Augustsson, Per; Karlsen, Jonas T.; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-01-01

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size. PMID:27180912

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

PubMed

Augustsson, Per; Karlsen, Jonas T; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-05-16

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2013-01-01

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways. PMID:23739767

AXL is a logical molecular target in head and neck squamous cell carcinoma

PubMed Central

Brand, Toni M.; Iida, Mari; Stein, Andrew P.; Corrigan, Kelsey L.; Braverman, Cara M.; Coan, John; Pearson, Hannah E.; Bahrar, Harsh; Fowler, Tyler L.; Bednarz, Bryan P.; Saha, Sandeep; Yang, David; Gill, Parkash S.; Lingen, Mark W.; Saloura, Vassiliki; Villaflor, Victoria M.; Salgia, Ravi; Kimple, Randall J.; Wheeler, Deric L.

2016-01-01

Purpose Head and neck squamous cell carcinoma (HNSCC) represents the eighth most common malignancy worldwide. Standard of care treatments for HNSCC patients include surgery, radiation and chemotherapy. Additionally, the anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab is often used in combination with these treatment modalities. Despite clinical success with these therapeutics, HNSCC remains a difficult to treat malignancy. Thus, identification of new molecular targets is critical. Experimental Design In the current study, the receptor tyrosine kinase AXL was investigated as a molecular target in HNSCC using established cell lines, HNSCC patient derived xenografts (PDXs), and human tumors. HNSCC dependency on AXL was evaluated with both anti-AXL siRNAs and the small molecule AXL inhibitor R428. Furthermore, AXL inhibition was evaluated with standard of care treatment regimes used in HNSCC. Results AXL was found to be highly overexpressed in several models of HNSCC, where AXL was significantly associated with higher pathologic grade, presence of distant metastases and shorter relapse free survival in patients with HNSCC. Further investigations indicated that HNSCC cells were reliant on AXL for cellular proliferation, migration, and invasion. Additionally, targeting AXL increased HNSCC cell line sensitivity to chemotherapy, cetuximab, and radiation. Moreover, radiation resistant HNSCC cell line xenografts and PDXs expressed elevated levels of both total and activated AXL, indicating a role for AXL in radiation resistance. Conclusion Collectively, this study provides evidence for the role of AXL in HNSCC pathogenesis and supports further pre-clinical and clinical evaluation of anti-AXL therapeutics for the treatment of patients with HNSCC. PMID:25767293

Solid Oxide Fuel Cell short stack performance testing - part B: Operation in carbon capture applications and degradation issues

NASA Astrophysics Data System (ADS)

Mastropasqua, L.; Campanari, S.; Brouwer, J.

2017-12-01

The need to experimentally understand the performance of Solid Oxide Fuel Cells (SOFC) stacks under Carbon Capture and Storage (CCS) mode operating conditions, hence with anode recirculation, has prompted this two-part study. The steady state performance of a 6-cell short stack of Y2O3 stabilised Zirconia (YSZ) with Ni/YSZ anodes and composite Sr-doped LaMnO3 (LSM)/YSZ cathodes is experimentally evaluated. In Part A, the electrical and environmental performance are assessed and the results are compared with the commercial full-scale micro-Combined Heat and Power system, which comprises the same cells. In Part B of this work, a specific set of stack operating conditions important to CCS applications is explored. The experimental inlet composition is changed in order to reproduce a simulated syngas in CCS mode operation for different fuel utilisation factors. Operation with the simulated anode recycle syngas leads to lower voltage when the anode recycle is lower, mainly due to higher internal reforming and polarisation losses. A clear voltage trend is observed when the amount of CO content in the inlet fuel is increased, signalling an improvement of the polarisation performance at constant current density and fixed inlet equivalent hydrogen content. Stack degradation is measured and results in line with manufacturer's data.

Vibrational dynamics (IR, Raman, NRVS) and DFT study of new antitumor tetranuclearstannoxanecluster, Sn(IV)$-$oxo$-$${di$$-$o$-$vanillin} dimethyl dichloride

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arjmand, F.; Sharma, S.; Usman, M.

