Systematic identification of combinatorial drivers and targets in cancer cell lines.

PubMed

Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation.

PubMed

Hayes, Tyler F; Benaich, Nathan; Goldie, Stephen J; Sipilä, Kalle; Ames-Draycott, Ashley; Cai, Wenjun; Yin, Guangliang; Watt, Fiona M

2016-12-01

Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

PubMed

Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

2014-01-01

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

PubMed

Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

2016-08-01

Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

PubMed Central

Cheng, J; Haas, M

1990-01-01

Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

PubMed

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A; Kim, Sungjoon; Wilson, Christopher J; Lehár, Joseph; Kryukov, Gregory V; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F; Monahan, John E; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H; Cheng, Jill; Yu, Guoying K; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P; Gabriel, Stacey B; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E; Weber, Barbara L; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L; Meyerson, Matthew; Golub, Todd R; Morrissey, Michael P; Sellers, William R; Schlegel, Robert; Garraway, Levi A

2012-03-28

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.

Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta.

PubMed

Qin, Sharon Q; Kusuma, Gina D; Al-Sowayan, Batla; Pace, Rishika A; Isenmann, Sandra; Pertile, Mark D; Gronthos, Stan; Abumaree, Mohamed H; Brennecke, Shaun P; Kalionis, Bill

2016-03-01

Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Metabolic Profile of Oral Squamous Carcinoma Cell Lines Relies on a Higher Demand of Lipid Metabolism in Metastatic Cells

PubMed Central

Sant’Anna-Silva, Ana Carolina B.; Santos, Gilson C.; Campos, Samir P. Costa; Oliveira Gomes, André Marco; Pérez-Valencia, Juan Alberto; Rumjanek, Franklin David

2018-01-01

Tumor cells are subjected to a broad range of selective pressures. As a result of the imposed stress, subpopulations of surviving cells exhibit individual biochemical phenotypes that reflect metabolic reprograming. The present work aimed at investigating metabolic parameters of cells displaying increasing degrees of metastatic potential. The metabolites present in cell extracts fraction of tongue fibroblasts and of cell lines derived from human tongue squamous cell carcinoma lineages displaying increasing metastatic potential (SCC9 ZsG, LN1 and LN2) were analyzed by 1H NMR (nuclear magnetic resonance) spectroscopy. Living, intact cells were also examined by the non-invasive method of fluorescence lifetime imaging microscopy (FLIM) based on the auto fluorescence of endogenous NADH. The cell lines reproducibly exhibited distinct metabolic profiles confirmed by Partial Least-Square Discriminant Analysis (PLS-DA) of the spectra. Measurement of endogenous free and bound NAD(P)H relative concentrations in the intact cell lines showed that ZsG and LN1 cells displayed high heterogeneity in the energy metabolism, indicating that the cells would oscillate between glycolysis and oxidative metabolism depending on the microenvironment’s composition. However, LN2 cells appeared to have more contributions to the oxidative status, displaying a lower NAD(P)H free/bound ratio. Functional experiments of energy metabolism, mitochondrial physiology, and proliferation assays revealed that all lineages exhibited similar energy features, although resorting to different bioenergetics strategies to face metabolic demands. These differentiated functions may also promote metastasis. We propose that lipid metabolism is related to the increased invasiveness as a result of the accumulation of malonate, methyl malonic acid, n-acetyl and unsaturated fatty acids (CH2)n in parallel with the metastatic potential progression, thus suggesting that the NAD(P)H reflected the lipid catabolic/anabolic pathways. PMID:29456966

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

PubMed Central

Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

2014-01-01

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

Characterizing Mystery Cell Lines: Student-driven Research Projects in an Undergraduate Neuroscience Laboratory Course.

PubMed

Lemons, Michele L

2012-01-01

Inquiry-based projects promote discovery and retention of key concepts, increase student engagement, and stimulate interest in research. Described here are a series of lab exercises within an undergraduate upper level neuroscience course that train students to design, execute and analyze their own hypothesis-driven research project. Prior to developing their own projects, students learn several research techniques including aseptic cell culture, cell line maintenance, immunocytochemistry and fluorescent microscopy. Working in groups, students choose how to use these techniques to characterize and identify a "mystery" cell line. Each lab group is given a unique cell line with either a neural, astrocyte, or Schwann cell origin. Working together, students plan and execute experiments to determine the cellular origin and other unique characteristics of their mystery cell line. Students generate testable hypotheses, design interpretable experiments, generate and analyze data, and report their findings in both oral and written formats. Students receive instructor and peer feedback throughout the entire project. In summary, these labs train students the process of scientific research. This series of lab exercises received very strong positive feedback from the students. Reflections on student feedback and plans for future improvements are discussed.

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.

Characterizing Mystery Cell Lines: Student-driven Research Projects in an Undergraduate Neuroscience Laboratory Course

PubMed Central

Lemons, Michele L.

2012-01-01

Inquiry-based projects promote discovery and retention of key concepts, increase student engagement, and stimulate interest in research. Described here are a series of lab exercises within an undergraduate upper level neuroscience course that train students to design, execute and analyze their own hypothesis-driven research project. Prior to developing their own projects, students learn several research techniques including aseptic cell culture, cell line maintenance, immunocytochemistry and fluorescent microscopy. Working in groups, students choose how to use these techniques to characterize and identify a “mystery” cell line. Each lab group is given a unique cell line with either a neural, astrocyte, or Schwann cell origin. Working together, students plan and execute experiments to determine the cellular origin and other unique characteristics of their mystery cell line. Students generate testable hypotheses, design interpretable experiments, generate and analyze data, and report their findings in both oral and written formats. Students receive instructor and peer feedback throughout the entire project. In summary, these labs train students the process of scientific research. This series of lab exercises received very strong positive feedback from the students. Reflections on student feedback and plans for future improvements are discussed. PMID:23504583

Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks

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

Smeets, M.F.M.A.; Mooren, E.H.M.; Abdel-Wahab, A.H.A.

1994-11-01

The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines,more » the t{sub 1/2} of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine). 31 refs., 8 figs.« less

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

PubMed Central

2011-01-01

Background Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. PMID:21767383

Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System

PubMed Central

Fredebohm, Johannes; Boettcher, Michael; Eisen, Christian; Gaida, Matthias M.; Heller, Anette; Keleg, Shereen; Tost, Jörg; Greulich-Bode, Karin M.; Hotz-Wagenblatt, Agnes; Lathrop, Mark; Giese, Nathalia A.; Hoheisel, Jörg D.

2012-01-01

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer. PMID:23152778

The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity

PubMed Central

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A.; Kim, Sungjoon; Wilson, Christopher J.; Lehár, Joseph; Kryukov, Gregory V.; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F.; Monahan, John E.; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A.; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H.; Cheng, Jill; Yu, Guoying K.; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D.; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C.; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P.; Gabriel, Stacey B.; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E.; Weber, Barbara L.; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L.; Meyerson, Matthew; Golub, Todd R.; Morrissey, Michael P.; Sellers, William R.; Schlegel, Robert; Garraway, Levi A.

2012-01-01

The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic avenues remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacologic annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacologic profiles for 24 anticancer drugs across 479 of the lines, this collection allowed identification of genetic, lineage, and gene expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Altogether, our results suggest that large, annotated cell line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of “personalized” therapeutic regimens2. PMID:22460905

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

PubMed

Narasimhan, Prakash Babu; Akabas, Leor; Tariq, Sameha; Huda, Naureen; Bennuru, Sasisekhar; Sabzevari, Helen; Hofmeister, Robert; Nutman, Thomas B; Tolouei Semnani, Roshanak

2018-04-01

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

Elliptical storm cell modeling of digital radar data

NASA Technical Reports Server (NTRS)

Altman, F. J.

1972-01-01

A model for spatial distributions of reflectivity in storm cells was fitted to digital radar data. The data were taken with a modified WSR-57 weather radar with 2.6-km resolution. The data consisted of modified B-scan records on magnetic tape of storm cells tracked at 0 deg elevation for several hours. The MIT L-band radar with 0.8-km resolution produced cross-section data on several cells at 1/2 deg elevation intervals. The model developed uses ellipses for contours of constant effective-reflectivity factor Z with constant orientation and eccentricity within a horizontal cell cross section at a given time and elevation. The centers of the ellipses are assumed to be uniformly spaced on a straight line, with areas linearly related to log Z. All cross sections are similar at different heights (except for cell tops, bottoms, and splitting cells), especially for the highest reflectivities; wind shear causes some translation and rotation between levels. Goodness-of-fit measures and parameters of interest for 204 ellipses are considered.

Phenotypic and Molecular Analysis of Mes-3, a Maternal-Effect Gene Required for Proliferation and Viability of the Germ Line in C. Elegans

PubMed Central

Paulsen, J. E.; Capowski, E. E.; Strome, S.

1995-01-01

mes-3 is one of four maternal-effect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells and no gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearance of the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmed cell death. The few germ cells that appear healthy in mes-3 worms do not differentiate into gametes, even after elimination of the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterization of mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins. PMID:8601481

Phase resolved and coherence gated en face reflection imaging of multilayered embryonal carcinoma cells

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Fukami, Tadashi; Iwai, Hidenao; Yamashita, Yutaka

2012-03-01

Embryonal carcinoma (EC) cells, which are cell lines derived from teratocarcinomas, have characteristics in common with stem cells and differentiate into many kinds of functional cells. Similar to embryonic stem (ES) cells, undifferentiated EC cells form multi-layered spheroids. In order to visualize the three-dimensional structure of multilayered EC cells without labeling, we employed full-field interference microscopy with the aid of a low-coherence quantitative phase microscope, which is a reflection-type interference microscope employing the digital holographic technique with a low-coherent light source. Owing to the low-coherency of the light-source (halogen lamp), only the light reflected from reflective surface at a specific sectioning height generates an interference image on the CCD camera. P19CL6 EC cells, derived from mouse teratocarcinomas, formed spheroids that are about 50 to 200 micrometers in diameter. Since the height of each cell is around 10 micrometers, it is assumed that each spheroid has 5 to 20 cell layers. The P19CL6 spheroids were imaged in an upright configuration and the horizontally sectioned reflection images of the sample were obtained by sequentially and vertically scanning the zero-path-length height. Our results show the threedimensional structure of the spheroids, in which plasma and nuclear membranes were distinguishably imaged. The results imply that our technique is further capable of imaging induced pluripotent stem (iPS) cells for the assessment of cell properties including their pluripotency.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

PubMed

Wrona, Dominik; Siler, Ulrich; Reichenbach, Janine

2017-03-13

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47 phox -deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47 phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47 phox -subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47 phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47 phox -deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy.

PubMed

Boyde, A; Vesely, P; Gray, C; Jones, S J

1994-01-01

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37 degrees C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.

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.

Monitoring Intracellular Oxygen Concentration: Implications for Hypoxia Studies and Real-Time Oxygen Monitoring.

PubMed

Potter, Michelle; Badder, Luned; Hoade, Yvette; Johnston, Iain G; Morten, Karl J

2016-01-01

The metabolic properties of cancer cells have been widely accepted as a hallmark of cancer for a number of years and have shown to be of critical importance in tumour development. It is generally accepted that tumour cells exhibit a more glycolytic phenotype than normal cells. In this study, we investigate the bioenergetic phenotype of two widely used cancer cell lines, RD and U87MG, by monitoring intracellular oxygen concentrations using phosphorescent Pt-porphyrin based intracellular probes. Our study demonstrates that cancer cell lines do not always exhibit an exclusively glycolytic phenotype. RD demonstrates a reliance on oxidative phosphorylation whilst U87MG display a more glycolytic phenotype. Using the intracellular oxygen sensing probe we generate an immediate readout of intracellular oxygen levels, with the glycolytic lines reflecting the oxygen concentration of the environment, and cells with an oxidative phenotype having significantly lower levels of intracellular oxygen. Inhibition of oxygen consumption in lines with high oxygen consumption increases intracellular oxygen levels towards environmental levels. We conclude that the use of intracellular oxygen probes provides a quantitative assessment of intracellular oxygen levels, allowing the manipulation of cellular bioenergetics to be studied in real time.

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.

Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics.

PubMed

Valtink, Monika; Gruschwitz, Rita; Funk, Richard H W; Engelmann, Katrin

2008-01-01

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Copyright 2008 S. Karger AG, Basel.

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

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.

LASER APPLICATIONS AND OTHER TOPICS IN QUANTUM ELECTRONICS: Selective reflection of obliquely incident polarised light

NASA Astrophysics Data System (ADS)

Fofanov, Ya A.

2009-06-01

A series of reflection resonances formed by the hyperfine components of the D2-lines in the spectrum of the natural mixture of rubidium isotopes is studied. Passages from resonantly frustrated total internal reflection to resonance Brewster reflection caused by the frequency tuning of the incident light are demonstrated experimentally. The contrast of the strongest refection resonances exceeds 500% at the moderate heating of reflecting cells. The intensity of the reflected light changes in this case by more than 20 times. A theory is developed which is based on a two-level model for resonance atoms and Fresnel formulas for reflection coefficients. Numerical calculations based on the proposed theory confirm main experimental results.

Mimicking Metastases Including Tumor Stroma: A New Technique to Generate a Three-Dimensional Colorectal Cancer Model Based on a Biological Decellularized Intestinal Scaffold.

PubMed

Nietzer, Sarah; Baur, Florentin; Sieber, Stefan; Hansmann, Jan; Schwarz, Thomas; Stoffer, Carolin; Häfner, Heide; Gasser, Martin; Waaga-Gasser, Ana Maria; Walles, Heike; Dandekar, Gudrun

2016-07-01

Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of β-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.

Proteomic Research Funding Opportunity | Office of Cancer Clinical Proteomics Research

Cancer.gov

To expand the understanding of how cells sense and respond to changes in their physical environment, the NCI is seeking to perform proteomic assays on the panel of cell lines grown on a variety of substrates. These assays will provide insight into changes in protein levels or phosphorylation changes that could reflect the activity of mechano-transduction pathways.

Farnesyltransferase inhibitor tipifarnib inhibits Rheb prenylation and stabilizes Bax in acute myelogenous leukemia cells

PubMed Central

Ding, Husheng; McDonald, Jennifer S.; Yun, Seongseok; Schneider, Paula A.; Peterson, Kevin L.; Flatten, Karen S.; Loegering, David A.; Oberg, Ann L.; Riska, Shaun M.; Huang, Shengbing; Sinicrope, Frank A.; Adjei, Alex A.; Karp, Judith E.; Meng, X. Wei; Kaufmann, Scott H.

2014-01-01

Although farnesyltransferase inhibitors have shown promising activity in relapsed lymphoma and sporadic activity in acute myelogenous leukemia, their mechanism of cytotoxicity is incompletely understood, making development of predictive biomarkers difficult. In the present study, we examined the action of tipifarnib in human acute myelogenous leukemia cell lines and clinical samples. In contrast to the Ras/MEK/ERK pathway-mediated Bim upregulation that is responsible for tipifarnib-induced killing of malignant lymphoid cells, inhibition of Rheb-induced mTOR signaling followed by dose-dependent upregulation of Bax and Puma occurred in acute myelogenous leukemia cell lines undergoing tipifarnib-induced apoptosis. Similar Bax and Puma upregulation occurred in serial bone marrow samples harvested from a subset of acute myelogenous leukemia patients during tipifarnib treatment. Expression of FTI-resistant Rheb M184L, like knockdown of Bax or Puma, diminished tipifarnib-induced killing. Further analysis demonstrated that increased Bax and Puma levels reflect protein stabilization rather than increased gene expression. In U937 cells selected for tipifarnib resistance, neither inhibition of signaling downstream of Rheb nor Bax and Puma stabilization occurred. Collectively, these results not only identify a pathway downstream from Rheb that contributes to tipifarnib cytotoxicity in human acute myelogenous leukemia cells, but also demonstrate that FTI-induced killing of lymphoid versus myeloid cells reflects distinct biochemical mechanisms downstream of different farnesylated substrates. (ClinicalTrials.gov identifier NCT00602771) PMID:23996484

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism.

PubMed

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-10-24

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism

PubMed Central

Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-01-01

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated “HL17NL10” and “HL18NL11.” All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin–Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses. PMID:27791106

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status

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

Vlashi, Erina, E-mail: evlashi@mednet.ucla.edu; Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, California; Chen, Allen M.

Purpose: To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Methods and Materials: Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positivemore » and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription–polymerase chain reaction for re-expression of reprogramming factors. Results: Patients with HPV-positive tumors have superior overall survival and local–regional control. Human papillomavirus–positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus–negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Conclusions: Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor.« less

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status.

PubMed

Vlashi, Erina; Chen, Allen M; Boyrie, Sabrina; Yu, Garrett; Nguyen, Andrea; Brower, Philip A; Hess, Clayton B; Pajonk, Frank

2016-04-01

To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positive and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription-polymerase chain reaction for re-expression of reprogramming factors. Patients with HPV-positive tumors have superior overall survival and local-regional control. Human papillomavirus-positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus-negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative analysis of cell columns in the cerebral cortex.

PubMed

Buxhoeveden, D P; Switala, A E; Roy, E; Casanova, M F

2000-04-01

We present a quantified imaging method that describes the cell column in mammalian cortex. The minicolumn is an ideal template with which to examine cortical organization because it is a basic unit of function, complete in itself, which interacts with adjacent and distance columns to form more complex levels of organization. The subtle details of columnar anatomy should reflect physiological changes that have occurred in evolution as well as those that might be caused by pathologies in the brain. In this semiautomatic method, images of Nissl-stained tissue are digitized or scanned into a computer imaging system. The software detects the presence of cell columns and describes details of their morphology and of the surrounding space. Columns are detected automatically on the basis of cell-poor and cell-rich areas using a Gaussian distribution. A line is fit to the cell centers by least squares analysis. The line becomes the center of the column from which the precise location of every cell can be measured. On this basis several algorithms describe the distribution of cells from the center line and in relation to the available surrounding space. Other algorithms use cluster analyses to determine the spatial orientation of every column.

A Preliminary Investigation into the Impact of a Pesticide Combination on Human Neuronal and Glial Cell Lines In Vitro

PubMed Central

Coleman, Michael D.; O'Neil, John D.; Woehrling, Elizabeth K.; Ndunge, Oscar Bate Akide; Hill, Eric J.; Menache, Andre; Reiss, Claude J.

2012-01-01

Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health. PMID:22880100

The functional genomic studies of curcumin.

PubMed

Huminiecki, Lukasz; Horbańczuk, Jarosław; Atanasov, Atanas G

2017-10-01

Curcumin is a natural plant-derived compound that has attracted a lot of attention for its anti-cancer activities. Curcumin can slow proliferation of and induce apoptosis in cancer cell lines, but the precise mechanisms of these effects are not fully understood. However, many lines of evidence suggested that curcumin has a potent impact on gene expression profiles; thus, functional genomics should be the key to understanding how curcumin exerts its anti-cancer activities. Here, we review the published functional genomic studies of curcumin focusing on cancer. Typically, a cancer cell line or a grafted tumor were exposed to curcumin and profiled with microarrays, methylation assays, or RNA-seq. Crucially, these studies are in agreement that curcumin has a powerful effect on gene expression. In the majority of the studies, among differentially expressed genes we found genes involved in cell signaling, apoptosis, and the control of cell cycle. Curcumin can also induce specific methylation changes, and is a powerful regulator of the expression of microRNAs which control oncogenesis. We also reflect on how the broader technological progress in transcriptomics has been reflected on the field of curcumin. We conclude by discussing the areas where more functional genomic studies are highly desirable. Integrated OMICS approaches will clearly be the key to understanding curcumin's anticancer and chemopreventive effects. Such strategies may become a template for elucidating the mode of action of other natural products; many natural products have pleiotropic effects that are well suited for a systems-level analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

PubMed

Havranek, Ondrej; Xu, Jingda; Köhrer, Stefan; Wang, Zhiqiang; Becker, Lisa; Comer, Justin M; Henderson, Jared; Ma, Wencai; Man Chun Ma, John; Westin, Jason R; Ghosh, Dipanjan; Shinners, Nicholas; Sun, Luhong; Yi, Allen F; Karri, Anusha R; Burger, Jan A; Zal, Tomasz; Davis, R Eric

2017-08-24

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL. © 2017 by The American Society of Hematology.

Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility.