2016-06-21

The vibrational dynamics of a newly synthesized tetrastannoxane was characterized with a combination of experimental (Raman, IR and tin-based nuclear resonance vibrational spectroscopy) and computational (DFT/B3LYP) methods, with an emphasis on the vibrations of the tin sites. The cytotoxic activity revealed a significant regression selectively against the human pancreatic cell lines.

Ontological representation, integration, and analysis of LINCS cell line cells and their cellular responses.

PubMed

Ong, Edison; Xie, Jiangan; Ni, Zhaohui; Liu, Qingping; Sarntivijai, Sirarat; Lin, Yu; Cooper, Daniel; Terryn, Raymond; Stathias, Vasileios; Chung, Caty; Schürer, Stephan; He, Yongqun

2017-12-21

Aiming to understand cellular responses to different perturbations, the NIH Common Fund Library of Integrated Network-based Cellular Signatures (LINCS) program involves many institutes and laboratories working on over a thousand cell lines. The community-based Cell Line Ontology (CLO) is selected as the default ontology for LINCS cell line representation and integration. CLO has consistently represented all 1097 LINCS cell lines and included information extracted from the LINCS Data Portal and ChEMBL. Using MCF 10A cell line cells as an example, we demonstrated how to ontologically model LINCS cellular signatures such as their non-tumorigenic epithelial cell type, three-dimensional growth, latrunculin-A-induced actin depolymerization and apoptosis, and cell line transfection. A CLO subset view of LINCS cell lines, named LINCS-CLOview, was generated to support systematic LINCS cell line analysis and queries. In summary, LINCS cell lines are currently associated with 43 cell types, 131 tissues and organs, and 121 cancer types. The LINCS-CLO view information can be queried using SPARQL scripts. CLO was used to support ontological representation, integration, and analysis of over a thousand LINCS cell line cells and their cellular responses.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

PubMed

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

A functional in vivo screen for regulators of tumor progression identifies HOXB2 as a regulator of tumor growth in breast cancer.

PubMed

Boimel, Pamela J; Cruz, Cristian; Segall, Jeffrey E

2011-09-01

Microarray profiling in breast cancer patients has identified genes correlated with prognosis whose functions are unknown. The purpose of this study was to develop an in vivo assay for functionally screening regulators of tumor progression using a mouse model. Transductant shRNA cell lines were made in the MDA-MB-231 breast cancer line. A pooled population of 25 transductants was injected into the mammary fat pads and tail veins of mice to evaluate tumor growth, and experimental metastasis. The proportions of transductants were evaluated in the tumor and metastases using barcodes specific to each shRNA transductant. We characterized the homeobox 2 transcription factor as a negative regulator, decreasing tumor growth in MDA-MB-231, T47D, and MTLn3 mammary adenocarcinoma cell lines. Homeobox genes have been correlated with cancer patient prognosis and tumorigenesis. Here we use a novel in vivo shRNA screen to identify a new role for a homeobox gene in human mammary adenocarcinoma. Copyright © 2011 Elsevier Inc. All rights reserved.

A functional in vivo screen for regulators of tumor progression identifies HOXB2 as a regulator of tumor growth in breast cancer

PubMed Central

Boimel, Pamela J.; Cruz, Cristian; Segall, Jeffrey E.

2011-01-01

Microarray profiling in breast cancer patients have identified genes correlated with prognosis whose functions are unknown. The purpose of this study was to develop an in vivo assay for functionally screening regulators of tumor progression using a mouse model. Transductant shRNA cell lines were made in the MDA-MB-231 breast cancer line. A pooled population of 25 transductants was injected into the mammary fat pads and tail veins of mice to evaluate tumor growth, and experimental metastasis. The proportions of transductants were evaluated in the tumor and metastases using barcodes specific to each shRNA transductant. We characterized the homeobox 2 transcription factor as a negative regulator, decreasing tumor growth in MDA-MB-231, T47D, and MTLn3 mammary adenocarcinoma cell lines. Homeobox genes have been correlated with cancer patient prognosis and tumorigenesis. Here we use a novel in vivo shRNA screen to identify a new role for a homeobox gene in human mammary adenocarcinoma. PMID:21672623

A read-in IC for infrared scene projectors with voltage drop compensation for improved uniformity of emitter current

NASA Astrophysics Data System (ADS)

Cho, Min Ji; Shin, Uisub; Lee, Hee Chul

2017-05-01

This paper proposes a read-in integrated circuit (RIIC) for infrared scene projectors, which compensates for the voltage drops in ground lines in order to improve the uniformity of the emitter current. A current output digital-to-analog converter is utilized to convert digital scene data into scene data currents. The unit cells in the array receive the scene data current and convert it into data voltage, which simultaneously self-adjusts to account for the voltage drop in the ground line in order to generate the desired emitter current independently of variations in the ground voltage. A 32 × 32 RIIC unit cell array was designed and fabricated using a 0.18-μm CMOS process. The experimental results demonstrate that the proposed RIIC can output a maximum emitter current of 150 μA and compensate for a voltage drop in the ground line of up to 500 mV under a 3.3-V supply. The uniformity of the emitter current is significantly improved compared to that of a conventional RIIC.

Creatine supplementation with methylglyoxal: a potent therapy for cancer in experimental models.

PubMed

Pal, Aparajita; Roy, Anirban; Ray, Manju

2016-08-01

The anti-cancer effect of methylglyoxal (MG) is now well established in the literature. The main aim of this study was to investigate the effect of creatine as a supplement in combination with MG both in vitro and in vivo. In case of the in vitro studies, two different cell lines, namely MCF-7 (human breast cancer cell line) and C2C12 (mouse myoblast cell line) were chosen. MG in combination with creatine showed enhanced apoptosis as well as higher cytotoxicity in the breast cancer MCF-7 cell line, compared to MG alone. Pre-treatment of well-differentiated C2C12 myotubes with cancerogenic 3-methylcholanthrene (3MC) induced a dedifferentiation of these myotubes towards cancerous cells (that mimic the effect of 3MC observed in solid fibro-sarcoma animal models) and subsequent exposure of these induced cancer cells with MG proved to be cytotoxic. Thus, creatine plus ascorbic acid enhanced the anti-cancer effects of MG. In contrast, when normal C2C12 muscle cells or myotubes (mouse normal myoblast cell line) were treated with MG or MG plus creatine and ascorbic acid, no detrimental effects were seen. This indicated that cytotoxic effects of MG are specifically limited towards cancer cells and are further enhanced when MG is used in combination with creatine and ascorbic acid. For the in vivo studies, tumors were induced by injecting Sarcoma-180 cells (2 × 10(6) cells/mouse) in the left hind leg. After 7 days of tumor inoculation, treatments were started with MG (20 mg/kg body wt/day, via the intravenous route), with or without creatine (150 mg/kg body wt/day, fed orally) and ascorbic acid (50 mg/kg body wt/day, fed orally) and continued for 10 consecutive days. Significant regression of tumor size was observed when Sarcoma-180 tumor-bearing mice were treated with MG and even more so with the aforesaid combination. The creatine-supplemented group demonstrated better overall survival in comparison with tumor-bearing mice without creatine. In conclusion, it may be stated that the anti-cancer effect of MG is enhanced by concomitant creatine supplementation, both in chemically transformed (by 3MC) muscle cells in vitro as well as in sarcoma animal model in vivo. These data strongly suggest that creatine supplementation may gain importance as a safe and effective supplement in therapeutic intervention with the anti-cancer agent MG.