PubMed

Knudsen, Erik S; Balaji, Uthra; Mannakee, Brian; Vail, Paris; Eslinger, Cody; Moxom, Christopher; Mansour, John; Witkiewicz, Agnieszka K

2018-03-01

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities. 27 primary PDAC tumours were employed for genetic analysis and development of tumour models. Tumour tissue was used for derivation of xenografts and cell lines. Exome sequencing was performed on the originating tumour and developed models. RNA sequencing, histological and functional analyses were employed to determine the relationship of the patient-derived models to clinical presentation of PDAC. The cohort employed captured the genetic diversity of PDAC. From most cases, both cell lines and xenograft models were developed. Exome sequencing confirmed preservation of the primary tumour mutations in developed cell lines, which remained stable with extended passaging. The level of genetic conservation in the cell lines was comparable to that observed with patient-derived xenograft (PDX) models. Unlike historically established PDAC cancer cell lines, patient-derived models recapitulated the histological architecture of the primary tumour and exhibited metastatic spread similar to that observed clinically. Detailed genetic analyses of tumours and derived models revealed features of ex vivo evolution and the clonal architecture of PDAC. Functional analysis was used to elucidate therapeutic vulnerabilities of relevance to treatment of PDAC. These data illustrate that with the appropriate methods it is possible to develop cell lines that maintain genetic features of PDAC. Such models serve as important substrates for analysing the significance of genetic variants and create a unique biorepository of annotated cell lines and xenografts that were established simultaneously from same primary tumour. These models can be used to infer genetic and empirically determined therapeutic sensitivities that would be germane to the patient. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Three-dimensional alginate spheroid culture system of murine osteosarcoma.

PubMed

Akeda, Koji; Nishimura, Akinobu; Satonaka, Haruhiko; Shintani, Ken; Kusuzaki, Katsuyuki; Matsumine, Akihiko; Kasai, Yuichi; Masuda, Koichi; Uchida, Atsumasa

2009-11-01

Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

High reflected cubic cavity as long path absorption cell for infrared gas sensing

NASA Astrophysics Data System (ADS)

Yu, Jia; Gao, Qiang; Zhang, Zhiguo

2014-10-01

One direct and efficient method to improve the sensitivity of infrared gas sensors is to increase the optical path length of gas cells according to Beer-Lambert Law. In this paper, cubic shaped cavities with high reflected inner coating as novel long path absorption cells for infrared gas sensing were developed. The effective optical path length (EOPL) for a single cubic cavity and tandem cubic cavities were investigated based on Tunable Diode Laser Absorption Spectroscopy (TDLAS) measuring oxygen P11 line at 763 nm. The law of EOPL of a diffuse cubic cavity in relation with the reflectivity of the coating, the port fraction and side length of the cavity was obtained. Experimental results manifested an increase of EOPL for tandem diffuse cubic cavities as the decrease of port fraction of the connecting aperture f', and the EOPL equaled to the sum of that of two single cubic cavities at f'<0.01. The EOPL spectra at infrared wavelength range for different inner coatings including high diffuse coatings and high reflected metallic thin film coatings were deduced.

Expression of Genes for Drug Transporters in the Human Female Genital Tract and Modulatory Effect of Antiretroviral Drugs.

PubMed

Hijazi, Karolin; Cuppone, Anna M; Smith, Kieron; Stincarelli, Maria A; Ekeruche-Makinde, Julia; De Falco, Giulia; Hold, Georgina L; Shattock, Robin; Kelly, Charles G; Pozzi, Gianni; Iannelli, Francesco

2015-01-01

Anti-retroviral (ARV) -based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues.

Expression of Genes for Drug Transporters in the Human Female Genital Tract and Modulatory Effect of Antiretroviral Drugs

PubMed Central

Hijazi, Karolin; Cuppone, Anna M.; Smith, Kieron; Stincarelli, Maria A.; Ekeruche-Makinde, Julia; De Falco, Giulia; Hold, Georgina L.; Shattock, Robin; Kelly, Charles G.; Pozzi, Gianni; Iannelli, Francesco

2015-01-01

Anti-retroviral (ARV) –based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug transporters involved in transport of tenofovir points to the possibility of combining these drugs to improve retention of individual drugs at target tissues. PMID:26102284

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.

Cytotoxic consequences of Halloysite nanotube/iron oxide nanocomposite and iron oxide nanoparticles upon interaction with bacterial, non-cancerous and cancerous cells.

PubMed

Abhinayaa, R; Jeevitha, G; Mangalaraj, D; Ponpandian, N; Vidhya, Kalieswaran; Angayarkanni, Jayaraman

2018-05-19

Cytotoxic effects of iron oxide (Fe 3 O 4 ) nanoparticles and Halloysite nanotube/iron oxide (HNT/Fe 3 O 4 ) nanocomposite are compared based on their interaction with Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis. Similarly, the action of these two nanomaterials on non-cancerous Vero cell lines and human lung cancerous (A-549) cell lines are compared. The cytotoxicity studies on Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite showed difference in the rate of killing of bacterial cells. This is reflected in differential cell growth, cell membrane integrity loss, lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production. These factors are measured over a range of concentrations of Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite and at specified time intervals, to test if there is any statistically significant difference between the toxicity of the two nanomaterials. Between the two nanomaterials, HNT/Fe 3 O 4 nanocomposite is found to be less toxic to bacterial cells than Fe 3 O 4 nanoparticles. HNT, when attached to the Fe 3 O 4 nanoparticles, changes their surface characteristics and suppresses their inherent toxicity on bacteria. In the study on the effect on cell lines, Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite are both seen to be biocompatible with Vero cell lines. However, HNT/Fe 3 O 4 nanocomposite showed more cytotoxicity than Fe 3 O 4 nanoparticles on A-549 cell lines. Copyright © 2018 Elsevier B.V. All rights reserved.

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

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

PubMed

Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

2016-03-01

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Evaluation of anti-apoptotic activity of different dietary antioxidants in renal cell carcinoma against hydrogen peroxide

PubMed Central

Garg, Neeraj K; Mangal, Sharad; Sahu, Tejram; Mehta, Abhinav; Vyas, Suresh P; Tyagi, Rajeev K

2011-01-01

Objective To evaluate the anti-apoptotic and radical scavenging activities of dietary phenolics, namely ascorbic acid,α-tocopherol acetate, citric acid, salicylic acid, and estimate H2O2-induced apoptosis in renal cell carcinoma cells. Methods The intracellular antioxidant potency of antioxidants was investigated. H2O2-induced apoptosis in RCC-26 was assayed with the following parameters: cell viability (% apoptosis), nucleosomal damage and DNA fragmentation, bcl-2 levels and flow cytometery analysis (ROS production evaluation). Results The anticancer properties of antioxidants such as ascorbic acid, α-tocopherol acetate, citric acid, salicylic acid with perdurable responses were investigated. It was observed that these antioxidants had protective effect (anti-apoptotic activity) against hydrogen peroxide (H2O2) in renal cell carcinoma (RCC-26) cell line. Conclusions This study reveals and proves the anticancer properties. However, in cancer cell lines anti-apoptotic activity can indirectly reflect the cancer promoter activity through radicals scavenging, and significantly protect nucleus and bcl-2. PMID:23569726

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

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.

Cell sources for in vitro human liver cell culture models

PubMed Central

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

2016-01-01

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. PMID:27385595

Transmittance of MCF-7 breast tumor cell line through visible and near infrared spectrum

NASA Astrophysics Data System (ADS)

Tabakoǧlu, H. Ã.-zgür

2016-03-01

In this study, light transmittance of MCF-7 tumor cells from 450 nm to 1100 nm has been measured in their growing medium and evaluated. Transmittance differences have been tried to be put forward in cancer cell line on visible (VIS) and near infrared (NIR) spectrum as well as in between different numbers of cells in medium. An absorption-reflection spectrophotometer was used in the experiments. System has a tungsten light source, optical chopper, a monochromator, sample chamber, silicon detectors, lock-in amplifier and computer. System was controlled by software in order to adjust scan range, scan steps and grating configuration. Cells were grown in medium, and measurements were taken from cells while they were in 5 ml medium. According to our findings, there are significant differences between VIS and NIR regions for the same number of cells. There were found no statistical difference among different numbers of cells. Increasing number of cells has not affected the transmittance. Transmittance of medium is not significantly different from different concentration of cells.

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

Role of the EGF-Related Growth Factor Cripto in Murine Mammary Tumorigenesis

DTIC Science & Technology

1999-10-01

release or disclosure of technical data (other than detailed manufacturing or process data) to, or use of such data by, a foreign government that is in...function, we have made constructs that express secreted processed NODAL protein in transfected mammalian cells. For this purpose, we have used a wild-type...that processing of unmodified NODAL protein is dependent upon the cell line used for expression, which presumably reflects differential expression of

Metabolic phenotyping of a model of adipocyte differentiation

PubMed Central

Roberts, Lee D.; Virtue, Sam; Vidal-Puig, Antonio; Nicholls, Andrew W.

2009-01-01

The 3T3-L1 murine cell line is a robust and widely used model for the study of adipogenesis and processes occurring in mature adipocytes. The fibroblastic like cells can be induced by hormones to differentiate into mature adipocytes. In this study, the metabolic phenotype associated with differentiation of the 3T3-L1 cell line has been studied using gas chromatography-mass spectrometry, 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry, direct infusion-mass spectrometry, and 13C substrate labeling in conjunction with multivariate statistics. The changes in metabolite concentrations at distinct periods during differentiation have been defined including alterations in the TCA cycle, glycolysis, the production of odd chain fatty acids by α-oxidation, fatty acid synthesis, fatty acid desaturation, polyamine biosynthesis, and trans-esterification to produce complex lipids. The metabolic changes induced during differentiation of the 3T3-L1 cell line were then compared with the metabolic differences between pre- and postdifferentiation primary adipocytes. These metabolic alterations reflect the changing role of the 3T3-L1 cells during differentiation, as well as possibly providing metabolic triggers to stimulate the processes which occur during differentiation. PMID:19602617

Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

PubMed

Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

2002-07-01

The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Mass spectrometric detection of 27-hydroxycholesterol in breast cancer exosomes.

PubMed

Roberg-Larsen, Hanne; Lund, Kaja; Seterdal, Kristina Erikstad; Solheim, Stian; Vehus, Tore; Solberg, Nina; Krauss, Stefan; Lundanes, Elsa; Wilson, Steven Ray

2017-05-01

Exosomes from cancer cells are rich sources of biomarkers and may contain elevated levels of lipids of diagnostic value. 27-Hydroxycholesterol (27-OHC) is associated with proliferation and metastasis in estrogen receptor positive (ER+) breast cancer. In this study, we investigated the levels of 27-OHC, and other sidechain-hydroxylated oxysterols in exosomes. To study both cytoplasmic and exosomal oxysterol samples of limited size, we have developed a capillary liquid chromatography-mass spectrometry platform that outperforms our previously published systems regarding chromatographic resolution, analysis time and sensitivity. In the analyzed samples, the quantified level of cytoplasmic 27-OHC using this platform fitted with mRNA levels of 27-OHC's corresponding enzyme, CYP27A1. We find clearly increased levels of 27-OHC in exosomes (i.e., enrichment) from an ER+ breast cancer cell line (MCF-7) compared to exosomes derived from an estrogen receptor (ER-) breast cancer cell line (MDA-MB-231) and other control exosomes (non-cancerous cell line (HEK293) and human pooled serum). The exosomal oxysterol profile did not reflect cytoplasmic oxysterol profiles in the cells of origin; cytoplasmic 27-OHC was low in ER+ MCF-7 cells while high in MDA-MB-231 cells. Other control cancer cells showed varied cytoplasmic oxysterol levels. Hence, exosome profiling in cancer cells might provide complementary information with the possibility of diagnostic value. Copyright © 2016 Elsevier Ltd. All rights reserved.

Aberrant patterns of X chromosome inactivation in a new line of human embryonic stem cells established in physiological oxygen concentrations.

PubMed

de Oliveira Georges, Juliana Andrea; Vergani, Naja; Fonseca, Simone Aparecida Siqueira; Fraga, Ana Maria; de Mello, Joana Carvalho Moreira; Albuquerque, Maria Cecília R Maciel; Fujihara, Litsuko Shimabukuro; Pereira, Lygia Veiga

2014-08-01

One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.

Proteins involved in neuronal differentiation of neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-Eun; Freilinger, Angelika; Gelpi, Ellen; Pollak, Arnold; Hengstschläger, Markus; Lubec, Gert

2007-06-01

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.

Lightning propagation and flash density in squall lines as determined with radar

NASA Technical Reports Server (NTRS)

Mazur, V.; Rust, W. D.

1983-01-01

Lightning echo rise times and range-time variations due to discharge propagation are determined using S and L band radars, and the evolution of precipitation reflectivity and the associated lightning activity in squall lines is investigated using VHF and L band radars. The rise time of radar echoes can be explained by ionized channel propagation through the radar beams. Speeds of at least 250,000 m/s are found from measurements of the radial velocity of streamer propagation along the antenna beam. The range-time variations in lightning echoes indicate that either new ionization occurs as streamers develop into different parts of the cloud, channel delay occurs during which adequate ionization exists for radar detection, or continuing current occurs. Determinations of the lightning flash density for a squall line in the U.S. show that the maximum lightning density tends to be near the leading edge of the precipitation cores in developing cells. Long discharges are produced as a cell in the squall line develops and the total lightning density increases, although short discharges predominate. As the cell dissipates, short flashes diminish or cease and the long flashes dominate the lightning activity.

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

A human thyroid cancer cell line, DH-14-3, newly established from poorly differentiated thyroid carcinoma.

PubMed

Teshima, Jin; Doi, Hideyuki; Fujimori, Keisei; Watanabe, Michio; Nakajima, Noriaki; Nakano, Tomoyuki; Takahashi, Yoshio; Ohuchi, Noriaki; Satomi, Susumu

2013-06-01

Poorly differentiated thyroid carcinoma (PDTC) is a newly recognized histological type of malignant thyroid tumor, accounting for about 2 - 13% of all thyroid carcinomas. PDTC is considered as a morphologically and biologically intermediate stage between well-differentiated thyroid carcinoma and anaplastic thyroid carcinoma. PDTC preferentially manifests bone metastases. We here established a cell line from a resected tumor specimen from a 70-year-old male patient with PDTC who presented with multiple bone metastases. This new thyroid tumor cell line was designated as DH-14-3 and was subsequently grown in culture for several years. DH-14-3 cells express thyroglobulin in the cytoplasm and thyroid transcription factor-1 in the nuclei, both proteins of which are specific markers for the thyroid gland. Importantly, triiodothyronine (T3) was detected in the cultured medium of DH-14-3 cells, in which, however, thyroxine (T4) was undetectable. Moreover, DH-14-3 cells secreted interleukin-8, transforming growth factor-β1, vascular endothelial growth factor, matrix metalloproteinase-1 and parathyroid hormone-related protein, all of which may be responsible for the aggressiveness or bone metastasis of PDTC. Thus, the production of these proteins may reflect the metastatic potential of this cell line. DH-14-3 cells also express CXC chemokine receptor-4 and epidermal growth factor receptor, and carry a missense mutation in the p53 tumor suppressor gene. In fact, transplantation of DH-14-3 cells into the back of nude mice resulted in the formation of tumors, thereby confirming the capability of tumorigenesis. DH-14-3 cells may be useful for investigating the biological features of PDTC and will contribute to the therapeutic study of thyroid cancer.

Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism

PubMed Central

Mills, Melody; Meysick, Karen C.; O'Brien, Alison D.

2000-01-01

Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli. PMID:10992497

Production of proinflammatory cytokines without invocation of cytotoxic effects by an Epstein-Barr virus-infected natural killer cell line established from a patient with hypersensitivity to mosquito bites.

PubMed

Suzuki, Daisuke; Tsuji, Kazuhide; Yamamoto, Takenobu; Fujii, Kazuyasu; Iwatsuki, Keiji

2010-10-01

Cumulative evidence supports that Epstein-Barr virus (EBV)-infected natural killer (NK) cells induce severe systemic and cutaneous inflammation in patients with hypersensitivity to mosquito bites (HMB). In order to understand the pathogenesis of HMB, we established an EBV-infected cell line and characterized the cytological profiles. A novel EBV-infected NK-cell line, designated NKED, was established from a patient with HMB and used for the present study along with two other NK-cell lines, KAI3 and KHYG-1. NKED expressed the latency II-related transcripts. NKED cells were positive for CD2 and CD161 antigens, and negative for CD3, CD16, CD34, CD56, and T-cell receptor α/β and γ/δ antigens. Although NKED cells contained several cytotoxic molecules, the cells had an extremely poor cytotoxic activity. The majority of NKED cells were negative for perforin, major histocompatibility complex class I-restricted NK-cell receptors, CD94 and KIR2D, and an activating receptor, NKG2D. NKED cells, however, secreted higher levels of tumor necrosis factor-α. Stimulation with phorbol 12-myristate 13-acetate or tumor necrosis factor-α induced expression of BZLF1 messenger RNA in the NKED and KAI3 cells, indicating the transition from the latent- to the lytic-cycle infection. These data suggested that NKED cells revealed a very low cytotoxic effect probably because of the low expression levels of perforin, but had the ability to release proinflammatory cytokines. NKED cells did not reflect the characteristics of HMB, as they were different from pathogenic NK cells proliferating in the HMB patient, but the difference indicated that pathogenic NK cells could change their character in the presence of interleukin-2. Copyright © 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection.

PubMed

Thang, P H; Ruffin, N; Brodin, D; Rethi, B; Cam, P D; Hien, N T; Lopalco, L; Vivar, N; Chiodi, F

2010-08-01

Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-gamma enhanced the expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant down-regulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL-1alpha,IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results are discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

PubMed Central

2004-01-01

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated. PMID:15324306

Monitoring longitudinal changes in irradiated head and neck cancer xenografts using diffuse reflectance spectroscopy

NASA Astrophysics Data System (ADS)

Vishwanath, Karthik; Jiang, Shudong; Gunn, Jason R.; Marra, Kayla; Andreozzi, Jacqueline M.; Pogue, Brian W.

2016-02-01

Radiation therapy is often used as the preferred clinical treatment for control of localized head and neck cancer. However, during the course of treatment (6-8 weeks), feedback about functional and/or physiological changes within impacted tissue are not obtained, given the onerous financial and/or logistical burdens of scheduling MRI, PET or CT scans. Diffuse optical sensing is well suited to address this problem since the instrumentation can be made low-cost and portable while still being able to non-invasively provide information about vascular oxygenation in vivo. Here we report results from studies that employed an optical fiber-based portable diffuse reflectance spectroscopy (DRS) system to longitudinally monitor changes in tumor vasculature within two head and neck cancer cell lines (SCC-15 and FaDu) xenografted in the flanks of nude mice, in two separate experiments. Once the tumor volumes were 100mm3, 67% of animals received localized (electron beam) radiation therapy in five fractions (8Gy/day, for 5 days) while 33% of the animals served as controls. DRS measurements were obtained from each animal on each day of treatment and then for two weeks post-treatment. Reflectance spectra were parametrized to extract total hemoglobin concentration and blood oxygen-saturation and the resulting time-trends of optical parameters appear to be dissimilar for the two cell-lines. These findings are also compared to previous animal experiments (using the FaDu line) that were irradiated using a photon beam radiotherapy protocol. These results and implications for the use of fiber-based DRS measurements made at local (irradiated) tumor site as a basis for identifying early radiotherapy-response are presented and discussed.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

PubMed Central

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

PubMed

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Space photovoltaic modules based on reflective optics

NASA Technical Reports Server (NTRS)

Andreev, V. M.; Larionov, V. R.; Rumyantsev, V. D.; Shvarts, M. Z.

1995-01-01

The conceptual design and experimental results for two types of space application concentrator photovoltaic modules, employing reflective optical elements, are presented. The first type is based on the use of compound parabolic concentrators, the second type is based on the use of line-focus parabolic troughs. Lightweight concentrators are formed with nickel foil coated silver with a diamond-like carbon layer protection. Secondary optical elements, including lenses and cones, are introduced for a better matching of concentrators and solar cells. Both types of modules are characterized by concentration ratios in the range 20x to 30x, depending on the chosen range of misorientation angles. The estimated specific parameters of these modules operating with single junction AlGaAs/GaAs solar cells are 240 W/sq m and 3 kg/sq m.

Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines

PubMed Central

Jacob, Francis; Guertler, Rea; Naim, Stephanie; Nixdorf, Sheri; Fedier, André; Hacker, Neville F.; Heinzelmann-Schwarz, Viola

2013-01-01

Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n = 25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ΔCt, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community. PMID:23554992

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

miR-370 regulates ISG15 expression and influences IFN-α sensitivity in hepatocellular carcinoma cells.

PubMed

Liu, Zhuo; Ma, Min; Yan, Lei; Chen, Shilin; Li, Sha; Yang, Darong; Wang, Xiaohong; Xiao, Hua; Deng, Hongyu; Zhu, Haizhen; Zuo, Chaohui; Xia, Man

2018-05-05

Interferon-α (IFN-α) is an adjuvant to chemotherapy and radiotherapy for hepatocellular carcinoma (HCC), but some HCC patients do not respond to treatment with IFN-α. We performed loss-of-function and gain-of-function experiments to examine the role of ISG15 in the IFN-α sensitivity of LH86, HLCZ01, SMMC7721, and Huh7 cell lines and tumor samples. The overexpression of ISG15 reduced apoptosis in Huh7 and LH86 cells in the presence of IFN-α, whereas the shRNA-mediated knock down of ISG15 expression increased apoptosis in both Huh7 and LH86 cells. We identified a putative miR-370 target site in the 3'-UTR in the ISG15 mRNA, and the level of miR-370 expression in HCC cell lines reflected the level of IFN-α-induced apoptosis exhibited by each. Both HCC cell lines and tumor samples had significantly lower levels of miR-370 than the control cells and tissues (P< 0.05). The overexpression of miR-370 in IFN-α-treated LH86 and Huh7 cells increased apoptosis and reduced the volume of LH86- and Huh7-derived xenograft tumors in mice treated with IFN-α compared with the control tumors. Our findings suggest that miR-370 functions as an HCC tumor suppressor and regulator of IFN-α sensitivity and that miR-370 might be a useful prognostic marker for HCC patients.

Differential Effector Engagement by Oncogenic KRAS.

PubMed

Yuan, Tina L; Amzallag, Arnaud; Bagni, Rachel; Yi, Ming; Afghani, Shervin; Burgan, William; Fer, Nicole; Strathern, Leslie A; Powell, Katie; Smith, Brian; Waters, Andrew M; Drubin, David; Thomson, Ty; Liao, Rosy; Greninger, Patricia; Stein, Giovanna T; Murchie, Ellen; Cortez, Eliane; Egan, Regina K; Procter, Lauren; Bess, Matthew; Cheng, Kwong Tai; Lee, Chih-Shia; Lee, Liam Changwoo; Fellmann, Christof; Stephens, Robert; Luo, Ji; Lowe, Scott W; Benes, Cyril H; McCormick, Frank

2018-02-13

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Nontransformed, GM-CSF-dependent macrophage lines are a unique model to study tissue macrophage functions.

PubMed

Fejer, György; Wegner, Mareike Dorothee; Györy, Ildiko; Cohen, Idan; Engelhard, Peggy; Voronov, Elena; Manke, Thomas; Ruzsics, Zsolt; Dölken, Lars; Prazeres da Costa, Olivia; Branzk, Nora; Huber, Michael; Prasse, Antje; Schneider, Robert; Apte, Ron N; Galanos, Chris; Freudenberg, Marina A

2013-06-11

Macrophages are diverse cell types in the first line of antimicrobial defense. Only a limited number of primary mouse models exist to study their function. Bone marrow-derived, macrophage-CSF-induced cells with a limited life span are the most common source. We report here a simple method yielding self-renewing, nontransformed, GM-CSF/signal transducer and activator of transcription 5-dependent macrophages (Max Planck Institute cells) from mouse fetal liver, which reflect the innate immune characteristics of alveolar macrophages. Max Planck Institute cells are exquisitely sensitive to selected microbial agents, including bacterial LPS, lipopeptide, Mycobacterium tuberculosis, cord factor, and adenovirus and mount highly proinflammatory but no anti-inflammatory IL-10 responses. They show a unique pattern of innate responses not yet observed in other mononuclear phagocytes. This includes differential LPS sensing and an unprecedented regulation of IL-1α production upon LPS exposure, which likely plays a key role in lung inflammation in vivo. In conclusion, Max Planck Institute cells offer an useful tool to study macrophage biology and for biomedical science.

Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

PubMed

Nagy, Zsolt; Acs, Bence; Butz, Henriett; Feldman, Karolina; Marta, Alexa; Szabo, Peter M; Baghy, Kornelia; Pazmany, Tamas; Racz, Karoly; Liko, Istvan; Patocs, Attila

2016-01-01

The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

PubMed Central

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

Left-handed compact MIMO antenna array based on wire spiral resonator for 5-GHz wireless applications

NASA Astrophysics Data System (ADS)

Alqadami, Abdulrahman Shueai Mohsen; Jamlos, Mohd Faizal; Soh, Ping Jack; Rahim, Sharul Kamal Abdul; Narbudowicz, Adam

2017-01-01

A compact coplanar waveguide-fed multiple-input multiple-output antenna array based on the left-handed wire loaded spiral resonators (SR) is presented. The proposed antenna consists of a 2 × 2 wire SR with two symmetrical microstrip feed lines, each line exciting a 1 × 2 wire SR. Left-handed metamaterial unit cells are placed on its reverse side and arranged in a 2 × 3 array. A reflection coefficient of less than -16 dB and mutual coupling of less than -28 dB are achieved at 5.15 GHz WLAN band.

Cabazitaxel overcomes cisplatin resistance in germ cell tumour cells.

PubMed

Gerwing, Mirjam; Jacobsen, Christine; Dyshlovoy, Sergey; Hauschild, Jessica; Rohlfing, Tina; Oing, Christoph; Venz, Simone; Oldenburg, Jan; Oechsle, Karin; Bokemeyer, Carsten; von Amsberg, Gunhild; Honecker, Friedemann

2016-09-01

Cisplatin-based chemotherapy is highly effective in metastasized germ cell tumours (GCT). However, 10-30 % of patients develop resistance to cisplatin, requiring salvage therapy. We investigated the in vitro activity of paclitaxel and the novel taxane cabazitaxel in cisplatin-sensitive and -resistant GCT cell lines. In vitro activity of paclitaxel and cabazitaxel was determined by proliferation assays, and mode of action of cabazitaxel was assessed by western blotting and two screening approaches, i.e. whole proteome analysis and a human apoptosis array. Activity of paclitaxel and cabazitaxel was not affected by cisplatin resistance, suggesting that there is no cross-resistance between these agents in vitro. Cabazitaxel treatment showed a strong inhibitory effect on colony formation capacity. Cabazitaxel induced classical apoptosis in all cell lines, reflected by cleavage of PARP and caspase 3, without inducing specific changes in the cell cycle distribution. Using the proteomic and human apoptosis array screening approaches, differential regulation of several proteins, including members of the bcl-2 family, was found, giving first insights into the mode of action of cabazitaxel in GCT. Cabazitaxel shows promising in vitro activity in GCT cells, independent of levels of cisplatin resistance.

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.

The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines

PubMed Central

2012-01-01

Background Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored. Material and methods In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53−/−) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (γH2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. Results Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 −/− cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ɣH2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 −/− cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 −/− tumors. PMID:22640402

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)

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.

Near Infrared Optical Visualization of Epidermal Growth Factor Receptors Levels in COLO205 Colorectal Cell Line, Orthotopic Tumor in Mice and Human Biopsies

PubMed Central

Cohen, Gadi; Lecht, Shimon; Oron-Herman, Mor; Momic, Tatjana; Nissan, Aviram; Lazarovici, Philip

2013-01-01

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner. PMID:23857061

Near infrared optical visualization of epidermal growth factor receptors levels in COLO205 colorectal cell line, orthotopic tumor in mice and human biopsies.

PubMed

Cohen, Gadi; Lecht, Shimon; Oron-Herman, Mor; Momic, Tatjana; Nissan, Aviram; Lazarovici, Philip

2013-07-12

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6-9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

Glioma Invasiveness Responds Variably to Irradiation in a Co-Culture Model

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

Nakamura, Jean L.; Haas-Kogan, Daphne A.; Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA

2007-11-01

Purpose: We developed a co-culture system to quantitate the growth and invasion of human malignant gliomas into a background of confluent normal human astrocytes, then used this assay to assess independently the effects of irradiating both cell types on glioma invasion. Methods and Materials: Enhanced green fluorescent protein (EGFP)-labeled immortalized human astrocytes, human malignant glioma cells, or transformed human astrocytes were focally plated onto a confluent layer of normal human astrocytes, and the invasiveness of EGFP-labeled cells was scored after 96 h. To address the consequences of irradiation on glioma invasion, the invasiveness of irradiated glioma cell lines and irradiatedmore » astrocytic backgrounds was assessed. Fluorescence-activated cell sorting was used to quantitate the total number of EGFP-labeled cells. Results: Growth in the co-culture assay consistently reflected transformation states of the plated cells. Immortalized, but untransformed human astrocytes failed even to establish growth on confluent normal human astrocytes. In contrast, all malignant human glioma cell lines and transformed human astrocytes demonstrated various degrees of infiltration into the astrocytic bed. Irradiation failed to alter the invasiveness of U87, A172, and U373. A 1-Gy dose slightly reduced the invasiveness of U251 MG by 75% (p < 0.05 by one-way analysis of variance and post hoc Neuman-Keuls), without reducing total cell numbers. Independently irradiating the human astrocytic bed did not alter the invasiveness of nonirradiated U251, whereas the matrix metalloproteinase (MMP) inhibitor GM6001 reduced U251 invasiveness in the co-culture assay. Conclusions: Growth in the co-culture assay reflects the transformation status and provides a useful in vitro model for assessing invasiveness. Human glioma invasiveness in the co-culture model responds variably to single low-dose fractions. MMP activity promotes invasiveness in the co-culture model. Reduced invasiveness in irradiated U251 appears to be mediated by MMP-independent mechanisms.« less

The anti-oncogenic influence of ellagic acid on colon cancer cells in leptin-enriched microenvironment.

PubMed

Yousef, Amany I; El-Masry, Omar S; Yassin, Eman H

2016-10-01

Ellagic acid (EA) has been proposed as a promising candidate for therapeutic use in colon cancer. Investigation of the effectiveness of EA in a leptin-enriched model might have been given a little interest. Here in, we investigated the anti-tumor effect of EA in the presence of leptin to reflect on therapeutic use of EA in obesity-linked colon cancer. Proven effective in leptin-enriched microenvironment, EA inhibited cell proliferation of HCT-116 and CaCo-2 cell lines, modulated cell cycle, translocated Bax to the mitochondrial fraction of cells, activated caspase-8, and reduced PCNA expression. The current study findings cast a beam of light on the potential therapeutic use of EA in obesity-related colon carcinogenesis.

Unique volatolomic signatures of TP53 and KRAS in lung cells

PubMed Central

Davies, M P A; Barash, O; Jeries, R; Peled, N; Ilouze, M; Hyde, R; Marcus, M W; Field, J K; Haick, H

2014-01-01

Background: Volatile organic compounds (VOCs) are potential biomarkers for cancer detection in breath, but it is unclear if they reflect specific mutations. To test this, we have compared human bronchial epithelial cell (HBEC) cell lines carrying the KRASV12 mutation, knockdown of TP53 or both with parental HBEC cells. Methods: VOC from headspace above cultured cells were collected by passive sampling and analysed by thermal desorption gas chromatography mass spectrometry (TD-GC–MS) or sensor array with discriminant factor analysis (DFA). Results: In TD-GC–MS analysis, individual compounds had limited ability to discriminate between cell lines, but by applying DFA analysis combinations of 20 VOCs successfully discriminated between all cell types (accuracies 80–100%, with leave-one-out cross validation). Sensor array detection DFA demonstrated the ability to discriminate samples based on their cell type for all comparisons with accuracies varying between 77% and 93%. Conclusions: Our results demonstrate that minimal genetic changes in bronchial airway cells lead to detectable differences in levels of specific VOCs identified by TD-GC–MS or of patterns of VOCs identified by sensor array output. From the clinical aspect, these results suggest the possibility of breath analysis for detection of minimal genetic changes for earlier diagnosis or for genetic typing of lung cancers. PMID:25051409

Chaetocin: a promising new antimyeloma agent with in vitro and in vivo activity mediated via imposition of oxidative stress

PubMed Central

Isham, Crescent R.; Tibodeau, Jennifer D.; Jin, Wendy; Xu, Ruifang; Timm, Michael M.

2007-01-01

Chaetocin, a thiodioxopiperazine natural product previously unreported to have anticancer effects, was found to have potent antimyeloma activity in IL-6–dependent and –independent myeloma cell lines in freshly collected sorted and unsorted patient CD138+ myeloma cells and in vivo. Chaetocin largely spares matched normal CD138− patient bone marrow leukocytes, normal B cells, and neoplastic B-CLL (chronic lymphocytic leukemia) cells, indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore, chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone, and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non–cross-resistant to chaetocin. Mechanistically, chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell, its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover, the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but, instead, heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively, chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. PMID:17090648

Resistance to etoposide-induced apoptosis in a Burkitt's lymphoma cell line.

PubMed

Zhao, E G; Song, Q; Cross, S; Misko, I; Lees-Miller, S P; Lavin, M F

1998-08-31

Burkitt's lymphoma cells that vary in their phenotypic characteristics show significantly different degrees of susceptibility to radiation-induced apoptosis. Propensity to undergo apoptosis is reflected in the degradation of substrates such as DNA-dependent protein kinase but the status of bcl-2, c-myc and p53 has been uninformative. In this study, we have focused on 2 Epstein-Barr virus (EBV)-associated Burkitt's cell lines, one (WW2) susceptible and the other (BL29) resistant to etoposide-induced apoptosis. Differences in expression of BHRF1, an EBV gene that is homologous to the Bcl-2 proto-oncogene and known to inhibit apoptosis, or changes in apoptosis inhibitory proteins (IAPs), did not appear to account for the difference in susceptibility in the 2 cell lines. Cytoplasmic extracts from etoposide-treated WW2 cells caused apoptotic changes in nuclei isolated from either BL29 or WW2 cells, whereas extracts from BL29 cells failed to do so. In addition, extracts from etoposide-treated WW2 cells degraded the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an important indicator of apoptosis, but this protein was resistant to degradation by BL29 extracts. It appears likely that caspase 3 (CPP32) is involved in this degradation since it was activated only in the apoptosis susceptible cells and the pattern of cleavage of DNA-PKcs was similar to that reported previously with recombinant caspase 3. As observed previously, addition of caspase 3 to nuclei failed to induce morphological changes indicative of apoptosis, but addition of caspase 3 to nuclei in the presence of extract from the resistant cells led to apoptotic changes. We conclude that resistance to apoptosis in BL29 cells is due to a failure of etoposide to activate upstream effectors of caspase activity.

Tumor cell differentiation by label-free microscopy

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Weber, Petra; Wagner, Michael

2013-05-01

Autofluorescence and Raman measurements of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from fluorescence spectra and nanosecond decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. Slight differences are also observed in the Raman spectra of these cell lines, mainly originating from small granules (lysosomes) surrounding the cell nucleus. While larger numbers of fluorescence and Raman spectra are evaluated by multivariate statistical methods, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme.

PubMed

Cowell, John K; Matsui, Sei-Ichi; Wang, Yong D; LaDuca, Jeffrey; Conroy, Jeffrey; McQuaid, Devin; Nowak, Norma J

2004-05-01

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately identify subtle, novel genetic abnormalities in tumors directly linked to the human genome sequence. CGHa far surpasses the resolution and information provided by conventional metaphase CGH, without relying on in vitro culture of tumors for metaphase spreads.

Chicken lines divergently selected for antibody responses to sheep red blood cells show line-specific differences in sensitivity to immunomodulation by diet. Part I: Humoral parameters.

PubMed

Adriaansen-Tennekes, R; de Vries Reilingh, G; Nieuwland, M G B; Parmentier, H K; Savelkoul, H F J

2009-09-01

Individual differences in nutrient sensitivity have been suggested to be related with differences in stress sensitivity. Here we used layer hens divergently selected for high and low specific antibody responses to SRBC (i.e., low line hens and high line hens), reflecting a genetically based differential immune competence. The parental line of these hens was randomly bred as the control line and was used as well. Recently, we showed that these selection lines differ in their stress reactivity; the low line birds show a higher hypothalamic-pituitary-adrenal (HPA) axis reactivity. To examine maternal effects and neonatal nutritional exposure on nutrient sensitivity, we studied 2 subsequent generations. This also created the opportunity to examine egg production in these birds. The 3 lines were fed 2 different nutritionally complete layer feeds for a period of 22 wk in the first generation. The second generation was fed from hatch with the experimental diets. At several time intervals, parameters reflecting humoral immunity were determined such as specific antibody to Newcastle disease and infectious bursal disease vaccines; levels of natural antibodies binding lipopolysaccharide, lipoteichoic acid, and keyhole limpet hemocyanin; and classical and alternative complement activity. The most pronounced dietary-induced effects were found in the low line birds of the first generation: specific antibody titers to Newcastle disease vaccine were significantly elevated by 1 of the 2 diets. In the second generation, significant differences were found in lipoteichoic acid natural antibodies of the control and low line hens. At the end of the observation period of egg parameters, a significant difference in egg weight was found in birds of the high line. Our results suggest that nutritional differences have immunomodulatory effects on innate and adaptive humoral immune parameters in birds with high HPA axis reactivity and affect egg production in birds with low HPA axis reactivity.

Comparison of sub-Doppler-width resonances in transmission and reflection spectra of micrometer-thick Cs vapor layers

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

Krasteva, A., E-mail: anna0kr0stz@gmail.com; Cartaleva, S., E-mail: stefka-c@ie.bas.bg; Vartanyan, T., E-mail: tigran@vartanyan.com

2016-03-25

We report experimental study of resonance sign reversal at the hyperfine F{sub g} = 4 → F{sub e} = 5 transition on D{sub 2} line of {sup 133}Cs. Alkali vapors are confined in an optical cell with micrometric thickness. For circular polarization of the irradiating light, transmission and reflection spectra, recorded at different laser intensities and different temperature of alkali vapor, are studied and compared. At low laser intensity, in the transmission spectra we observe a narrow resonance of increased absorption (bright resonance) at the F{sub g} = 4 → F{sub e} = 5 transition, however in reflection spectra, recordedmore » at the same experimental conditions, this bright resonance transforms into a dark resonance of reduced absorption. A simple explanation is proposed in order to explain the observed difference between the transmission and reflection spectra.« less

Synthesis of Bioactive Chlorogenic Acid-Silica Hybrid Materials via the Sol–Gel Route and Evaluation of Their Biocompatibility

PubMed Central

Pacifico, Severina

2017-01-01

Natural phenol compounds are gaining a great deal of attention because of their potential use as prophylactic and therapeutic agents in many diseases, as well as in applied science for their preventing role in oxidation deterioration. With the aim to synthetize new phenol-based materials, the sol–gel method was used to embed different content of the phenolic antioxidant chlorogenic acid (CGA) within silica matrices to obtain organic-inorganic hybrid materials. Fourier transform infrared (FTIR) measurements were used to characterize the prepared materials. The new materials were screened for their bioactivity and antioxidant potential. To this latter purpose, direct DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) methods were applied: radical scavenging capability appeared strongly dependent on the phenol amount in investigated hybrids, and became pronounced, mainly toward the ABTS radical cation, when materials with CGA content equal to 15 wt% and 20 wt% were analyzed. The in vitro biocompatibility of the synthetized materials was estimated by using the MTT assay towards fibroblast NIH 3T3 cells, human keratinocyte HaCaT cells, and the neuroblastoma SH-SY5Y cell line. As cell viability and morphology of tested cell lines seemed to be unaffected by new materials, the attenuated total reflectance (ATR)-FTIR method was applied to deeply measure the effects of the hybrids in the three different cell lines. PMID:28773198

Stochastic E2F activation and reconciliation of phenomenological cell-cycle models.

PubMed

Lee, Tae J; Yao, Guang; Bennett, Dorothy C; Nevins, Joseph R; You, Lingchong

2010-09-21

The transition of the mammalian cell from quiescence to proliferation is a highly variable process. Over the last four decades, two lines of apparently contradictory, phenomenological models have been proposed to account for such temporal variability. These include various forms of the transition probability (TP) model and the growth control (GC) model, which lack mechanistic details. The GC model was further proposed as an alternative explanation for the concept of the restriction point, which we recently demonstrated as being controlled by a bistable Rb-E2F switch. Here, through a combination of modeling and experiments, we show that these different lines of models in essence reflect different aspects of stochastic dynamics in cell cycle entry. In particular, we show that the variable activation of E2F can be described by stochastic activation of the bistable Rb-E2F switch, which in turn may account for the temporal variability in cell cycle entry. Moreover, we show that temporal dynamics of E2F activation can be recast into the frameworks of both the TP model and the GC model via parameter mapping. This mapping suggests that the two lines of phenomenological models can be reconciled through the stochastic dynamics of the Rb-E2F switch. It also suggests a potential utility of the TP or GC models in defining concise, quantitative phenotypes of cell physiology. This may have implications in classifying cell types or states.

Synthesis of Bioactive Chlorogenic Acid-Silica Hybrid Materials via the Sol-Gel Route and Evaluation of Their Biocompatibility.

PubMed

Catauro, Michelina; Pacifico, Severina

2017-07-21

Natural phenol compounds are gaining a great deal of attention because of their potential use as prophylactic and therapeutic agents in many diseases, as well as in applied science for their preventing role in oxidation deterioration. With the aim to synthetize new phenol-based materials, the sol-gel method was used to embed different content of the phenolic antioxidant chlorogenic acid (CGA) within silica matrices to obtain organic-inorganic hybrid materials. Fourier transform infrared (FTIR) measurements were used to characterize the prepared materials. The new materials were screened for their bioactivity and antioxidant potential. To this latter purpose, direct DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) methods were applied: radical scavenging capability appeared strongly dependent on the phenol amount in investigated hybrids, and became pronounced, mainly toward the ABTS radical cation, when materials with CGA content equal to 15 wt% and 20 wt% were analyzed. The in vitro biocompatibility of the synthetized materials was estimated by using the MTT assay towards fibroblast NIH 3T3 cells, human keratinocyte HaCaT cells, and the neuroblastoma SH-SY5Y cell line. As cell viability and morphology of tested cell lines seemed to be unaffected by new materials, the attenuated total reflectance (ATR)-FTIR method was applied to deeply measure the effects of the hybrids in the three different cell lines.