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

PubMed

Jin, Xun; Lee, Joong-Seob; Kwak, Sungwook; Lee, Soo-Yeon; Jung, Ji-Eun; Kim, Tae-Kyung; Xu, Chenxiong; Hong, Zhongshan; Li, Zhehu; Kim, Sun-Myung; Pian, Xumin; Lee, Dong-Hee; Yoon, Jong-Taek; You, Seungkwon; Choi, Yun-Jaie; Kim, Huunggee

2006-02-28

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Investigation of the expression of RIF1 gene on head and neck, pancreatic and brain cancer and cancer stem cells.

PubMed

GursesCila, Hacer E; Acar, Muradiye; Barut, Furkan B; Gunduz, Mehmet; Grenman, Reidar; Gunduz, Esra

2016-12-01

Recent studies have shown that cancer stem cells are resistant to chemotherapy. The aim of this study was to compare RIF1 gene expression in head and neck, pancreatic cancer and glioma cell lines and the cancer stem cells isolated from these cell lines. UT-SCC-74 from Turku University and UT-SCC-74B primary tumor metastasis and neck cancer cell lines, YKG-1 glioma cancer cell line from RIKEN, pancreatic cancer cell lines and ASPC-1 cells from ATCC were grown in cell culture. To isolate cancer stem cells, ALDH-1 for UT-SCC-74 and UT-SCC-74B cell line, CD-133 for YKG-1 cell line and CD-24 for ASPC-1 cell line, were used as markers of cancer stem cells. RNA isolation was performed for both cancer lines and cancer stem cells. RNAs were converted to cDNA. RIF1 gene expression was performed by qRT-PCR analysis. RIF1 gene expression was compared with cancer cell lines and cancer stem cells isolated from these cell lines. The possible effect of RIF1 gene was evaluated. In the pancreatic cells, RIF1 gene expression in the stem cell-positive cell line was 256 time that seen in the stem cell-negative cell line. Considering the importance of RIF1 in NHEJ and of NHEJ in pancreatic cancer, RIF1 may be one of the genes that plays an important role in the diagnoses and therapeutic treatment of pancreatic cancer. The results of head and neck and brain cancers are inconclusive and further studies are required to elucidate the connection between RIF1 gene and these other types of cancers.

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle.

PubMed

Ono, Takeshi; Tadakuma, Takushi; Rodriguez, Ana

2007-03-01

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.

The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH.

PubMed

Barbero-Becerra, Varenka J; Giraudi, Pablo J; Chávez-Tapia, Norberto C; Uribe, Misael; Tiribelli, Claudio; Rosso, Natalia

2015-10-01

A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact. The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR. FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system. The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators. Copyright © 2015 Elsevier Ltd. All rights reserved.

Apigenin inhibits NF-κB and snail signaling, EMT and metastasis in human hepatocellular carcinoma.

PubMed

Qin, Yuan; Zhao, Dong; Zhou, Hong-Gang; Wang, Xing-Hui; Zhong, Wei-Long; Chen, Shuang; Gu, Wen-Guang; Wang, Wei; Zhang, Chun-Hong; Liu, Yan-Rong; Liu, Hui-Juan; Zhang, Qiang; Guo, Yuan-Qiang; Sun, Tao; Yang, Cheng

2016-07-05

Apigenin is a naturally occurring compound with anti-inflammatory, antioxidant, and anticancer properties. In this study, we investigated the effects of apigenin on migration and metastasis in experimental human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo. Apigenin dose-dependently inhibited proliferation, migration, and invasion by PLC and Bel-7402 human HCC cells. It also suppressed tumor growth in PLC cell xenografts without altering body weight, thereby prolonging survival. Apigenin reduced Snai1 and NF-κB expression, reversed increases in epithelial-mesenchymal transition (EMT) marker levels, increased cellular adhesion, regulated actin polymerization and cell migration, and inhibited invasion and migration by HCC cells. Apigenin may therefore inhibit EMT by inhibiting the NF-κB/Snail pathway in human HCC.