Transcriptional and functional defects of dendritic cells derived from the MUTZ-3 leukaemia line

PubMed Central

Rasaiyaah, Jane; Noursadeghi, Mahdad; Kellam, Paul; Chain, Benjamin

2009-01-01

Dendritic cells (DC) generated from MUTZ-3, an immortalized acute myeloid leukaemia-derived cell line, have potential application as a model for the study of human DC, and as a tool with which to stimulate immunotherapeutic responses to cancer. However, the relationship of MUTZ-3 DC to their non-transformed counterparts remains incompletely understood. Immunoselected CD14+ MUTZ-3 cells were used to generate a homogeneous population of DC (M3DC). These cells had a cell surface phentoype and morphology characteristic of conventional monocyte-derived DC (MDDC). Whole genome transcriptome comparison of M3DC and MDDC however, revealed extensive differences between these two cell types. Functional ontology-based data analysis revealed three enriched clusters of genes downregulated in M3DC, with functions in pathogen recognition, DC maturation and cytokine/chemokine signalling. Downregulation of protein expression was confirmed for several of these genes. The molecular differences were accompanied by a profoundly impaired phenotypic and functional response of M3DC to microbial stimulation. The immortalized phenotype of MUTZ-3 therefore reflects not only deregulated proliferative capacity, but substantial perturbation of normal antigen-presenting cell function. These results have important implications for studies using MUTZ-3 as a model of MDDC or for cancer immunotherapy. PMID:19538250

Radiation-hardened microwave communications system

NASA Astrophysics Data System (ADS)

Smith, S. F.; Bible, D. W.; Crutcher, R. I.; Hannah, J. H.; Moore, J. A.; Nowlin, C. H.; Vandermolen, R. I.; Chagnot, D.; Leroy, A.

1993-03-01

To develop a wireless communication system to meet the stringent requirements for a nuclear hot cell and similar environments, including control of advanced servomanipulators, a microwave signal transmission system development program was established to produce a demonstration prototype for the Consolidated Fuel Reprocessing Program at Oak Ridge National Laboratory (ORNL). Proof-of-principle tests in a partially metal lined enclosure at ORNL successfully demonstrated the feasibility of directed microwave signal transmission techniques for remote systems applications. The potential for much more severe radio-frequency (RF) multipath propagation conditions in fully metal lined cells led to a programmatic decision to conduct additional testing in more typical hot-cell environments at other sites. Again, the test results were excellent. Based on the designs of the earlier systems, an advanced microwave signal transmission system configuration was subsequently developed that, in highly reflective environments, will support both high-performance video channels and high baud-rate digital data links at total gamma dose tolerance levels exceeding 10(exp 7) rads and at elevated ambient temperatures.

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

PubMed

Strobel, Oliver; Dadabaeva, Nigora; Felix, Klaus; Hackert, Thilo; Giese, Nathalia A; Jesenofsky, Ralf; Werner, Jens

2016-02-01

Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

PubMed

Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T

2000-02-14

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

PubMed

Walz, Jenna A; Mace, Charles R

2018-06-05

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

PubMed Central

Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq; Povirk, Lawrence F.; Hendrickson, Eric A.; Gewirtz, David A.

2016-01-01

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. PMID:26934368

Expression of Ki-67 in odontogenic cysts: A comparative study between odontogenic keratocysts, radicular cysts and dentigerous cysts.

PubMed

Modi, Tapan G; Chalishazar, Monali; Kumar, Malay

2018-01-01

Odontogenic cysts are the most common cysts of the jaws and are formed from the remnants of the odontogenic apparatus. Among these odontogenic cysts, radicular cysts (RCs) (about 60% of all diagnosed jaw cysts), dentigerous cysts (DCs) (16.6% of all jaw cysts) and odontogenic keratocysts (OKCs) (11.2% of all developmental odontogenic cysts) are the most common. The behavior of any lesion is generally reflected by its growth potential. Growth potential is determined by measuring the cell proliferative activity. The cell proliferative activity is measured by various methods among which immunohistochemistry (IHC) is the commonly used technique. Most of the IHC studies on cell proliferation have been based on antibodies such as Ki-67 and proliferating cell nuclear antigen. In the present study, the total sample size comprised of 45 cases of odontogenic cysts, with 15 cases each of OKC, RC and DC. Here, an attempt is made to study immunohistochemical (streptavidin-biotin detection system HRP-DAB) method to assess the expression of Ki-67 in different layers of the epithelial lining of OKCs, RCs and DCs. Ki-67 positive cells were highest in epithelium of OKC as compared to DC and RC. The increased Ki-67 labeling index and its expression in suprabasal cell layers of epithelial lining in OKC and its correlation with suprabasal cell layers of epithelial lining in DC and RC could contribute toward its clinically aggressive behavior. OKC is of more significance to the oral pathologist and oral surgeon because of its specific histopathological features, high recurrence rate and aggressive behavior.

Epigenetic Changes and Suppression of the Nuclear Factor of Activated T Cell 1 (NFATC1) Promoter in Human Lymphomas with Defects in Immunoreceptor Signaling

PubMed Central

Akimzhanov, Askar; Krenacs, Laszlo; Schlegel, Timm; Klein-Hessling, Stefan; Bagdi, Enikö; Stelkovics, Eva; Kondo, Eisaku; Chuvpilo, Sergei; Wilke, Philipp; Avots, Andris; Gattenlöhner, Stefan; Müller-Hermelink, Hans-Konrad; Palmetshofer, Alois; Serfling, Edgar

2008-01-01

The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin’s lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a “window of hypomethylation,” which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing. PMID:18156209

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

PubMed Central

Krauss, R S; Guadagno, S N; Weinstein, I B

1992-01-01

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation. Images PMID:1535685

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Microwave Detection of Chemical Agents: A Review

DTIC Science & Technology

1986-06-01

Health (NIOSH).8’l1 This instrument was designed to detect acetonitrile, acetaldehyde , acetone, carbonyl sulfide, ethanol, ethylene oxide , isopropyl...absolute temperature mij - the dipole matrix element connecting the upper and lower energy states vo = the absorption line center v - transition...from multiple reflections through the cell. The Q of a cavity is defined as the electro - magnetic energy in the cavity divided by the energy lost per

mTORC2 activation is regulated by the urokinase receptor (uPAR) in bladder cancer.

PubMed

Hau, Andrew M; Leivo, Mariah Z; Gilder, Andrew S; Hu, Jing-Jing; Gonias, Steven L; Hansel, Donna E

2017-01-01

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer. Copyright © 2016. Published by Elsevier Inc.

One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells

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

Protic-Sabljic, M.; Kraemer, K.H.

1985-10-01

The authors have developed a host cell reactivation assay of DNA repair utilizing UV-treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. They studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfectionmore » resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines than in the other human cell lines tested. The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV-induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA.« less

Intracellular pH gradients in migrating cells.

PubMed

Martin, Christine; Pedersen, Stine F; Schwab, Albrecht; Stock, Christian

2011-03-01

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.

Examining FKBP5 mRNA expression in human iPSC-derived neural cells

PubMed Central

Lieberman, Richard; Kranzler, Henry R.; Levine, Eric S.; Covault, Jonathan

2016-01-01

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual. PMID:27915167

Relativistic Iron Emission and Disk Reflection in Galactic Microquasar XTE J1748-288

NASA Technical Reports Server (NTRS)

Miller, J. M.; Fox, D. W.; Matteo, T. DI; Wijnands, R.; Belloni, T.; Pooley, D.; Kouveliotou, C.; Lewin, W. H. G.

2001-01-01

We report evidence for an Fe K(alpha) fluorescence line feature and disk reflection in the very high, high-, and low-state X-ray spectra of the Galactic microquasar XTE J1748-288 during its 1998 June outburst. Spectral analyses are made on data gathered throughout the outburst by the Rossi X-Ray Timing Explorer Proportional Counter Array. Gaussian line, relativistic disk emission line, and ionized disk reflection models are fitted to the data. In the very high state the line profile appears strongly redshifted, consistent with disk emission from the innermost stable orbits around a maximally rotating Kerr black hole. In the high state the line profile is less redshifted and increasingly prominent. The low-state line profile is very strong (approx. 0.5 keV equivalent width) and centered at 6.7 +/- 0.10 keV; disk line emission model fits indicate that the inner edge of the disk fluctuates between approx. 20Rg and approx. 100Rg in this state. The disk reflection fraction is traced through the outburst; reflection from an ionized disk is preferred in the very high and high states, and reflection from a relatively neutral disk is preferred in the low state. We discuss the implications of our findings for the binary system dynamics and accretion flow geometry in XTE J1748-288.

Relativistic Iron Emission and Disk Reflection in Galactic Microquasar XTE J1748-288

NASA Technical Reports Server (NTRS)

Miller, J. M.; Fox, D. W.; DiMatteo, T.; Wijnands, R.; Belloni, T.; Pooley, D.; Kouveliotou, C.; Lewin, W. H. G.

2001-01-01

We report evidence for an Fe K-alpha fluorescence line feature and disk reflection in the very high, high-, and low-state X-ray spectra of the Galactic microquasar XTE J1748 - 288 during its 1998 June outburst. Spectral analyses are made on data gathered throughout the outburst by the Rossi X-Ray Timing Explorer Proportional Counter Array. Gaussian line, relativistic disk emission line, and ionized disk reflection models are fitted to the data. In the very high state the line profile appears strongly redshifted, consistent with disk emission from the innermost stable orbits around a maximally rotating Kerr black hole. In the high state the line profile is less redshifted and increasingly prominent. The low-state line profile is very strong (approx. 0.5 keV equivalent width) and centered at 6.7 +/- 0.10 keV; disk line emission model fits indicate that the inner edge of the disk fluctuates between approx. 20R(sub g) and - approx. 100R(sub g) in this state. The disk reflection fraction is traced through the outburst; reflection from an ionized disk is preferred in the very high and high states, and reflection from a relatively neutral disk is preferred in the low state. We discuss the implications of our findings for the binary system dynamics and accretion flow geometry in XTE J1748 - 288.

Direct evidence for the partial reflection of hyaluronan molecules by the lining of rabbit knee joints during trans-synovial flow

PubMed Central

Scott, D; Coleman, P J; Mason, R M; Levick, J R

1998-01-01

In synovial joints hydraulic and turnover studies indicate that the synovial lining may partially reflect large macromolecules like hyaluronan, despite discontinuities in the lining cell layer. The reflection hypothesis was tested directly in the present study. Solutions of high molecular weight hyaluronan were infused at controlled pressures into the cavity of rabbit knees under anaesthesia, at concentrations of 0.2 g l−1 (n = 5), 2 g l−1 (n = 5) and 4 g l−1 (n = 6). Time-averaged trans-synovial flows were 9.6, 4.8 and 2.9 μl min−1, respectively. After 5 h infusion the intra-articular fluid was mixed and sampled. Hyaluronan concentration was determined by size-exclusion chromatography. In all sixteen experiments the hyaluronan concentration in the aspirate was greater than that in the infusate (P = 0.0001, Student's paired t test). The increases averaged 2.28 ± 0.04 times at high filtration rates (0.2 g l−1 infusates; mean ± s.e.m.), 1.60 ± 0.09 times at intermediate filtration rates (2 g l−1 infusates) and 1.26 ± 0.08 times at low filtration rates (4 g l−1 infusates). Between 48 and 95% of the hyaluronan in the filtrand was retained in the joint cavity. The greater retention at 2 g l−1, viz.95%, than at 0.2 g l−1, viz.48%, was attributed to interactions between overlapping molecular domains in the more concentrated solution. It is concluded that synovial interstitial matrix can partially reflect hyaluronan molecules, and thus conserve intra-articular lubricant. PMID:9508822

PGE2 suppresses NK activity in vivo directly and through adrenal hormones: Effects that cannot be reflected by ex-vivo assessment of NK cytotoxicity

PubMed Central

Meron, G.; Tishler, Y.; Shaashua, L.; Rosenne, E.; Levi, B.; Melamed, R.; Gotlieb, N.; Matzner, P.; Sorski, L.; Ben-Eliyahu, S.

2013-01-01

Surgery can suppress in vivo levels of NK cell cytotoxicity (NKCC) through various mechanisms, including catecholamine-, glucocorticoid (CORT)-, and prostaglandin (PG)-mediated responses. However, PGs are synthesized locally following tissue damage, driving proinflammatory and CORT responses, while their systemic levels are often unaffected. Thus, we herein studied the role of adrenal factors in mediating in vivo effects of PGs on NKCC, using adrenalectomized and sham-operated F344 rats subjected to surgery or PGE2 administration. In vivo and ex-vivo approaches were employed, based on intravenous administration of the NK-sensitive MADB106 tumor line, and based on ex-vivo assessment of YAC-1 and MADB106 target-line lysis. Additionally, in vitro studies assessed the kinetics of the impact of epinephrine, CORT, and PGE2 on NKCC. The results indicated that suppression of NKCC by epinephrine and PGE2 are short lasting, and cannot be evident when these compounds are removed from the in vitro assay milieu, or in the context of ex-vivo assessment of NKCC. In contrast, the effects of CORT are long-lasting and are reflected in both conditions even after its removal. Marginating-pulmonary NKCC was less susceptible to suppression than circulating NKCC, when tested against the xenogeneic YAC-1 target line, but not against the syngeneic MADB106 line, which seems to involve different cytotoxicity mechanisms. Overall, these findings indicate that elevated systemic PG levels can directly suppress NKCC in vivo, but following laparotomy adrenal hormones mediate most of the effects of endogenously-released PGs. Additionally, the ex-vivo approach seems limited in reflecting the short-lasting NK-suppressive effects of catecholamines and PGs. PMID:23153554

PGE2 suppresses NK activity in vivo directly and through adrenal hormones: effects that cannot be reflected by ex vivo assessment of NK cytotoxicity.

PubMed

Meron, G; Tishler, Y; Shaashua, L; Rosenne, E; Levi, B; Melamed, R; Gotlieb, N; Matzner, P; Sorski, L; Ben-Eliyahu, S

2013-02-01

Surgery can suppress in vivo levels of NK cell cytotoxicity (NKCC) through various mechanisms, including catecholamine-, glucocorticoid (CORT)-, and prostaglandin (PG)-mediated responses. However, PGs are synthesized locally following tissue damage, driving proinflammatory and CORT responses, while their systemic levels are often unaffected. Thus, we herein studied the role of adrenal factors in mediating in vivo effects of PGs on NKCC, using adrenalectomized and sham-operated F344 rats subjected to surgery or PGE(2) administration. In vivo and ex vivo approaches were employed, based on intravenous administration of the NK-sensitive MADB106 tumor line, and based on ex vivo assessment of YAC-1 and MADB106 target-line lysis. Additionally, in vitro studies assessed the kinetics of the impact of epinephrine, CORT, and PGE(2) on NKCC. The results indicated that suppression of NKCC by epinephrine and PGE(2) are short lasting, and cannot be evident when these compounds are removed from the in vitro assay milieu, or in the context of ex vivo assessment of NKCC. In contrast, the effects of CORT are long-lasting and are reflected in both conditions even after its removal. Marginating-pulmonary NKCC was less susceptible to suppression than circulating NKCC, when tested against the xenogeneic YAC-1 target line, but not against the syngeneic MADB106 line, which seems to involve different cytotoxicity mechanisms. Overall, these findings indicate that elevated systemic PG levels can directly suppress NKCC in vivo, but following laparotomy adrenal hormones mediate most of the effects of endogenously-released PGs. Additionally, the ex vivo approach seems limited in reflecting the short-lasting NK-suppressive effects of catecholamines and PGs. Copyright © 2012 Elsevier Inc. All rights reserved.

Cytotoxic and genotoxic studies of essential oil from Rosa damascene Mill., Kashan, Iran.

PubMed

Shokrzadeh, Mohammad; Habibi, Emran; Modanloo, Mona

2017-08-01

Aim Rosa damascene Mill. belongs to the family of Roseaceae and its essential oil is produced in large amounts in Iran. The wide application of rose oil has raised questions about potential adverse health effects. We have investigated cytotoxic activity and genotoxic effects of Rosa oil from Kashan, Iran. Methods The cytotoxic effect and IC50 of the essential oil on the cell lines was studied followed by MTT assay. In this assay mitochondrial oxidoreductase enzymes with reducing the tetrazolium dye MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reflect the number of viable cells. Genotoxic effect of the oil was evaluated by micronucleus assay by evaluating produced micronuclei due to cytogenetic damage in binucleated lymphocytes. Results The results showed that essential oil significantly had cytotoxic and genotoxic effects at doses over 10µg/mL (p<0.05). Also, essential oil of Rose showed lower IC50 in cancer cell line (A549) in comparison with the normal cell line (NIH3T3). Conclusion Cytotoxic and genotoxic properties of essential oil of Rose in Kashan, Iran, are safe at a dose of 10µg/mL. Also, a good cytotoxic effect was shown and could be introduced as an anticancer compound. Further studies are needed with regard to anti-cancer effects of Rose essential oil. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

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

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Epigenetics-related genes in prostate cancer: expression profile in prostate cancer tissues, androgen-sensitive and -insensitive cell lines.

PubMed

Shaikhibrahim, Zaki; Lindstrot, Andreas; Ochsenfahrt, Jacqueline; Fuchs, Kerstin; Wernert, Nicolas

2013-01-01

Epigenetic changes have been suggested to drive prostate cancer (PCa) development and progression. Therefore, in this study, we aimed to identify novel epigenetics-related genes in PCa tissues, and to examine their expression in metastatic PCa cell lines. We analyzed the expression of epigenetics-related genes via a clustering analysis based on gene function in moderately and poorly differentiated PCa glands compared to normal glands of the peripheral zone (prostate proper) from PCa patients using Whole Human Genome Oligo Microarrays. Our analysis identified 12 epigenetics-related genes with a more than 2-fold increase or decrease in expression and a p-value <0.01. In modera-tely differentiated tumors compared to normal glands of the peripheral zone, we found the genes, TDRD1, IGF2, DICER1, ADARB1, HILS1, GLMN and TRIM27, to be upregulated, whereas TNRC6A and DGCR8 were found to be downregulated. In poorly differentiated tumors, we found TDRD1, ADARB and RBM3 to be upregulated, whereas DGCR8, PIWIL2 and BC069781 were downregulated. Our analysis of the expression level for each gene in the metastatic androgen-sensitive VCaP and LNCaP, and -insensitive PC3 and DU-145 PCa cell lines revealed differences in expression among the cell lines which may reflect the different biological properties of each cell line, and the potential role of each gene at different metastatic sites. The novel epigenetics-related genes that we identified in primary PCa tissues may provide further insight into the role that epigenetic changes play in PCa. Moreover, some of the genes that we identified may play important roles in primary PCa and metastasis, in primary PCa only, or in metastasis only. Follow-up studies are required to investigate the functional role and the role that the expression of these genes play in the outcome and progression of PCa using tissue microarrays.

Coherent optical transients observed in rubidium atomic line filtered Doppler velocimetry experiments

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

Fajardo, Mario E., E-mail: mario.fajardo@eglin.af.mil; Molek, Christopher D.; Vesely, Annamaria L.

2015-10-14

We report the first successful results from our novel Rubidium Atomic Line Filtered (RALF) Doppler velocimetry apparatus, along with unanticipated oscillatory signals due to coherent optical transients generated within pure Rb vapor cells. RALF is a high-velocity and high-acceleration extension of the well-known Doppler Global Velocimetry (DGV) technique for constructing multi-dimensional flow velocity vector maps in aerodynamics experiments [H. Komine, U.S. Patent No. 4,919,536 (24 April 1990)]. RALF exploits the frequency dependence of pressure-broadened Rb atom optical absorptions in a heated Rb/N{sub 2} gas cell to encode the Doppler shift of reflected near-resonant (λ{sub 0} ≈ 780.24 nm) laser light onto the intensitymore » transmitted by the cell. The present RALF apparatus combines fiber optic and free-space components and was built to determine suitable operating conditions and performance parameters for the Rb/N{sub 2} gas cells. It yields single-spot velocities of thin laser-driven-flyer test surfaces and incorporates a simultaneous Photonic Doppler Velocimetry (PDV) channel [Strand et al., Rev. Sci. Instrum. 77, 083108 (2006)] for validation of the RALF results, which we demonstrate here over the v = 0 to 1 km/s range. Both RALF and DGV presume the vapor cells to be simple Beer's Law optical absorbers, so we were quite surprised to observe oscillatory signals in experiments employing low pressure pure Rb vapor cells. We interpret these oscillations as interference between the Doppler shifted reflected light and the Free Induction Decay (FID) coherent optical transient produced within the pure Rb cells at the original laser frequency; this is confirmed by direct comparison of the PDV and FID signals. We attribute the different behaviors of the Rb/N{sub 2} vs. Rb gas cells to efficient dephasing of the atomic/optical coherences by Rb-N{sub 2} collisions. The minimum necessary N{sub 2} buffer gas density ≈0.3 amagat translates into a smallest useful velocity range of 0 to 2 km/s, which can readily be extended to cover the 0 to 10 km/s range, and beyond. The recognition that coherent optical transients can be produced within low pressure vapor cells during velocimetry experiments may offer new insights into some quantitative discrepancies reported in earlier DGV studies. Future plans include “line-RALF” experiments with streak camera detection, and two-dimensional surface velocity mapping using pulsed laser illumination and/or gated intensified CCD camera detection.« less

Thermally switchable photonic band-edge to random laser emission in dye-doped cholesteric liquid crystals

NASA Astrophysics Data System (ADS)

Ye, Lihua; Wang, Yan; Feng, Yangyang; Liu, Bo; Gu, Bing; Cui, Yiping; Lu, Yanqing

2018-03-01

By changing the doping concentration of the chiral agent to adjust the relative position of the reflection band of cholesteric liquid crystals and the fluorescence emission spectrum of the dye, photonic band-edge and random lasing were observed, respectively. The reflection band of the cholesteric phase liquid crystal can also be controlled by adjusting the temperature: the reflection band is blue-shifted with increasing temperature, and a reversible switch from photonic band-edge to random lasing is obtained. Furthermore, the laser line width can be thermally adjusted from 1.1 nm (at 27 °C) to 4.6 nm (at 32.1 °C). A thermally tunable polarization state of a random laser from dual cells was observed, broadening the field of application liquid crystal random lasers.

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

NASA Astrophysics Data System (ADS)

Chishti, Arif A.; Hellweg, Christine E.; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther

2015-01-01

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Clinical Benefit of Everolimus as Second-Line Therapy in Metastatic Renal Cell Carcinoma: The French Retrospective SECTOR Study.

PubMed

Oudard, Stéphane; Joly, Florence; Geoffrois, Lionnel; Laguerre, Brigitte; Houede, Nadine; Barthelemy, Philippe; Gross-Goupil, Marine; Vano, Yann; Lucidarme, Oliver; Bidault, Francois; Kelkouli, Nadia; Slimane, Khemaies; Escudier, Bernard

2016-12-01

Real-world data of everolimus after vascular endothelial growth factor receptor (VEGFR)-tyrosine kinase inhibitor (TKI) therapy in metastatic renal cell carcinoma (mRCC) are limited. The retrospective, noninterventional SECTOR (SECond line with afiniTOR) study (N = 165) assessed outcomes of second-line everolimus after initial VEGFR-TKI (TKI-everolimus, n = 144) and of third-line VEGFR-TKI after everolimus (TKI-everolimus-TKI, n = 59) in patients with mRCC. The primary end point was duration of everolimus therapy for both populations. Median duration was 4.0 months (range, 0.0-33.0 months) for second-line everolimus and 18.0 months (range, 2-78 months) for sequential VEGFR-TKI and everolimus. Median overall survival (OS) for this sequence was 36.0 months (95% confidence interval [CI], 27.0-56.0 months) and was longer for patients who received a first-line TKI for ≥ 9 months (not reached) than for < 9 months (28.0 months; P < .001). During second-line everolimus treatment, commonly reported adverse events (all grades) were fatigue (n = 66, 40.7%), anemia (n = 58, 35.8%), and stomatitis (n = 41, 25.3%). Median duration from initiation of first-line TKI to the end of the third-line TKI was 24.0 months (95% CI, 19.0-29.0 months). Median OS for this sequence was 41.0 months (95% CI, 25.0-57.0 months) and was significantly longer for patients who received the first-line TKI for ≥ 9 months (37.5 months) than for < 9 months (19.0 months; P < .0001). These results reflect clinical use of sequential TKI-everolimus and TKI-everolimus-TKI and provide additional evidence that everolimus could be an option in second-line therapy in mRCC. Results of the CheckMate-025 (Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma) and METEOR (Metastatic RCC Phase 3 Study Evaluating Cabozantinib versus Everolimus) studies might change the treatment landscape. Copyright © 2016 Elsevier Inc. All rights reserved.

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

NASA Astrophysics Data System (ADS)

Zhou, Ming-Da; Hao, Sijie; Williams, Anthony J.; Harouaka, Ramdane A.; Schrand, Brett; Rawal, Siddarth; Ao, Zheng; Brennaman, Randall; Gilboa, Eli; Lu, Bo; Wang, Shuwen; Zhu, Jiyue; Datar, Ram; Cote, Richard; Tai, Yu-Chong; Zheng, Si-Yang

2014-12-01

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 103), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Genotoxic activity and induction of biotransformation enzymes in two human cell lines after treatment by Erika fuel extract.

PubMed

Amat-Bronnert, Agnès; Castegnaro, Marcel; Pfohl-Leszkowicz, Annie

2007-01-01

On 12 December 1999, the tanker Erika broke in two parts at about 60km from the Brittany French coasts (Point of Penmarc'h, Sud Finistère, France). About 10,000tonnes of heavy oil fuel were released in the sea. DNA adduct have been detected in fish liver and mussels digestive gland exposed to the Erika oil spill. In order to investigate the mechanism by which Erika fuel extract exhibits genotoxic effects the induction of DNA adducts by an Erika fuel extract have been analysed on two cell lines, human epithelial bronchial cells (WI) and human hepatoma cells. DNA adducts, reflected by a diagonal radioactive zone and individual adducts are detected only in hepatoma cells indicating biotransformation via CYP 1A2 and CYP 1B1. In addition, Erika fuel extract induces some metabolizing enzymes such CYP 1A2, COX2 and 5-LOX, the two later are involved in cancer processes. Formation of leucotrienes B4 (LTB(4)), a mediator playing a role in inflammation, is induced in epithelial bronchial cells. Since inhalation is one of the ways of contamination for human, the above results are important for human health and prevention. Copyright © 2006 Elsevier B.V. All rights reserved.

FLIM data analysis of NADH and Tryptophan autofluorescence in prostate cancer cells

NASA Astrophysics Data System (ADS)

O'Melia, Meghan J.; Wallrabe, Horst; Svindrych, Zdenek; Rehman, Shagufta; Periasamy, Ammasi

2016-03-01

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes -- rather than whole cell averages - makes this assay particularly valuable.

A meta-analysis of HLA peptidome composition in different hematological entities: entity-specific dividing lines and “pan-leukemia” antigens

PubMed Central

Walz, Simon; Schuster, Heiko; Berlin, Claudia; Neidert, Marian Christoph; Schemionek, Mirle; Brümmendorf, Tim H.; Vucinic, Vladan; Niederwieser, Dietger; Kanz, Lothar; Salih, Helmut Rainer; Kohlbacher, Oliver; Weisel, Katja; Rammensee, Hans-Georg; Stevanović, Stefan; Walz, Juliane Sarah

2017-01-01

Hematological malignancies (HM) are highly amenable targets for immunotherapeutic intervention and may be effectively treated by antigen-specific T-cell based treatment. Recent studies demonstrate that physiologically occurring anti-cancer T-cell responses in certain HM entities target broadly presented non-mutated epitopes. HLA ligands are thus implied as prime targets for broadly applicable and antigen-specific off-the-shelf compounds. With the aim of assessing the presence of common targets shared among different HM which may enable addressing a larger patient collective we conducted a meta-analysis of 83 mass spectrometry-based HLA peptidome datasets (comprising 40,361 unique peptide identifications) across four major HM (19 AML, 16 CML, 35 CLL, and 13 MM/MCL samples) and investigated similarities and differences within the HLA presented antigenic landscape. We found the cancer HLA peptidome datasets to cluster specifically along entity and lineage lines, suggesting that the immunopeptidome directly reflects the differences in the underlying (tumor-)biology. In line with these findings, we only detected a small set of entity-spanning antigens, which were predominantly characterized by low presentation frequencies within the different patient cohorts. These findings suggest that design of T-cell immunotherapies for the treatment of HM should ideally be conducted in an entity-specific fashion. PMID:28159928

Abstract - Cooperative Research and Development Agreement between Oxergy, Inc. and National Energy Technology Laboratory

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

Chorpening, Benjamin T.; Kamler, Jonathan

The Raman Gas Analyzer (RGA) has been demonstrated to have an extremely fast response (<1 second), pressurized, multi-gas analysis capability. All but the noble gases are Raman active, although the Raman interaction is weak. The RGA uses a reflectively lined capillary as the optical cell, providing both a small sample volume for fast gas exchange, and a much greater Raman signal collection than traditional instrument configurations.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

PubMed

Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

2016-06-01

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Nonimaging optical illumination system

DOEpatents

Winston, R.; Ries, H.

1996-12-17

A nonimaging illumination optical device for producing a selected far field illuminance over an angular range. The optical device includes a light source, a light reflecting surface, and a family of light edge rays defined along a reference line with the reflecting surface defined in terms of the reference line as a parametric function R(t) where t is a scalar parameter position and R(t)=k(t)+Du(t) where k(t) is a parameterization of the reference line, and D is a distance from a point on the reference line to the reflection surface along the desired edge ray through the point. 35 figs.

Nonimaging optical illumination system

DOEpatents

Winston, R.; Ries, H.

1998-10-06

A nonimaging illumination optical device for producing a selected far field illuminance over an angular range. The optical device includes a light source a light reflecting surface, and a family of light edge rays defined along a reference line with the reflecting surface defined in terms of the reference lines a parametric function R(t) where t is a scalar parameter position and R(t)=k(t)+Du(t) where k(t) is a parameterization of the reference line, and D is a distance from a point on the reference line to the reflection surface along the desired edge ray through the point. 35 figs.

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

PubMed Central

Lee, John K.; Bangayan, Nathanael J.; Chai, Timothy; Smith, Bryan A.; Pariva, Tiffany E.; Yun, Sangwon; Vashisht, Ajay; Zhang, Qingfu; Park, Jung Wook; Corey, Eva; Huang, Jiaoti; Wohlschlegel, James; Witte, Owen N.

2018-01-01

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting. PMID:29686080

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line.

PubMed

Niu, Chao; Chauhan, Uday; Gargus, Matthew; Shaker, Anisa

2016-01-01

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.

Systemic Therapy for Stage IV Non–Small-Cell Lung Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update

PubMed Central

Masters, Gregory A.; Temin, Sarah; Azzoli, Christopher G.; Giaccone, Giuseppe; Baker, Sherman; Brahmer, Julie R.; Ellis, Peter M.; Gajra, Ajeet; Rackear, Nancy; Schiller, Joan H.; Smith, Thomas J.; Strawn, John R.; Trent, David; Johnson, David H.

2015-01-01

Purpose To provide evidence-based recommendations to update the American Society of Clinical Oncology guideline on systemic therapy for stage IV non–small-cell lung cancer (NSCLC). Methods An Update Committee of the American Society of Clinical Oncology NSCLC Expert Panel based recommendations on a systematic review of randomized controlled trials from January 2007 to February 2014. Results This guideline update reflects changes in evidence since the previous guideline. Recommendations There is no cure for patients with stage IV NSCLC. For patients with performance status (PS) 0 to 1 (and appropriate patient cases with PS 2) and without an EGFR-sensitizing mutation or ALK gene rearrangement, combination cytotoxic chemotherapy is recommended, guided by histology, with early concurrent palliative care. Recommendations for patients in the first-line setting include platinum-doublet therapy for those with PS 0 to 1 (bevacizumab may be added to carboplatin plus paclitaxel if no contraindications); combination or single-agent chemotherapy or palliative care alone for those with PS 2; afatinib, erlotinib, or gefitinib for those with sensitizing EGFR mutations; crizotinib for those with ALK or ROS1 gene rearrangement; and following first-line recommendations or using platinum plus etoposide for those with large-cell neuroendocrine carcinoma. Maintenance therapy includes pemetrexed continuation for patients with stable disease or response to first-line pemetrexed-containing regimens, alternative chemotherapy, or a chemotherapy break. In the second-line setting, recommendations include docetaxel, erlotinib, gefitinib, or pemetrexed for patients with nonsquamous cell carcinoma; docetaxel, erlotinib, or gefitinib for those with squamous cell carcinoma; and chemotherapy or ceritinib for those with ALK rearrangement who experience progression after crizotinib. In the third-line setting, for patients who have not received erlotinib or gefitinib, treatment with erlotinib is recommended. There are insufficient data to recommend routine third-line cytotoxic therapy. Decisions regarding systemic therapy should not be made based on age alone. Additional information can be found at http://www.asco.org/guidelines/nsclc and http://www.asco.org/guidelineswiki. PMID:26324367

Expression of Ki-67 in odontogenic cysts: A comparative study between odontogenic keratocysts, radicular cysts and dentigerous cysts

PubMed Central

Modi, Tapan G; Chalishazar, Monali; Kumar, Malay

2018-01-01

Introduction and Objectives: Odontogenic cysts are the most common cysts of the jaws and are formed from the remnants of the odontogenic apparatus. Among these odontogenic cysts, radicular cysts (RCs) (about 60% of all diagnosed jaw cysts), dentigerous cysts (DCs) (16.6% of all jaw cysts) and odontogenic keratocysts (OKCs) (11.2% of all developmental odontogenic cysts) are the most common. The behavior of any lesion is generally reflected by its growth potential. Growth potential is determined by measuring the cell proliferative activity. The cell proliferative activity is measured by various methods among which immunohistochemistry (IHC) is the commonly used technique. Most of the IHC studies on cell proliferation have been based on antibodies such as Ki-67 and proliferating cell nuclear antigen. Materials and Method: In the present study, the total sample size comprised of 45 cases of odontogenic cysts, with 15 cases each of OKC, RC and DC. Here, an attempt is made to study immunohistochemical (streptavidin-biotin detection system HRP-DAB) method to assess the expression of Ki-67 in different layers of the epithelial lining of OKCs, RCs and DCs. Observations and Results: Ki-67 positive cells were highest in epithelium of OKC as compared to DC and RC. Conclusion: The increased Ki-67 labeling index and its expression in suprabasal cell layers of epithelial lining in OKC and its correlation with suprabasal cell layers of epithelial lining in DC and RC could contribute toward its clinically aggressive behavior. OKC is of more significance to the oral pathologist and oral surgeon because of its specific histopathological features, high recurrence rate and aggressive behavior. PMID:29731577

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates.

PubMed

Ozgür, Burak; Saaby, Lasse; Langthaler, Kristine; Brodin, Birger

2018-01-15

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm 2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14 C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [ 3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. K m and V max of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm -2 ·min -1 (n=3, total N=6), respectively. V max and K m of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC 50 ) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC 50 values of inhibition, K i values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function. Copyright © 2017 Elsevier B.V. All rights reserved.

Ethnically diverse pluripotent stem cells for drug development.

PubMed

Fakunle, Eyitayo S; Loring, Jeanne F

2012-12-01

Genetic variation is an identified factor underlying drug efficacy and toxicity, and adverse drug reactions, such as liver toxicity, are the primary reasons for post-marketing drug failure. Genetic predisposition to toxicity might be detected early in the drug development pipeline by introducing cell-based assays that reflect the genetic and ethnic variation of the expected treatment population. One challenge for this approach is obtaining a collection of suitable cell lines derived from ethnically diverse populations. Induced pluripotent stem cells (iPSCs) seem ideal for this purpose. They can be obtained from any individual, can be differentiated into multiple relevant cell types, and their self-renewal capability makes it possible to generate large quantities of quality-controlled cell types. Here, we discuss the benefits and challenges of using iPSCs to introduce genetic diversity into the drug development process. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed

Doersen, C J; Stanbridge, E J

1981-04-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

In vivo and ex vivo cetuximab sensitivity assay using three-dimensional primary culture system to stratify KRAS mutant colorectal cancer

PubMed Central

Tashiro, Takahiro; Okuyama, Hiroaki; Endo, Hiroko; Kawada, Kenji; Ashida, Yasuko; Ohue, Masayuki; Sakai, Yoshiharu; Inoue, Masahiro

2017-01-01

In clinic, cetuximab, an anti-EGFR antibody, improves treatment outcomes in colorectal cancer (CRC). KRAS-mutant CRC is generally resistant to cetuximab, although difference of the sensitivity among KRAS-mutants has not been studied in detail. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method for cancer cells. We applied CTOS method to investigate whether ex vivo cetuximab sensitivity assays reflect the difference in sensitivity in the xenografts. Firstly, in vivo cetuximab treatment was performed with xenografts derived from 10 CTOS lines (3 KRAS-wildtype and 7 KRAS mutants). All two CTOS lines which exhibited tumor regression were KRAS-wildtype, meanwhile all KRAS-mutant CTOS lines grew more than the initial size: were resistant to cetuximab according to the clinical evaluation criteria, although the sensitivity was quite diverse. We divided KRAS-mutants into two groups; partially responsive group in which cetuximab had a substantial growth inhibitory effect, and resistant group which exhibited no effect. The ex vivo signaling assay with EGF stimulation revealed that the partially responsive group, but not the resistant group, exhibited suppressed ERK phosphorylation ex vivo. Furthermore, two lines from the partially responsive group, but none of the lines in the resistant group, exhibited a combinatory effect of cetuximab and trametinib, a MEK inhibitor, ex vivo and in vivo. Taken together, the results indicate that ex vivo signaling assay reflects the difference in sensitivity in vivo and stratifies KRAS mutant CTOS lines by sensitivity. Therefore, coupling the in vivo and ex vivo assays with CTOS can be a useful platform for understanding the mechanism of diversity in drug sensitivity. PMID:28301591

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy

PubMed Central

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

Background: The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Methods: Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Results: Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the “map” area, mainly in the basal epithelial cell layer. In “fingerprint” lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. Conclusion: HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities. PMID:22888214

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

PubMed

Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

2009-04-01

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

On-line observation of cell growth in a three-dimensional matrix on surface-modified microelectrode arrays.

PubMed

Lin, Shu-Ping; Kyriakides, Themis R; Chen, Jia-Jin J

2009-06-01

Despite many successful applications of microelectrode arrays (MEAs), typical two-dimensional in-vitro cultures do not project the full scale of the cell growth environment in the three-dimensional (3D) in-vivo setting. This study aims to on-line monitor in-vitro cell growth in a 3D matrix on the surface-modified MEAs with a dynamic perfusion culture system. A 3D matrix consisting of poly(ethylene glycol) hydrogel supplemented with poly-D-lysine was subsequently synthesized in situ on the self-assembled monolayer modified MEAs. FTIR spectrum analysis revealed a peak at 2100 cm(-1) due to the degradation of the structure of the 3D matrix. After 2 wks, microscopic examination revealed that the non-degraded area was around 1500 microm(2) and provided enough space for cell growth. Fluorescence microscopy revealed that the degraded 3D matrix was non-cytotoxic allowing the growth of NIH3T3 fibroblasts and cortical neurons in vitro. Time-course changes of total impedance including resistance and reactance were recorded for 8 days to evaluate the cell growth in the 3D matrix on the MEA. A consistent trend reflecting changes of reactance and total impedance was observed. These in-vitro assays demonstrate that our 3D matrix can construct a biomimetic system for cell growth and analysis of cell surface interactions.

Diffuse Reflectance Spectroscopy (DRS) of radiation-induced re-oxygenation in sensitive and resistant head and neck tumor xenografts

NASA Astrophysics Data System (ADS)

Dadgar, Sina; Rodríguez Troncoso, Joel; Rajaram, Narasimhan

2018-02-01

Currently, anatomical assessment of tumor volume performed several weeks after completion of treatment is the clinical standard to determine whether a cancer patient has responded to a treatment. However, functional changes within the tumor could potentially provide information regarding treatment resistance or response much earlier than anatomical changes. We have used diffuse reflectance spectroscopy to assess the short and long-term re-oxygenation kinetics of a human head and neck squamous cell carcinoma xenografts in response to radiation therapy. First, we injected UM-SCC-22B cell line into the flank of 50 mice to grow xenografts. Once the tumor volume reached 200 mm3 (designated as Day 1), the mice were distributed into radiation and control groups. Members of radiation group underwent a clinical dose of radiation of 2 Gy/day on Days 1, 4, 7, and 10 for a cumulative dose of 8 Gy. DRS spectra of these tumors were collected for 14 days during and after therapy, and the collected spectra of each tumor were converted to its optical properties using a lookup table-base inverse model. We found statistically significant differences in tumor growth rate between two groups which is in indication of the sensitivity of this cell line to radiation. We further acquired significantly different contents of hemoglobin and scattering magnitude and size in two groups. The scattering has previously been associated with necrosis. We furthermore found significantly different time-dependent changes in vascular oxygenation and tumor hemoglobin concentration in post-radiation days.

Therapeutic cloning in the mouse

PubMed Central

Mombaerts, Peter

2003-01-01

Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice. PMID:12949262

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo.