Apigenin inhibits NF-κB and Snail signaling, EMT and metastasis in human hepatocellular carcinoma

PubMed Central

Zhong, Wei-long; Chen, Shuang; Gu, Wen-guang; Wang, Wei; Zhang, Chun-hong; Liu, Yan-rong; Liu, Hui-juan; Zhang, Qiang; Guo, Yuan-qiang; Sun, Tao; Yang, Cheng

2016-01-01

Apigenin is a naturally occurring compound with anti-inflammatory, antioxidant, and anticancer properties. In this study, we investigated the effects of apigenin on migration and metastasis in experimental human hepatocellular carcinoma (HCC) cell lines in vitro and in vivo. Apigenin dose-dependently inhibited proliferation, migration, and invasion by PLC and Bel-7402 human HCC cells. It also suppressed tumor growth in PLC cell xenografts without altering body weight, thereby prolonging survival. Apigenin reduced Snai1 and NF-κB expression, reversed increases in epithelial-mesenchymal transition (EMT) marker levels, increased cellular adhesion, regulated actin polymerization and cell migration, and inhibited invasion and migration by HCC cells. Apigenin may therefore inhibit EMT by inhibiting the NF-κB/Snail pathway in human HCC. PMID:27203387

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm

PubMed Central

Philippe, Laure; Ceroi, Adam; Bôle-Richard, Elodie; Jenvrin, Alizée; Biichle, Sabeha; Perrin, Sophie; Limat, Samuel; Bonnefoy, Francis; Deconinck, Eric; Saas, Philippe; Garnache-Ottou, Francine; Angelot-Delettre, Fanny

2017-01-01

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals’ survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged. PMID:28798071

Pharmacodynamic Modeling of Cell Cycle Effects for Gemcitabine and Trabectedin Combinations in Pancreatic Cancer Cells

PubMed Central

Miao, Xin; Koch, Gilbert; Ait-Oudhia, Sihem; Straubinger, Robert M.; Jusko, William J.

2016-01-01

Combinations of gemcitabine and trabectedin exert modest synergistic cytotoxic effects on two pancreatic cancer cell lines. Here, systems pharmacodynamic (PD) models that integrate cellular response data and extend a prototype model framework were developed to characterize dynamic changes in cell cycle phases of cancer cell subpopulations in response to gemcitabine and trabectedin as single agents and in combination. Extensive experimental data were obtained for two pancreatic cancer cell lines (MiaPaCa-2 and BxPC-3), including cell proliferation rates over 0–120 h of drug exposure, and the fraction of cells in different cell cycle phases or apoptosis. Cell cycle analysis demonstrated that gemcitabine induced cell cycle arrest in S phase, and trabectedin induced transient cell cycle arrest in S phase that progressed to G2/M phase. Over time, cells in the control group accumulated in G0/G1 phase. Systems cell cycle models were developed based on observed mechanisms and were used to characterize both cell proliferation and cell numbers in the sub G1, G0/G1, S, and G2/M phases in the control and drug-treated groups. The proposed mathematical models captured well both single and joint effects of gemcitabine and trabectedin. Interaction parameters were applied to quantify unexplainable drug-drug interaction effects on cell cycle arrest in S phase and in inducing apoptosis. The developed models were able to identify and quantify the different underlying interactions between gemcitabine and trabectedin, and captured well our large datasets in the dimensions of time, drug concentrations, and cellular subpopulations. PMID:27895579

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

USDA-ARS?s Scientific Manuscript database

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

PubMed Central

2011-01-01

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest. PMID:21435248

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.

PubMed

Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N; Glenn, Sean T; Liu, Song; Trump, Donald L; Johnson, Candace S

2015-04-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of ethylnitrosoureas of substituted naphthalimides as anticancer compounds.

PubMed

Pain, Anindita; Samanta, Suva; Dutta, Sushanta; Saxena, Ajit K; Shanmugavel, Mutiah; Sharma, Madhunika; Qazi, Gulam N; Sanyal, Utpal

2007-01-01

Four new ethylnitrosourea derivatives of substituted naphthalimides 3a-d have been synthesized from the respective N-(2-ethylamino) naphthalimides. Their chemical alkylating activity compared with the clinical drug CCNU and an experimental compound Mitonafide indicated that they possess lower alkylating activity than CCNU and comparable activity with the latter. Their anti-tumor efficacies were assessed in vivo in two murine ascites tumors namely Sarcoma-180 (S-180) and Ehrlich ascites carcinoma (EAC) by measuring the increase in median survival times (MST) of drug treated (T) over untreated control (C) mice. CCNU and Mitonafide were used as positive controls for comparison. The representative compound 3a has displayed marginal anti-tumoral activity in these tumors. Three compounds were further screened in vitro in 4 different human tumor cell lines but no significant activity was observed in those lines. These compounds moderately inhibit the synthesis of DNA and RNA in S-180 tumor cells.

IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling

PubMed Central

Lu, Jinchang; Song, Guohui; Tang, Qinglian; Zou, Changye; Han, Feng; Zhao, Zhiqiang; Yong, Bicheng; Yin, Junqiang; Xu, Huaiyuan; Xie, Xianbiao; Kang, Tiebang; Lam, YingLee; Yang, Huiling; Shen, Jingnan; Wang, Jin

2015-01-01

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis–free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis. PMID:25822025

Classification of bladder cancer cell lines using Raman spectroscopy: a comparison of excitation wavelength, sample substrate and statistical algorithms

NASA Astrophysics Data System (ADS)

Kerr, Laura T.; Adams, Aine; O'Dea, Shirley; Domijan, Katarina; Cullen, Ivor; Hennelly, Bryan M.

2014-05-01

Raman microspectroscopy can be applied to the urinary bladder for highly accurate classification and diagnosis of bladder cancer. This technique can be applied in vitro to bladder epithelial cells obtained from urine cytology or in vivo as an optical biopsy" to provide results in real-time with higher sensitivity and specificity than current clinical methods. However, there exists a high degree of variability across experimental parameters which need to be standardised before this technique can be utilized in an everyday clinical environment. In this study, we investigate different laser wavelengths (473 nm and 532 nm), sample substrates (glass, fused silica and calcium fluoride) and multivariate statistical methods in order to gain insight into how these various experimental parameters impact on the sensitivity and specificity of Raman cytology.

Placental transfer and metabolism: an overview of the experimental models utilizing human placental tissue.

PubMed

Myllynen, Päivi; Vähäkangas, Kirsi

2013-02-01

Over the decades several ex vivo and in vitro models which utilize delivered human placenta have been developed to study various placental functions. The use of models originating from human placenta to study transplacental transfer and related mechanisms is an attractive option because human placenta is relatively easily available for experimental studies. After delivery placenta has served its purpose and is usually disposed of. The purpose of this review is to give an overview of the use of human placental models for the studies on human placental transfer and related mechanisms such as transporter functions and xenobiotic metabolism. Human placental perfusion, the most commonly used continuous cell lines, primary cells and tissue culture, as well as subcellular fractions are briefly introduced and their major advantages and disadvantages are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular effects.

PubMed

Oostingh, Gertie J; Papaioannou, Eleni; Chasapidis, Leonidas; Akritidis, Theofylaktos; Konstandopoulos, Athanasios G; Duschl, Albert

2013-09-01

Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

PubMed

Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

2016-01-01

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

How do heterogeneities in single cell rigidity influence the mechanical behavior at the tissue level?

NASA Astrophysics Data System (ADS)

Bi, Dapeng; Wetzel, Franziska; Fritsch, Anatol; Marchetti, M. Cristina; Manning, M. Lisa; Kaes, Josef

It has been long recognized that solid tumor tissues are mechanically more rigid than surrounding healthy tissues. However recent experiments have shown that in primary tumor samples from patients with mammary and cervix carcinomas, cells exhibit a broad distribution of rigidities, with a higher fraction of softer and more contractile cells compared to normal tissues. This gives rise to a paradox: does softness emerge from adaptation to mechanical and chemical cues in the external microenvironment, or are soft cells already present inside a primary solid tumor? Motivated by these observations, we study a model of dense tissues that incorporates the experimental data for cell stiffness variations to reveal that, surprisingly, tumors with a significant fraction of very soft cells can still remain rigid. Moreover, in tissues with the observed distributions of cell stiffnesses, softer cells spontaneously self-organize into lines or streams, possibly facilitating cancer metastasis.