PubMed

Takamatsu, Yuki; Morita, Kouichi; Hayasaka, Daisuke

2015-06-01

We identified a unique amino acid of NS2A113, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nonimaging optical illumination system

DOEpatents

Winston, Roland; Ries, Harald

2000-01-01

A nonimaging illumination optical device for producing a selected far field illuminance over an angular range. The optical device includes a light source 102, a light reflecting surface 108, and a family of light edge rays defined along a reference line 104 with the reflecting surface 108 defined in terms of the reference line 104 as a parametric function R(t) where t is a scalar parameter position and R(t)=k(t)+Du(t) where k(t) is a parameterization of the reference line 104, and D is a distance from a point on the reference line 104 to the reflection surface 108 along the desired edge ray through the point.

Nonimaging optical illumination system

DOEpatents

Winston, Roland; Ries, Harald

1998-01-01

A nonimaging illumination optical device for producing a selected far field illuminance over an angular range. The optical device includes a light source 102, a light reflecting surface 108, and a family of light edge rays defined along a reference line 104 with the reflecting surface 108 defined in terms of the reference line 104 as a parametric function R(t) where t is a scalar parameter position and R(t)=k(t)+Du(t) where k(t) is a parameterization of the reference line 104, and D is a distance from a point on the reference line 104 to the reflection surface 108 along the desired edge ray through the point.

Nonimaging optical illumination system

DOEpatents

Winston, Roland; Ries, Harald

1996-01-01

A nonimaging illumination optical device for producing a selected far field illuminance over an angular range. The optical device includes a light source 102, a light reflecting surface 108, and a family of light edge rays defined along a reference line 104 with the reflecting surface 108 defined in terms of the reference line 104 as a parametric function R(t) where t is a scalar parameter position and R(t)=k(t)+Du(t) where k(t) is a parameterization of the reference line 104, and D is a distance from a point on the reference line 104 to the reflection surface 108 along the desired edge ray through the point.

Extended Abstracts. The Electrochemical Society Fall Meeting (174th) Held in Chicago, Illinois on 9-14 October 1988. Volume 88-2

DTIC Science & Technology

1988-10-14

Ion Ream Lithography Using Novolak Based Resist S. Matsui, Y. Kojima, and .7. Dchiai ......................................... 0, 448 FIB Direct Ion...to the line length, reflection effects occur. The impedance oscillates with small changes in frequency as constructive or destructive interference ...Materials and Chemical Sciences Division discharge rates by a factor of five. Na/ DMDS cell 1 Cyclotron Road, Berkeley, CA 94720 having I wt% CoPc were

Melatonin exerts anti-oral cancer effect via suppressing LSD1 in patient-derived tumor xenograft models

PubMed Central

Yang, Cheng-Yu; Lin, Chih-Kung; Tsao, Chang-Huei; Hsieh, Cheng-Chih; Lin, Gu-Jiun; Ma, Kuo-Hsing; Shieh, Yi-Shing; Sytwu, Huey-Kang; Chen, Yuan-Wu

2017-01-01

Aberrant activation of histone lysine-specific demethylase (LSD1) increases tumorigenicity; hence, LSD1 is considered a therapeutic target for various human cancers. Although melatonin, an endogenously produced molecule, may defend against various cancers, the precise mechanism involved in its anti-oral cancer effect remains unclear. Patient-derived tumor xenograft (PDTX) models are preclinical models that can more accurately reflect human tumor biology compared with cell line xenograft models. Here, we evaluated the anticancer activity of melatonin by using LSD1-overexpressing oral cancer PDTX models. By assessing oral squamous cell carcinoma (OSCC) tissue arrays through immunohistochemistry, we examined whether aberrant LSD1 overexpression in OSCC is associated with poor prognosis. We also evaluated the action mechanism of melatonin against OSCC with lymphatic metastases by using the PDTX models. Our results indicated that melatonin, at pharmacological concentrations, significantly suppresses cell proliferation in a dose- and time-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of LSD1 in oral cancer PDTXs and oral cancer cell lines. In conclusion, we determined that the beneficial effects of melatonin in reducing oral cancer cell proliferation are associated with reduced LSD1 expression in vivo and in vitro. PMID:28422711

Students Reflecting on Test Performance and Feedback: An On-Line Approach

ERIC Educational Resources Information Center

Fyfe, Georgina; Fyfe, Sue; Meyer, Jan; Ziman, Mel; Sanders, Kathy; Hill, Julie

2014-01-01

Undergraduate students accessing on-line tests in Human Biology in three Western Australian universities were asked to complete an on-line post-test reflective survey about their perceptions of their test performance in light of automated feedback. The survey allowed pre-determined choices and comment text boxes relating to students' perceptions…

Reflectance infrared spectroscopy for in-line monitoring of nicotine during a coating process for an oral thin film.

PubMed

Hammes, Florian; Hille, Thomas; Kissel, Thomas

2014-02-01

A process analytical method using reflectance infrared spectrometry was developed for the in-line monitoring of the amount of the active pharmaceutical ingredient (API) nicotine during a coating process for an oral thin film (OTF). In-line measurements were made using a reflectance infrared (RI) sensor positioned after the last drying zone of the coating line. Real-time spectra from the coating process were used for modelling the nicotine content. Partial least squares (PLS1) calibration models with different data pre-treatments were generated. The calibration model with the most comparable standard error of calibration (SEC) and the standard error of cross validation (SECV) was selected for an external validation run on the production coating line with an independent laminate. Good correlations could be obtained between values estimated from the reflectance infrared data and the reference HPLC test method, respectively. With in-line measurements it was possible to allow real-time adjustments during the production process to keep product specifications within predefined limits hence avoiding loss of material and batch. Copyright © 2013 Elsevier B.V. All rights reserved.

Age and genetic selection affect auto-immune profiles of chickens.

PubMed

Parmentier, Henk K; Harms, Elmer; Lammers, Aart; Nieuwland, Mike G B

2014-12-01

Specificity, antibody isotype distribution and levels, of natural autoantibodies (NAAb) may be potential informative parameters for immune mediated natural disease resistance, immune modulation, and maintenance of physiological homeostasis. In a previous study we detected IgM and IgG antibodies to liver antigens in plasma from 1 year old chickens. Auto-immune profiles directed towards liver antigens differed between chicken lines divergently selected for specific antibody responses to sheep red blood cells. In the present study we measured the presence and typed levels and antibody isotypes (IgG and IgM) of NAAb binding the 'auto-antigen' complex chicken liver cell lysate (CLL) in plasma samples obtained from chickens at 5 weeks and at 1-year of age, respectively, by quantitative western blotting. Extensive staining patterns of plasma antibodies binding CLL were found for both isotypes and at both ages in all birds. At both ages, IgM and IgG bound similar numbers of CLL antigens, which remained almost constant for IgM, whereas the number of IgG stained bands in time was enhanced. Significant differences of binding patterns of NAAb (stained antigen fragments of CLL and staining intensity) were detected between the three different chicken lines at both ages and between both ages, and lines could be clustered on the basis of their auto-antibody profile. The present results indicate that analysis of the plasma NAAb repertoire of poultry like in mammals could provide a way of distinguishing differences of immune competence (as reflected by the selection criterion of antibody responses) between individuals and lines, and could provide tools to select individual birds for health and other traits. The age-dependency of the auto-immune profile suggest that such profiles may also reflect immune maturation, which should be taken into account when relating an auto-immune profile with other traits. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interleukin-1 or tumor necrosis factor-alpha augmented the cytotoxic effect of mycobacteria on human fibroblasts: application to evaluation of pathogenesis of clinical isolates of Mycobacterium tuberculosis and M. avium complex.

PubMed

Takii, T; Abe, C; Tamura, A; Ramayah, S; Belisle, J T; Brennan, P J; Onozaki, K

2001-03-01

Mycobacteria-induced in vitro events reflecting human tuberculosis can contribute to the evaluation of the pathogenesis of Mycobacterium tuberculosis (MTB). In this study, we propose such an in vitro method based on live mycobacteria-induced cytotoxicity to human cell lines. When human lung-derived normal fibroblast cell line MRC-5 was infected with various strains of mycobacteria (M. tuberculosis H(37)Rv and H(37) Ra, Mycobacterium avium 427S and 2151SmO, and Mycobacterium bovis BCG Pasteur and Tokyo), the fibroblasts were killed by mycobacteria according to the degree of virulence. Other human originated macrophage (U-937, THP-1), myeloid (HL-60), and epithelial carcinoma (A549) cell lines exhibited a similar cytotoxic response to virulent mycobacteria. MRC-5 was most susceptible to virulent mycobacteria among various human cell lines examined. The cytotoxicity was enhanced by the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-alpha), which in the absence of mycobacteria stimulate the growth of normal human fibroblasts. This in vitro evaluation system was applied to clinical isolates of drug-sensitive MTB (DS-MTB), drug-resistant MTB (DR-MTB) including multidrug-resistant (MDR-MTB), and M. avium complex (MAC). MTB strains (n = 24) exhibited strong cytotoxic activity, but MAC strains (n = 5) had only weak activity. Furthermore, there was no significant difference in cytotoxicity between DS-MTB (n = 11) and DR-MTB (n = 13). Collectively, these results suggest that this new in vitro system is useful for evaluating the pathogenesis of mycobacteria and that there was no difference in the pathogenesis between drug-susceptible and drug-resistant clinical isolates.

A reliable Raman-spectroscopy-based approach for diagnosis, classification and follow-up of B-cell acute lymphoblastic leukemia

NASA Astrophysics Data System (ADS)

Managò, Stefano; Valente, Carmen; Mirabelli, Peppino; Circolo, Diego; Basile, Filomena; Corda, Daniela; de Luca, Anna Chiara

2016-04-01

Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.

PubMed

Lovelock, Paul K; Wong, Ee Ming; Sprung, Carl N; Marsh, Anna; Hobson, Karen; French, Juliet D; Southey, Melissa; Sculley, Tom; Pandeya, Nirmala; Brown, Melissa A; Chenevix-Trench, Georgia; Spurdle, Amanda B; McKay, Michael J

2007-09-01

Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting. Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls. Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status. Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

NASA Astrophysics Data System (ADS)

Paudel, Hari P.; Jung, Yookyung; Raphael, Anthony; Alt, Clemens; Wu, Juwell; Runnels, Judith; Lin, Charles P.

2018-02-01

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient's blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

Silica-gold nanoshells as potential intraoperative molecular probes for HER2-overexpression in ex vivo breast tissue using near-infrared reflectance confocal microscopy.

PubMed

Bickford, Lissett R; Agollah, Germaine; Drezek, Rebekah; Yu, Tse-Kuan

2010-04-01

Obtaining negative margins is critical for breast cancer patients undergoing conservation therapy in order to reduce the reemergence of the original cancer. Currently, breast cancer tumor margins are examined in a pathology lab either while the patient is anesthetized or after the surgical procedure has been terminated. These current methods often result in cancer cells present at the surgical resection margin due to inadequate margin assessment at the point of care. Due to such limitations evident in current diagnoses, tools for increasing the accuracy and speed of tumor margin detection directly in the operating room are still needed. We are exploring the potential of using a nano-biophotonics system to facilitate intraoperative tumor margin assessment ex vivo at the cellular level. By combining bioconjugated silica-based gold nanoshells, which scatter light in the near-infrared, with a portable FDA-approved reflectance confocal microscope, we first validate the use of gold nanoshells as effective reflectance-based imaging probes by evaluating the contrast enhancement of three different HER2-overexpressing cell lines. Additionally, we demonstrate the ability to detect HER2-overexpressing cells in human tissue sections within 5 min of incubation time. This work supports the use of targeted silica-based gold nanoshells as potential real-time molecular probes for HER2-overexpression in human tissue.

New insights on shallow and deep crustal geological structures of BABEL line 7 marine reflection seismic data revealed from reprocessing

NASA Astrophysics Data System (ADS)

Shahrokhi, H.; Malehmir, A.; Sopher, D.

2012-04-01

The BABEL project (Baltic And Bothnian Echoes from the Lithosphere) was a collaboration among British, Danish, Finnish, German and Swedish geoscientists to collect deep-crustal reflection and wide-angle refraction profiles in Baltic Shield and Gulf of Bothnia. The acquisition of 2,268km of deep marine reflection seismic data was carried out in 1989. The BABEL line 7 runs in E-W direction in the Bothnian Sea, north of the Åland islands and east of the city of Gävle. Several authors presented the seismic results but with a main focus of imaging and interpreting deep crustal geological structures and the nature and the depth of Moho discontinuity along line 7. Based on this seismic data, several publications about velocity distributions within the crust, the depth and texture of Moho discontinuity and seismic reflectivity patterns in the crust were presented. Some evidence from the reflection seismic data was also presented to suggest Early Proterozoic plate tectonics in the Baltic Shield. Previous seismic images of the BABEL line 7 reflection data show a dramatic change in the reflectivity pattern from weakly reflective lower crust in the west to a more reflective lower crust in the east, which was attributed to a change from a rigid crust to a plastic crust from the west to the east. The BABEL line 7 reflection data were acquired with a total profile length of 174km, a set of 48 airguns towed at 7.5m depth, and 3000m long streamer with 60 channels spaced with 50m intervals and towed at 15m depth. Seismic data were recorded for 25s using 4ms sampling interval and 75m shot interval. Seismic data is characterized by strong source-generated noise at shallow travel times and strong but randomly distributed spurious spikes at later arrival times. In this study, we have recovered and reprocessed the seismic data along BABEL line 7. Using modern processing and imaging techniques, which were not available at the time, and with a focus on the shallow parts of the seismic data, we have managed to reveal reflections as shallow as 1s in the data. Some of these reflections appear to be a continuation of deeper reflections but now they appear to reach to the surface, allowing correlation with the near-surface geology. At least two major moderately dipping shear zones are visible in the reprocessed data in comparison with the previous results. Deeper reflections are also improved which together with the improvements in the shallow parts of the data should allow small-scale geological structures encounter along the BABEL line 7 to be refined.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

PubMed

Irshad, Shazia; Bansal, Mukesh; Guarnieri, Paolo; Davis, Hayley; Al Haj Zen, Ayman; Baran, Brygida; Pinna, Claudia Maria Assunta; Rahman, Haseeb; Biswas, Sujata; Bardella, Chiara; Jeffery, Rosemary; Wang, Lai Mun; East, James Edward; Tomlinson, Ian; Lewis, Annabelle; Leedham, Simon John

2017-06-01

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

PubMed

Kudinov, Alexander E; Deneka, Alexander; Nikonova, Anna S; Beck, Tim N; Ahn, Young-Ho; Liu, Xin; Martinez, Cathleen F; Schultz, Fred A; Reynolds, Samuel; Yang, Dong-Hua; Cai, Kathy Q; Yaghmour, Khaled M; Baker, Karmel A; Egleston, Brian L; Nicolas, Emmanuelle; Chikwem, Adaeze; Andrianov, Gregory; Singh, Shelly; Borghaei, Hossein; Serebriiskii, Ilya G; Gibbons, Don L; Kurie, Jonathan M; Golemis, Erica A; Boumber, Yanis

2016-06-21

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

A novel shock and heat tolerant gyrosensor utilizing a one-port surface acoustic wave reflective delay line

NASA Astrophysics Data System (ADS)

Oh, Haekwan; Fu, Chen; Yang, Sang Sik; Wang, Wen; Lee, Keekeun

2012-04-01

A surface acoustic wave (SAW)-based gyroscope with an 80 MHz central frequency was fabricated on a 128° YX LiNbO3 piezoelectric substrate. The fabricated gyroscope is composed of a SAW resonator, metallic dots and a SAW reflective delay line. The SAW resonator, which is activated by a voltage-controlled oscillator, generates a stable standing wave with a large amplitude at an 80 MHz resonant frequency, and the metallic dots induce a Coriolis force and generate a secondary SAW in the direction orthogonal to the propagating standing wave. The SAW reflective delay line is employed to measure the Coriolis effect by analyzing the deviations in the resonant frequency of the SAW reflective delay line. A combined finite element method/boundary element method was utilized to extract the optimal device parameters prior to fabrication. The device was fabricated according to the modeling results and then measured on a rate table. When the device was subjected to an angular rotation, a secondary SAW from the vibrating metallic dots was generated owing to the Coriolis force, resulting in a perturbation of the propagating SAW in the SAW reflective delay line. Depending on the angular velocity, the reflection peak of SAW reflective delay line was changed linearly, and this change was measured by the network analyzer. The measured results matched the modeling results well. The obtained sensitivity was approximately 1.23 deg/(deg/s) in an angular rate range of 0-2000 deg s-1. Good thermal and shock stabilities were observed during the evaluation process proving the shock and heat robustness of the fabricated SAW gyroscope.

BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathways.

PubMed

Marsch-Martinez, Nayelli; Greco, Raffaella; Becker, Jörg D; Dixit, Shital; Bergervoet, Jan H W; Karaba, Aarati; de Folter, Stefan; Pereira, Andy

2006-12-01

The BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves.

Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders.

PubMed

Gurwitz, David

2016-09-01

The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

The Reference Forward Model (RFM)

NASA Astrophysics Data System (ADS)

Dudhia, Anu

2017-01-01

The Reference Forward Model (RFM) is a general purpose line-by-line radiative transfer model, currently supported by the UK National Centre for Earth Observation. This paper outlines the algorithms used by the RFM, focusing on standard calculations of terrestrial atmospheric infrared spectra followed by a brief summary of some additional capabilities and extensions to microwave wavelengths and extraterrestrial atmospheres. At its most basic level - the 'line-by-line' component - it calculates molecular absorption cross-sections by applying the Voigt lineshape to all transitions up to ±25 cm-1 from line-centre. Alternatively, absorptions can be directly interpolated from various forms of tabulated data. These cross-sections are then used to construct infrared radiance or transmittance spectra for ray paths through homogeneous cells, plane-parallel or circular atmospheres. At a higher level, the RFM can apply instrumental convolutions to simulate measurements from Fourier transform spectrometers. It can also calculate Jacobian spectra and so act as a stand-alone forward model within a retrieval scheme. The RFM is designed for robustness, flexibility and ease-of-use (particularly by the non-expert), and no claims are made for superior accuracy, or indeed novelty, compared to other line-by-line codes. Its main limitations at present are a lack of scattering and simplified modelling of surface reflectance and line-mixing.

Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

PubMed Central

Keibler, Mark A.; Park, Donglim Esther; Molla, Vadim; Cheng, Jingwei; Stephanopoulos, Gregory

2016-01-01

Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis. PMID:27880818

Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

PubMed

Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

2016-08-01

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Upregulation of IRS1 Enhances IGF1 Response in Y537S and D538G ESR1 Mutant Breast Cancer Cells.

PubMed

Li, Zheqi; Levine, Kevin M; Bahreini, Amir; Wang, Peilu; Chu, David; Park, Ben Ho; Oesterreich, Steffi; Lee, Adrian V

2018-01-01

Increased evidence suggests that somatic mutations in the ligand-binding domain of estrogen receptor [ER (ERα/ESR1)] are critical mediators of endocrine-resistant breast cancer progression. Insulinlike growth factor-1 (IGF1) is an essential regulator of breast development and tumorigenesis and also has a role in endocrine resistance. A recent study showed enhanced crosstalk between IGF1 and ERα in ESR1 mutant cells, but detailed mechanisms are incompletely understood. Using genome-edited MCF-7 and T47D cell lines harboring Y537S and D538G ESR1 mutations, we characterized altered IGF1 signaling. RNA sequencing revealed upregulation of multiple genes in the IGF1 pathway, including insulin receptor substrate-1 (IRS1), consistent in both Y537S and D538G ESR1 mutant cell line models. Higher IRS1 expression was confirmed by quantitative reverse transcription polymerase chain reaction and immunoblotting. ESR1 mutant cells also showed increased levels of IGF-regulated genes, reflected by activation of an IGF signature. IGF1 showed increased sensitivity and potency in growth stimulation of ESR1 mutant cells. Analysis of downstream signaling revealed the phosphoinositide 3-kinase (PI3K)-Akt axis as a major pathway mediating the enhanced IGF1 response in ESR1 mutant cells. Decreasing IRS1 expression by small interfering RNA diminished the increased sensitivity to IGF1. Combination treatment with inhibitors against IGF1 receptor (IGF1R; OSI-906) and ER (fulvestrant) showed synergistic growth inhibition in ESR1 mutant cells, particularly at lower effective concentrations. Our study supports a critical role of enhanced IGF1 signaling in ESR1 mutant cell lines, pointing toward a potential for cotargeting IGF1R and ERα in endocrine-resistant breast tumors with mutant ESR1. Copyright © 2018 Endocrine Society.

Problems for the standard black hole/accretion disk models in Cygnus X-1?

NASA Technical Reports Server (NTRS)

Done, C.; Mulchaey, J. S.; Mushotzky, R. F.; Arnaud, K. A.

1992-01-01

Archival EXOSAT and HEAO1-A2 data from Cyg X-1 show the 'high energy excess' above 10 keV seen in X-ray observations of AGN. Using a likelihood ratio test, we are for the first time able to distinguish conclusively in favor of Compton reflection rather than partial covering as the origin of the high energy excess. This supports the idea of an X-ray illuminated accretion disk in Cyg X-1, but the line equivalent width is smaller by a factor of 2-3 than that expected from such a disk. While the larger optical depth required for reflection as opposed to line emission admit the possibility of seeing line without reflection, the converse is not possible. To see a reflection spectrum, including the strong iron absorption edge, implies that strong iron emission must be observed as the line and edge are causally linked.

Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

PubMed

Soh, JunYi; Chueng, Adeline; Adio, Aminat; Cooper, Alan J; Birch, Brian R; Lwaleed, Bashir A

2013-04-01

Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

Quantitative Characterization of Cell Behaviors through Cell Cycle Progression via Automated Cell Tracking

PubMed Central

Wang, Yuliang; Jeong, Younkoo; Jhiang, Sissy M.; Yu, Lianbo; Menq, Chia-Hsiang

2014-01-01

Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells. PMID:24911281

Tumours can act as adjuvants for humoral immunity

PubMed Central

Brown, D M; Fisher, T L; Wei, C; Frelinger, J G; Lord, E M

2001-01-01

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen – green fluorescence protein (GFP) – also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c × C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect. PMID:11328383

Extracellular calcium controls the expression of two different forms of ripple-like hippocampal oscillations.

PubMed

Aivar, Paloma; Valero, Manuel; Bellistri, Elisa; Menendez de la Prida, Liset

2014-02-19

Hippocampal high-frequency oscillations (HFOs) are prominent in physiological and pathological conditions. During physiological ripples (100-200 Hz), few pyramidal cells fire together coordinated by rhythmic inhibitory potentials. In the epileptic hippocampus, fast ripples (>200 Hz) reflect population spikes (PSs) from clusters of bursting cells, but HFOs in the ripple and the fast ripple range are vastly intermixed. What is the meaning of this frequency range? What determines the expression of different HFOs? Here, we used different concentrations of Ca(2+) in a physiological range (1-3 mM) to record local field potentials and single cells in hippocampal slices from normal rats. Surprisingly, we found that this sole manipulation results in the emergence of two forms of HFOs reminiscent of ripples and fast ripples recorded in vivo from normal and epileptic rats, respectively. We scrutinized the cellular correlates and mechanisms underlying the emergence of these two forms of HFOs by combining multisite, single-cell and paired-cell recordings in slices prepared from a rat reporter line that facilitates identification of GABAergic cells. We found a major effect of extracellular Ca(2+) in modulating intrinsic excitability and disynaptic inhibition, two critical factors shaping network dynamics. Moreover, locally modulating the extracellular Ca(2+) concentration in an in vivo environment had a similar effect on disynaptic inhibition, pyramidal cell excitability, and ripple dynamics. Therefore, the HFO frequency band reflects a range of firing dynamics of hippocampal networks.

Femtoelectron-Based Terahertz Imaging of Hydration State in a Proton Exchange Membrane Fuel Cell

NASA Astrophysics Data System (ADS)

Buaphad, P.; Thamboon, P.; Kangrang, N.; Rhodes, M. W.; Thongbai, C.

2015-08-01

Imbalanced water management in a proton exchange membrane (PEM) fuel cell significantly reduces the cell performance and durability. Visualization of water distribution and transport can provide greater comprehension toward optimization of the PEM fuel cell. In this work, we are interested in water flooding issues that occurred in flow channels on cathode side of the PEM fuel cell. The sample cell was fabricated with addition of a transparent acrylic window allowing light access and observed the process of flooding formation (in situ) via a CCD camera. We then explore potential use of terahertz (THz) imaging, consisting of femtoelectron-based THz source and off-angle reflective-mode imaging, to identify water presence in the sample cell. We present simulations of two hydration states (water and nonwater area), which are in agreement with the THz image results. A line-scan plot is utilized for quantitative analysis and for defining spatial resolution of the image. Implementing metal mesh filtering can improve spatial resolution of our THz imaging system.

Oxidative stress modulates the expression of genes involved in cell survival in ΔF508 cystic fibrosis airway epithelial cells.

PubMed

Voisin, Grégory; Bouvet, Guillaume F; Legendre, Pierre; Dagenais, André; Massé, Chantal; Berthiaume, Yves

2014-09-01

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients. Copyright © 2014 the American Physiological Society.

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed Central

Doersen, C J; Stanbridge, E J

1981-01-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis. PMID:6965101

The Relative Biological Effectiveness for Carbon and Oxygen Ion Beams Using the Raster-Scanning Technique in Hepatocellular Carcinoma Cell Lines

PubMed Central

Habermehl, Daniel; Ilicic, Katarina; Dehne, Sarah; Rieken, Stefan; Orschiedt, Lena; Brons, Stephan; Haberer, Thomas; Weber, Klaus-Josef; Debus, Jürgen; Combs, Stephanie E.

2014-01-01

Background Aim of this study was to evaluate the relative biological effectiveness (RBE) of carbon (12C) and oxygen ion (16O)-irradiation applied in the raster-scanning technique at the Heidelberg Ion beam Therapy center (HIT) based on clonogenic survival in hepatocellular carcinoma cell lines compared to photon irradiation. Methods Four human HCC lines Hep3B, PLC, HepG2 and HUH7 were irradiated with photons, 12C and 16O using a customized experimental setting at HIT for in-vitro trials. Cells were irradiated with increasing physical photon single doses of 0, 2, 4 and 6 Gy and heavy ionsingle doses of 0, 0.125, 0.5, 1, 2, 3 Gy (12C and 16O). SOBP-penetration depth and extension was 35 mm +/−4 mm and 36 mm +/−5 mm for carbon ions and oxygen ions respectively. Mean energy level and mean linear energy transfer (LET) were 130 MeV/u and 112 keV/um for 12C, and 154 MeV/u and 146 keV/um for 16O. Clonogenic survival was computated and realtive biological effectiveness (RBE) values were defined. Results For all cell lines and both particle modalities α- and β-values were determined. As expected, α-values were significantly higher for 12C and 16O than for photons, reflecting a steeper decline of the initial slope of the survival curves for high-LET beams. RBE-values were in the range of 2.1–3.3 and 1.9–3.1 for 12C and 16O, respectively. Conclusion Both irradiation with 12C and 16O using the rasterscanning technique leads to an enhanced RBE in HCC cell lines. No relevant differences between achieved RBE-values for 12C and 16O were found. Results of this work will further influence biological-adapted treatment planning for HCC patients that will undergo particle therapy with 12C or 16O. PMID:25460352

Electromagnetic Spectrum Analysis and Its Influence on the Photoelectric Conversion Efficiency of Solar Cells.

PubMed

Hu, Kexiang; Ding, Enjie; Wangyang, Peihua; Wang, Qingkang

2016-06-01

The electromagnetic spectrum and the photoelectric conversion efficiency of the silicon hexagonal nanoconical hole (SiHNH) arrays based solar cells is systematically analyzed according to Rigorous Coupled Wave Analysis (RCWA) and Modal Transmission Line (MTL) theory. An ultimate efficiency of the optimized SiHNH arrays based solar cell is up to 31.92% in consideration of the absorption spectrum, 4.52% higher than that of silicon hexagonal nanoconical frustum (SiHNF) arrays. The absorption enhancement of the SiHNH arrays is due to its lower reflectance and more supported guided-mode resonances, and the enhanced ultimate efficiency is insensitive to bottom diameter (D(bot)) of nanoconical hole and the incident angle. The result provides an additional guideline for the nanostructure surface texturing fabrication design for photovoltaic applications.

Molecular Characterization of Acquired Tolerance of Tumor Cells to Picropodophyllin (PPP)

PubMed Central

Hashemi, Jamileh; Worrall, Claire; Vasilcanu, Daiana; Fryknäs, Mårten; Sulaiman, Luqman; Karimi, Mohsen; Weng, Wen-Hui; Lui, Weng-Onn; Rudduck, Christina; Axelson, Magnus; Jernberg-Wiklund, Helena; Girnita, Leonard; Larsson, Olle; Larsson, Catharina

2011-01-01

Background Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Methodology/Principal Findings Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/ amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Conclusions Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions. PMID:21423728

Molecular characterization of acquired tolerance of tumor cells to picropodophyllin (PPP).

PubMed

Hashemi, Jamileh; Worrall, Claire; Vasilcanu, Daiana; Fryknäs, Mårten; Sulaiman, Luqman; Karimi, Mohsen; Weng, Wen-Hui; Lui, Weng-Onn; Rudduck, Christina; Axelson, Magnus; Jernberg-Wiklund, Helena; Girnita, Leonard; Larsson, Olle; Larsson, Catharina

2011-03-14

Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

PubMed

Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

2014-11-01

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. © 2014 FEBS.

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

Hirvonen, Liisa M.; Le Marois, Alix; Suhling, Klaus, E-mail: klaus.suhling@kcl.ac.uk

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reducedmore » lifetime near the coverslip in TIR compared to epifluorescence FLIM.« less

Non-hazardous anticancerous and antibacterial colloidal 'green' silver nanoparticles.

PubMed

Barua, Shaswat; Konwarh, Rocktotpal; Bhattacharya, Satya Sundar; Das, Pallabi; Devi, K Sanjana P; Maiti, Tapas K; Mandal, Manabendra; Karak, Niranjan

2013-05-01

Poly(ethylene glycol) stabilized colloidal silver nanoparticles were prepared using the reductive potency of the aqueous extract of Thuja occidentalis leaves under ambient conditions. The nanoparticles were well dispersed within a narrow size spectrum (7-14 nm) and displayed characteristic surface plasmon resonance peak at around 420 nm and Bragg's reflection planes of fcc structure. MTT assay revealed the dose-dependent cytocompatibility and toxicity of the nanoparticles with the L929 normal cell line. On the other hand, the antiproliferative action of the nanoparticles was evaluated on HeLa cell (cancerous cells) line. Fluorescence and phase contrast microscopic imaging indicated the appearance of multinucleate stages with aggregation and nuclear membrane disruption of the HeLa cells post treatment with the nanoparticles. The interaction at the prokaryotic level was also assessed via differential antibacterial efficacy against Staphylococcus aureus (MTCC 3160) and Escherichia coli (MTCC 40). Under these perspectives, it is also necessary to observe the environmental impact of the prepared silver nanoparticles. Hence, the dose dependent toxicity of silver nanoparticles was evaluated upon the earthworm species Eisenia fetida. Neither the survival nor the reproduction was affected by the addition of silver nanoparticles up to 1000 ppm. Thus these 'green' silver nanoparticles have promising potential as future materials. Copyright © 2012 Elsevier B.V. All rights reserved.

Method for characterization of a spherically bent crystal for K.alpha. X-ray imaging of laser plasmas using a focusing monochromator geometry

DOEpatents

Kugland, Nathan; Doeppner, Tilo; Glenzer, Siegfried; Constantin, Carmen; Niemann, Chris; Neumayer, Paul

2015-04-07

A method is provided for characterizing spectrometric properties (e.g., peak reflectivity, reflection curve width, and Bragg angle offset) of the K.alpha. emission line reflected narrowly off angle of the direct reflection of a bent crystal and in particular of a spherically bent quartz 200 crystal by analyzing the off-angle x-ray emission from a stronger emission line reflected at angles far from normal incidence. The bent quartz crystal can therefore accurately image argon K.alpha. x-rays at near-normal incidence (Bragg angle of approximately 81 degrees). The method is useful for in-situ calibration of instruments employing the crystal as a grating by first operating the crystal as a high throughput focusing monochromator on the Rowland circle at angles far from normal incidence (Bragg angle approximately 68 degrees) to make a reflection curve with the He-like x-rays such as the He-.alpha. emission line observed from a laser-excited plasma.

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

NASA Technical Reports Server (NTRS)

Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

2000-01-01

The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

PubMed

Farley, J R; Stilt-Coffing, B

2001-01-01

Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P <0.005 for each), with associated decreases in cell layer protein (P <0.05 for each) and concomitant increases in the release of sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P <0.001 for each). In contrast, caspase and calpain inhibitors reduced apoptosis, increased cell layer protein, and decreased the release of sALP activity (P <0.05 for each). Exposure to IGF-I also decreased apoptosis, in a time- and dose-dependent manner (e.g., r = 0.93, P <0.001 for IGF-I doses), with associated proportional effects to increase cell layer protein (P <0.001) and decrease the release of sALP activity (P <0.001). IGF-I also inhibited the actions of TNF-a and okadiac acid to increase apoptosis and sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

PubMed

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A; Voorhoeve, P Mathijs

2012-05-01

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

PubMed Central

Beillard, Emmanuel; Ong, Siau Chi; Giannakakis, Antonis; Guccione, Ernesto; Vardy, Leah A.; Voorhoeve, P. Mathijs

2012-01-01

MicroRNA–mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3′ UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3′ UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3′ UTR–mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs. PMID:22417692

VARIABLE TIME DELAY MEANS

DOEpatents

Clemensen, R.E.

1959-11-01

An electrically variable time delay line is described which may be readily controlled simuitaneously with variable impedance matching means coupied thereto such that reflections are prevented. Broadly, the delay line includes a signal winding about a magnetic core whose permeability is electrically variable. Inasmuch as the inductance of the line varies directly with the permeability, the time delay and characteristic impedance of the line both vary as the square root of the permeability. Consequently, impedance matching means may be varied similariy and simultaneously w:th the electrically variable permeability to match the line impedance over the entire range of time delay whereby reflections are prevented.

Metallothionein turnover in mammalian cell lines: implications in drug resistance

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

Monia, B.P.; Butt, T.R.; Ecker, D.J.

1986-05-01

Metallothioneins (MT) are low molecular weight, cysteine-rich proteins believed to participate in metal detoxification. A wide variety of cells in culture have been shown to accumulate MT in response to metal administration. These metal-induced increases in MT levels result from an increased rate of MT gene transcription, MT mRNA accumulation, and MT synthesis. Turnover of Cd-, Zn- and Au-induced MT was studied in a Chinese Hamster Ovary (CHO) cell line which was resistant to Cd and the Au-containing drug Auranofin (AF). Cd, Zn and Au were potent inducers of MT mRNA and accumulated approximately equal amounts of mRNA under themore » conditions employed in this study. Pulse-chase studies utilizing (/sup 35/S)cysteine revealed that the half-life of Au-, Zn- and Cd-induced MT was 0.75, 10 and 24 hrs. respectively. The reported differences in the tertiary structure of Au-MT from that of Cd-MT lead us to propose that the differences in half-lives observed reflect differences in subceptibility to intracellular proteolysis, which in turn, may effect the ability of MT to confer resistance to various metals.« less

Production of Xylella fastidiosa diffusible signal factor in transgenic grape causes pathogen confusion and reduction in severity of Pierce's disease.

PubMed

Lindow, Steven; Newman, Karyn; Chatterjee, Subhadeep; Baccari, Clelia; Lavarone, Anthony T; Ionescu, Michael

2014-03-01

The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and mobility within the plant. Symptoms in such plants were restricted to near the point of inoculation and incidence of disease was two- to fivefold lower than in the parental line. Both the longitudinal and lateral movement of X. fastidiosa in the xylem was also much lower. DSF was detected in both leaves and xylem sap of RpfF-expressing plants using biological sensors, and both 2-Z-tetradecenoic acid, previously identified as a component of X. fastidiosa DSF, and cis-11-methyl-2-dodecenoic acid were detected in xylem sap using electrospray ionization mass spectrometry. A higher proportion of X. fastidiosa cells adhered to xylem vessels of the RpfF-expressing line than parental 'Freedom' plants, reflecting a higher adhesiveness of the pathogen in the presence of DSF. Disease incidence in RpfF-expressing plants in field trials in which plants were either mechanically inoculated with X. fastidiosa or subjected to natural inoculation by sharpshooter vectors was two- to fourfold lower in than that of the parental line. The number of symptomatic leaves on infected shoots was reduced proportionally more than the incidence of infection, reflecting a decreased ability of X. fastidiosa to move within DSF-producing plants.

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

Antiproliferative and proapoptotic effects of bisphenol A on human trophoblastic JEG-3 cells.

PubMed

Morice, Lucie; Benaîtreau, Delphine; Dieudonné, Marie-Noëlle; Morvan, Corinne; Serazin, Valérie; de Mazancourt, Philippe; Pecquery, René; Dos Santos, Esther

2011-07-01

Different studies performed in rodents revealed that bisphenol-A (BPA), an environmental compound, altered early embryonic development. However, little is known concerning the direct effects of BPA on human implantation process. Thus, we decided to study in vitro BPA's effects on proliferative capacities of the human trophoblastic cell line, JEG-3. For this purpose, we first have shown that JEG-3 cells express the specific BPA receptor, namely estrogen-related receptor γ1 (ERRγ1). Secondly, we demonstrated that BPA did not exert any cytotoxic action in JEG-3 cells up to 10(-6)M. Moreover [(3)H]-thymidine incorporation experiments revealed that BPA significantly reduced cell proliferation. The results also showed that BPA induced JEG-3 apoptosis capacity as reflected by DNA fragmentation experiments. In conclusion, we describe here the direct impact of BPA on trophoblastic cell number mediated through both anti-proliferative and pro-apoptotic effects. Copyright © 2011 Elsevier Inc. All rights reserved.

Nonlinear refraction and reflection travel time tomography

USGS Publications Warehouse

Zhang, Jiahua; ten Brink, Uri S.; Toksoz, M.N.

1998-01-01

We develop a rapid nonlinear travel time tomography method that simultaneously inverts refraction and reflection travel times on a regular velocity grid. For travel time and ray path calculations, we apply a wave front method employing graph theory. The first-arrival refraction travel times are calculated on the basis of cell velocities, and the later refraction and reflection travel times are computed using both cell velocities and given interfaces. We solve a regularized nonlinear inverse problem. A Laplacian operator is applied to regularize the model parameters (cell slownesses and reflector geometry) so that the inverse problem is valid for a continuum. The travel times are also regularized such that we invert travel time curves rather than travel time points. A conjugate gradient method is applied to minimize the nonlinear objective function. After obtaining a solution, we perform nonlinear Monte Carlo inversions for uncertainty analysis and compute the posterior model covariance. In numerical experiments, we demonstrate that combining the first arrival refraction travel times with later reflection travel times can better reconstruct the velocity field as well as the reflector geometry. This combination is particularly important for modeling crustal structures where large velocity variations occur in the upper crust. We apply this approach to model the crustal structure of the California Borderland using ocean bottom seismometer and land data collected during the Los Angeles Region Seismic Experiment along two marine survey lines. Details of our image include a high-velocity zone under the Catalina Ridge, but a smooth gradient zone between. Catalina Ridge and San Clemente Ridge. The Moho depth is about 22 km with lateral variations. Copyright 1998 by the American Geophysical Union.

Novel approaches to models of Alzheimer's disease pathology for drug screening and development.

PubMed

Shaughnessy, Laura; Chamblin, Beth; McMahon, Lori; Nair, Ayyappan; Thomas, Mary Beth; Wakefield, John; Koentgen, Frank; Ramabhadran, Ram

2004-01-01

Development of therapeutics for Alzheimer's disease (AD) requires appropriate cell culture models that reflect the errant biochemical pathways and animal models that reflect the pathological hallmarks of the disease as well as the clinical manifestations. In the past two decades AD research has benefited significantly from the use of genetically engineered cell lines expressing components of the amyloid-generating pathway, as well as from the study of transgenic mice that develop the pathological hallmarks of the disease, mainly neuritic plaques. The choice of certain cell types and the choice of mouse as the model organism have been mandated by the feasibility of introduction and expression of foreign genes into these model systems. We describe a universal and efficient gene-delivery system, using lentiviral vectors, that permits the development of relevant cell biological systems using neuronal cells, including primary neurons and animal models in mammalian species best suited for the study of AD. In addition, lentiviral gene delivery provides avenues for creation of novel models by direct and prolonged expression of genes in the brain in any vertebrate animal. TranzVector is a lentiviral vector optimized for efficiency and safety that delivers genes to cells in culture, in tissue explants, and in live animals regardless of the dividing or differentiated status of the cells. Genes can also be delivered efficiently to fertilized single-cell-stage embryos of a wide range of mammalian species, broadening the range of the model organism (from rats to nonhuman primates) for the study of disease mechanism as well as for development of therapeutics. Copyright 2004 Humana Press Inc.

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.

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.

Method and apparatus for imaging a sample on a device

DOEpatents

Trulson, Mark; Stern, David; Fiekowsky, Peter; Rava, Richard; Walton, Ian; Fodor, Stephen P. A.

2001-01-01

A method and apparatus for imaging a sample are provided. An electromagnetic radiation source generates excitation radiation which is sized by excitation optics to a line. The line is directed at a sample resting on a support and excites a plurality of regions on the sample. Collection optics collect response radiation reflected from the sample I and image the reflected radiation. A detector senses the reflected radiation and is positioned to permit discrimination between radiation reflected from a certain focal plane in the sample and certain other planes within the sample.