Concepts of Cell Lineage in Mammalian Embryos.

PubMed

Papaioannou, Virginia E

2016-01-01

Cell lineage is the framework for understanding cellular diversity, stability of differentiation, and its relationship to pluripotency. The special condition of in utero development in mammals has presented challenges to developmental biologists in tracing cell lineages but modern imaging and cell marking techniques have allowed the gradual elucidation of lineage relationships. Early experimental embryology approaches had limited resolution and relied of suboptimal cell markers and considerable disturbance to the embryos. Transgenic technology introduced genetic markers, particularly fluorescent proteins that, combined with sophisticated imaging modalities, greatly increase resolution and allow clonal analysis within lineages. The concept of cell lineage has also undergone evolution as it became possible to trace the lineage of cells based not only on their physical location or attributes but also on their gene expression pattern, thus opening up mechanistic lines of investigation into the determinants of cell lineage. © 2016 Elsevier Inc. All rights reserved.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

PubMed

Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C; Raghuram, Viswanathan; Knepper, Mark A

2015-12-15

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa. Copyright © 2015 the American Physiological Society.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions

PubMed Central

Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C.; Raghuram, Viswanathan

2015-01-01

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out “deep” proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate “virtual Western blots” for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa. PMID:26310816

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Apoptotic effect of α-Fe2O3 and SiO2 nanoparticles in human rhabdomyosarcoma cell line

NASA Astrophysics Data System (ADS)

Fatima, Mahvish; Fakhar-e-Alam, Muhammad; Atif, M.; Nadeem Shakoor, Muhammad; Afzal, Muhammad; Waseem, Muhammad; Hammad Aziz, Muhammad

2014-12-01

Nanotechnology provides the opportunity for the development of new materials in the nanometer size range, with many potential applications in biological sciences and clinical medicine. It has been reported that RD (muscle cancer cell line) is the most common soft tissue sarcoma in children originating from immature cells, comprising 2.9% of all malignancies in patients younger than 20 years old, with 350 cases diagnosed annually in the United States. Soft tissue is the most common target organ for nanoparticles after they gain significant entry into the target site through any of the possible routes. RD cell lines have been used as an experimental biological model in this article. A suitable environment was provided until 75% of RD cell confluence was reached. Prior to determination of toxicity of hematite (α-Fe2O3) and SiO2 nanoparticles, the sizes and shapes were confirmed using scanning electron microscopy (SEM), and the sizes were about 66 and 250 nm respectively. Moreover, 10-80 μg ml-1 of α-Fe2O3 and SiO2 nanoparticles dispersed in solution were labeled for each row of 96 well plates. The present study evaluates the suppression factor of the said particles, which leads to cell killing phenomena. After successful measurements in the above mentioned experiment, the author will be able to give the actual cause of cell killing effects. The given study has provided valuable insights into a feasible mechanism of apoptosis caused by α-Fe2O3 and SiO2 nanoparticles. An underlying promising mechanism of apoptosis due to α-Fe2O3 and SiO2 nanoparticle exposure should be further investigated at the in vivo level.

Molecularly Targeted Dose-Enhancement Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model

DTIC Science & Technology

2013-10-01

cell lines, such as cervix cancer cell line (HeLa) and breast cancer cell line (MDA-MB-231), were also employed. The experiments with other cell lines...breast cancer cell line (MDA-MB- 231), and cervix cancer cell line (HeLa). Different from our hypothesis, prostate cancer cell lines did not present...Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model PRINCIPAL INVESTIGATOR: Kwang Song