Meeting Report: Using Stem Cells for Biological and Therapeutics Discovery in Mental Illness, April 2012

PubMed Central

2013-01-01

This report synthesizes the discussions during a workshop convened April 24–25, 2012, by the National Institute of Mental Health and the Foundation for the NIH in Bethesda, Maryland, that focused on progress and challenges in the use of patient-derived reprogrammed cells for basic biological discovery, target identification, screening, and drug development for mental illnesses such as schizophrenia, bipolar disorder, and autism spectrum disorders. The workshop revealed that the greatest progress has been made in reprogramming methods and agreed-upon standards for validating the resulting induced pluripotent stem cell lines. However, challenges remain in several areas, including efficiently generating and validating specific neural cell types with respect to regional identity, establishing assays with predictive validity to mental illness pathophysiology, and generating sufficient statistical power and data reproducibility across laboratories. A brainstorming session yielded a number of suggestions, including calls to (a) facilitate the replication of results by standardizing protocols and samples used across laboratories; (b) improve technology by generating cheaper/faster targeting methods, reporters, and assays; and (c) improve resource sharing and collaboration, with an emphasis on rapid sharing of new cell lines, technologies, and best practices, possibly incorporated into a public-private partnership. The meeting provided an important venue for academic, government, and private sector scientists to address potential opportunities for translational and clinical applications of reprogrammed cell research. A number of activities since the workshop have reflected the feedback from meeting participants. PMID:23408104

Meeting report: using stem cells for biological and therapeutics discovery in mental illness, April 2012.

PubMed

Panchision, David M

2013-03-01

This report synthesizes the discussions during a workshop convened April 24-25, 2012, by the National Institute of Mental Health and the Foundation for the NIH in Bethesda, Maryland, that focused on progress and challenges in the use of patient-derived reprogrammed cells for basic biological discovery, target identification, screening, and drug development for mental illnesses such as schizophrenia, bipolar disorder, and autism spectrum disorders. The workshop revealed that the greatest progress has been made in reprogramming methods and agreed-upon standards for validating the resulting induced pluripotent stem cell lines. However, challenges remain in several areas, including efficiently generating and validating specific neural cell types with respect to regional identity, establishing assays with predictive validity to mental illness pathophysiology, and generating sufficient statistical power and data reproducibility across laboratories. A brainstorming session yielded a number of suggestions, including calls to (a) facilitate the replication of results by standardizing protocols and samples used across laboratories; (b) improve technology by generating cheaper/faster targeting methods, reporters, and assays; and (c) improve resource sharing and collaboration, with an emphasis on rapid sharing of new cell lines, technologies, and best practices, possibly incorporated into a public-private partnership. The meeting provided an important venue for academic, government, and private sector scientists to address potential opportunities for translational and clinical applications of reprogrammed cell research. A number of activities since the workshop have reflected the feedback from meeting participants.

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

Genome-edited human stem cell-derived beta cells: a powerful tool for drilling down on type 2 diabetes GWAS biology.

PubMed

Beer, Nicola L; Gloyn, Anna L

2016-01-01

Type 2 diabetes (T2D) is a disease of pandemic proportions, one defined by a complex aetiological mix of genetic, epigenetic, environmental, and lifestyle risk factors. Whilst the last decade of T2D genetic research has identified more than 100 loci showing strong statistical association with disease susceptibility, our inability to capitalise upon these signals reflects, in part, a lack of appropriate human cell models for study. This review discusses the impact of two complementary, state-of-the-art technologies on T2D genetic research: the generation of stem cell-derived, endocrine pancreas-lineage cells and the editing of their genomes. Such models facilitate investigation of diabetes-associated genomic perturbations in a physiologically representative cell context and allow the role of both developmental and adult islet dysfunction in T2D pathogenesis to be investigated. Accordingly, we interrogate the role that patient-derived induced pluripotent stem cell models are playing in understanding cellular dysfunction in monogenic diabetes, and how site-specific nucleases such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are helping to confirm genes crucial to human endocrine pancreas development. We also highlight the novel biology gleaned in the absence of patient lines, including an ability to model the whole phenotypic spectrum of diabetes phenotypes occurring both in utero and in adult cells, interrogating the non-coding 'islet regulome' for disease-causing perturbations, and understanding the role of other islet cell types in aberrant glycaemia. This article aims to reinforce the importance of investigating T2D signals in cell models reflecting appropriate species, genomic context, developmental time point, and tissue type.

Mutations participating in interallelic complementation in propionic acidemia

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

Gravel, R.A.; Akerman, B.R.; Lamhonwah, A.M.

1994-07-01

Deficiency of propionyl-CoA carboxylase (PCC; [alpha][sub 4][beta][sub 4]) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA ([alpha]-subunit) and PCCB ([beta]-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, the authors have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound hetrozygote of Pro228Leu andmore » Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS+1 G[yields]T, located after Lys466. The authors suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, they previously had proposed that [Delta]Ile408 was the complementing allele. They now show that its second allele, [open quotes]Ins[center dot]Del[close quotes], a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. The authors conclude that the interallelic complementation results from mutations in domains that can interact between [beta]-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, they suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site. 37 refs., 5 figs., 2 tabs.« less

The lateral line system in anuran tadpoles: neuromast morphology, arrangement, and innervation.

PubMed

Quinzio, Silvia; Fabrezi, Marissa

2014-08-01

Anuran larvae have been classified into four morphological types which reflect intraordinal macroevolution. At present, complete characterizations of the lateral line system are only available for Xenopus laevis (Type I) and Discoglossus pictus (Type III). We analyzed the morphology, arrangement, and innervation of neuromasts related to the anterodorsal and anteroventral lateral line nerves in 10 anuran species representing Types I, II, and IV with the aim of interpreting the existing variation and discussing the evolution of the lateral line in anuran larvae. We found: (1) the presence of two orbital and three mandibular neuromast lines in all anuran larvae studied, (2) the ventral arrangement of mandibular neuromast lines appears to have evolved convergently in Larval Types I and II, and the lateroventral arrangement of mandibular lines of neuromasts appears to have evolved in Larval Types III and IV; (3) interspecific variation in the organization, size, and number of sensory cells per neuromast within the lines; and (4) the supralabial extension of the Angular line in Lepidobatrachus spp. and the tentacular location of the Oral neuromasts in X. laevis are concomitant with their particular morphologies. Based on the variation described we find that the lateral line system in anuran larvae seems to have been maintained without significant changes, with the exception of Lepidobatrachus spp. and Xenopus. These unique features added to other of Lepidobatrachus tadpoles are sufficient to propose a new Larval Type (V). Copyright © 2014 Wiley Periodicals, Inc.

Aptamer-Targeted Gold Nanoparticles As Molecular-Specific Contrast Agents for Reflectance Imaging

PubMed Central

2008-01-01

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer−gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(−) cell lines treated with the anti-PSMA aptamer−gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications. PMID:18512972

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.

Seismic reflection response from cross-correlations of ambient vibrations on non-conventional hidrocarbon reservoir

NASA Astrophysics Data System (ADS)

Huerta, F. V.; Granados, I.; Aguirre, J.; Carrera, R. Á.

2017-12-01

Nowadays, in hydrocarbon industry, there is a need to optimize and reduce exploration costs in the different types of reservoirs, motivating the community specialized in the search and development of alternative exploration geophysical methods. This study show the reflection response obtained from a shale gas / oil deposit through the method of seismic interferometry of ambient vibrations in combination with Wavelet analysis and conventional seismic reflection techniques (CMP & NMO). The method is to generate seismic responses from virtual sources through the process of cross-correlation of records of Ambient Seismic Vibrations (ASV), collected in different receivers. The seismic response obtained is interpreted as the response that would be measured in one of the receivers considering a virtual source in the other. The acquisition of ASV records was performed in northern of Mexico through semi-rectangular arrays of multi-component geophones with instrumental response of 10 Hz. The in-line distance between geophones was 40 m while in cross-line was 280 m, the sampling used during the data collection was 2 ms and the total duration of the records was 6 hours. The results show the reflection response of two lines in the in-line direction and two in the cross-line direction for which the continuity of coherent events have been identified and interpreted as reflectors. There is certainty that the events identified correspond to reflections because the time-frequency analysis performed with the Wavelet Transform has allowed to identify the frequency band in which there are body waves. On the other hand, the CMP and NMO techniques have allowed to emphasize and correct the reflection response obtained during the correlation processes in the frequency band of interest. The results of the processing and analysis of ASV records through the seismic interferometry method have allowed us to see interesting results in light of the cross-correlation process in combination with the Wavelet analysis and conventional seismic reflection techniques. Therefore it was possible to recover the seismic response on each analyzed source-receiver pair, allowing us to obtain the reflection response of each analyzed seismic line.

Russian Activities in Space Photovoltaic Power Modules with Concentrators

NASA Technical Reports Server (NTRS)

Andreev, Vyacheslav M.; Rumyantsev, Valeri D.

2004-01-01

Space concentrator modules with point-and line-focus Fresnel lenses and with reflective parabolic troughs have been developed recently at Ioffe Physico-Technical Institute. PV receivers for these modules are based: on the single junction LPE and MOCVD AlGaAs/GaAs solar cells characterized by AM0 efficiencies of 23.5 - 24% at 20 - 50 suns and 24 - 24.75 at 50 - 200 suns; on the mechanically stacked tandem AlGaAs/GaAs-GaSb cells with efficiency of 27 - 28 at 20 - 100 suns. MOCVD AlGaAs/GaAs cells with internal Bragg reflector have shown a higher radiation resistance as compared to a traditional structure. Monolithic two-terminal tandems AlGaAs (top)-GaAs (bottom) for space application and GaSb (top) - InGaAsSb (bottom) for TRV application are under development as well.

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.

Thermophotovoltaic in-situ mirror cell

DOEpatents

Campbell, Brian C.

1997-01-01

A photovoltaic cell used in a direct energy conversion generator for converting heat to electricity includes a reflective layer disposed within the cell between the active layers of the cell and the cell substrate. The reflective layer reflects photons of low energy back to a photon producing emitter for reabsorption by the emitter, or reflects photons with energy greater than the cell bandgap back to the cell active layers for conversion into electricity. The reflective layer can comprise a reflective metal such as gold while the substrate can comprise heavily doped silicon or a metal.

Nicotinamide augments the survival and incidence of apoptosis in glioma cells following photodynamic therapy in vitro

NASA Astrophysics Data System (ADS)

Bisland, Stuart K.; Modi, Nayan; Wilson, Brian C.

2004-10-01

The ability to customize photodynamic therapy (PDT) parameters with regards to timing and dosing of administered drug and light can be beneficial in determining target specificity and mode of cell death. Sustained, low level PDT or metronomic PDT (mPDT) may afford enhanced apoptotic cell death. This is of particular importance when considering PDT for the treatment of brain tumors as unlike apoptosis, necrotic cell death often leads to inflammation with increased intracranial pressure. The ability, therefore, to 'fine tune' PDT in favour of apoptosis is paramount. We have studied both acute (one time treatment) PDT (aPDT) and mPDT delivery strategies in combination with nicotinamide (NA) in an attempt to maximize the number of tumor cells dieing by apoptosis. Using several different glioma cell lines (9L, U87-MG and CNS-1) we now confirm that NA provides a dose-dependent (0.1-0.5 mM) increase in apoptotic cells following d-aminolevulinic acid-mediated aPDT or mPDT. Furthermore, using the 9L cell line stably transfected with the luciferase gene, NA was shown to delay the depletion of bioluminscence signal in aPDT and mPDT treated cells, inferring that adenosine triphosphate levels are maintained for longer following NA treatment. NA has previously been reported as promoting neuronal and vascular cell survival in normal brain following a number of neurological insults in which reactive oxygen species are implicated including, stroke, Alzheimer's disease and toxin-induced lesions. It is likely that the effects of NA reflect its capacity as an antioxidant as well as its ability to inhibit poly (adenosine diphosphate-ribose) polymerase-mediated depletion of ATP. Our results indicate that NA may prove therapeutically advantageous when used in combination with PDT treatment of brain tumors.

Genome-wide analysis of human constitutive androstane receptor (CAR) transcriptome in wild-type and CAR-knockout HepaRG cells.

PubMed

Li, Daochuan; Mackowiak, Bryan; Brayman, Timothy G; Mitchell, Michael; Zhang, Lei; Huang, Shiew-Mei; Wang, Hongbing

2015-11-01

The constitutive androstane receptor (CAR) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Dexamethasone enhances agonist induction of tissue factor in monocytes but not in endothelial cells.

PubMed

Bottles, K D; Morrissey, J H

1993-06-01

Stimulation of monocytic cells by inflammatory agents such as bacterial lipopolysaccharide or tumour necrosis factor-alpha leads to the rapid and transient expression of tissue factor, the major cellular initiator of the extrinsic coagulation cascade in both haemostasis and tissue inflammation. In this study we investigated whether the synthetic anti-inflammatory glucocorticoid, dexamethasone, would inhibit agonist induction of tissue factor expression in both monocytes and endothelial cells. Surprisingly, dexamethasone significantly enhanced the induction of tissue factor expression by peripheral blood mononuclear cells and an established monocytic cell line, THP-1, in response to lipopolysaccharide or tumour necrosis factor-alpha. However, unlike monocytic cells, dexamethasone did not enhance agonist induction of tissue factor in endothelial cells. Synergistic enhancement of tissue factor expression by dexamethasone was also reflected in tissue factor mRNA levels in THP-1 cells, but was not the result of improved TF mRNA stability. Synergism between bacterial lipopolysaccharide and glucocorticoid in the induction of monocyte effector function is extremely unusual and may help to explain the variable outcome of glucocorticoid treatment of septic shock.

The metabolism of testosterone by dermal papilla cells cultured from human pubic and axillary hair follicles concurs with hair growth in 5 alpha-reductase deficiency.

PubMed

Hamada, K; Thornton, M J; Laing, I; Messenger, A G; Randall, V A

1996-05-01

Androgens regulate the growth of many human hair follicles, but only pubic, axillary, and scalp hair growth occur in men with 5 alpha-reductase deficiency. This suggests that 5 alpha-dihydrotestosterone is the active intracellular androgen in androgen-dependent follicles, except in the axilla and pubis. Since the dermal papilla plays a major regulatory role in hair follicles and may be the site of androgen action, we have investigated androgen metabolism in six primary lines of cultured dermal papilla cells from pubic and axillary hair follicles; previous studies have shown that beard cells take up and metabolize testosterone, retaining and secreting 5 alpha-dihydrotestosterone. After 24 h preincubation in serum-free Eagle's medium 199, 100-mm dishes of confluent cells were incubated for 2 h with 5 nM [1,2,6,7-3H]testosterone. Media were collected and the cells washed with phosphate-buffered saline and extracted with chloroform: methanol (2:1). After the addition of unlabeled and 14C-labeled marker steroids, the extracts were analyzed by a two-step thin-layer chromatography system; steroid identity was confirmed by recrystallization to a constant 3H/14C ratio. Beard and pubic dermal papilla cells were also incubated for 24 h, and the medium was analyzed at various times. The results from pubic and axillary primary cell lines were similar. In both cells and media the major steroid identified was testosterone, but significant amounts of androstenedione were present, indicating 17 beta-hydroxysteroid dehydrogenase activity; androstenedione was also identified within the cells, but a small amount of 5 alpha-dihydrotestosterone was only identified in one pubic cell line. Beard dermal papilla cells secreted large amounts of 5 alpha-dihydrotestosterone into the medium over 24 h in contrast to pubic cells, which produced only very small amounts. The pubic and axillary cell results contrasts with the observations of pronounced 5 alpha-dihydrotestosterone in beard cells and confirm that androgen metabolism in cultured dermal papilla cells reflects the parent follicle's ability to respond to androgen in the absence of 5 alpha-reductase type II in vivo. This supports our hypothesis that androgen acts on hair follicles via the dermal papilla and suggests that cultured dermal papilla cells may offer an important model system for studies of androgen action.

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.

Mix-ups and mycoplasma: the enemies within.

PubMed

Drexler, Hans G; Uphoff, Cord C; Dirks, Willy G; MacLeod, Roderick A F

2002-04-01

Human leukemia-lymphoma (LL) cell lines represent important tools for experimental research. Among the various problems associated with cell lines, the two most common concern contaminations: (1) cross-contamination with unrelated cells and (2) contamination with microorganisms, in particular mycoplasma. The bad news is that about one-third of the cell lines are either cross-contaminated or mycoplasma-infected or both. The good news is that there are means to recognize and overcome these problems. In cases where, during attempts to establish new LL cell lines, primary LL cultures are cross-contaminated with continuous cell lines, intended new cell lines simply cannot be established ("early" cross-contamination). In cases of "late" cross-contamination of existing LL cell lines where the intrusive cells have a growth advantage, the original ("uncontaminated") cell lines may still be available elsewhere. DNA fingerprinting and cytogenetic analysis appear to be the most suitable approaches to detect cross-contaminations and to authenticate LL cell lines. A different but related aspect of "false" LL cell lines is the frequent misclassification of cell lines whereby the actual cell type of the cell line does not correspond to the purported model character of the cell line. Mycoplasma infection can have a multitude of effects on the eukaryotic cells which, due to the variety of infecting mycoplasma species and many other contributing parameters, cannot be predicted, rendering resulting data questionable at best. Practical procedures for the detection and elimination of mycoplasma contamination have been developed. Diagnostic and preventive strategies in order to hem the alarming increase in "false" and mycoplasma-positive LL cell lines are recommended.

Relativistic Effects on Reflection X-ray Spectra of AGN

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

Lee, Khee-Gan; /University Coll. London; Fuerst, Steven V.

2007-01-05

We have calculated the reflection component of the X-ray spectra of active galactic nuclei (AGN) and shown that they can be significantly modified by the relativistic motion of the accretion flow and various gravitational effects of the central black hole. The absorption edges in the reflection spectra suffer severe energy shifts and smearing. The degree of distortion depends on the system parameters, and the dependence is stronger for some parameters such as the inner radius of the accretion disk and the disk viewing inclination angles. The relativistic effects are significant and are observable. Improper treatment of the reflection component ofmore » the X-ray continuum in spectral fittings will give rise to spurious line-like features, which will mimic the fluorescent emission lines and mask the relativistic signatures of the lines.« less

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...

Microscopic visualization of metabotropic glutamate receptors on the surface of living cells using bifunctional magnetic resonance imaging probes.

PubMed

Mishra, Anurag; Mishra, Ritu; Gottschalk, Sven; Pal, Robert; Sim, Neil; Engelmann, Joern; Goldberg, Martin; Parker, David

2014-02-19

A series of bimodal metabotropic glutamate-receptor targeted MRI contrast agents has been developed and evaluated, based on established competitive metabotropic Glu receptor subtype 5 (mGluR5) antagonists. In order to directly visualize mGluR5 binding of these agents on the surface of live astrocytes, variations in the core structure were made. A set of gadolinium conjugates containing either a cyanine dye or a fluorescein moiety was accordingly prepared, to allow visualization by optical microscopy in cellulo. In each case, surface receptor binding was compromised and cell internalization observed. Another approach, examining the location of a terbium analogue via sensitized emission, also exhibited nonspecific cell uptake in neuronal cell line models. Finally, biotin derivatives of two lead compounds were prepared, and the specificity of binding to the mGluR5 cell surface receptors was demonstrated with the aid of their fluorescently labeled avidin conjugates, using both total internal reflection fluorescence (TIRF) and confocal microscopy.

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

[The factors involved in invasive ability of endometrial carcinoma cells].

PubMed

Mori, Y; Mizuuchi, H; Sato, K; Okamura, N; Kudo, R

1994-06-01

The in vitro invasive ability, the expression of cell adhesion molecule E-cadherin, activity of matrix metalloproteinase (MMP) and K-ras point mutation were investigated in eight human endometrial carcinoma cell lines. 1) In vitro invasive abilities of endometrial carcinoma cell lines depend on the degree of cell differentiation and the origin of cell lines. A poorly-differentiated carcinoma cell line (NUE-1) and a cell line derived from metastatic lymph node (SNG-M) were more invasive than moderately-(HEC-1A, HEC-1BE) and well-differentiated (HEC-6, Ishikawa) cell lines. 2) Immunohistochemically, less or non-invasive cell lines expressed E-cadherin strongly, whereas a highly invasive cell line (NUE-1) expressed E-cadherin weakly. 3) When cultured on Matrigel-coated dishes, the tumor cells derived from moderately- and well-differentiated carcinoma aggregated with each other and did not invade Matrigel in the invasion assay. The aggregated cells expressed E-cadherin more strongly when cultured on Matrigel. 4) 72-kD gelatinase (MMP-2) was secreted in serum-free conditioned medium of all cell lines. In an invasive cell line (NUE-1,SNG-M), the activity of MMP-2 was stronger than in other cell lines. And the activity of 92-kDa gelatinase (MMP-9) was detected in most invasive cell line (NUE-1). 5) Point mutation of K-ras codon 12 was detected in four of eight (50%) cell lines by the PCR-RFLP method. The changes in the DNA sequence were identified, but K-ras point mutation was not correlated with in vitro invasiveness of the tumor cells.

Introduction of a Ground Penetrating Radar System for Subsurface Investigation in Balik Pulau, Penang Island

NASA Astrophysics Data System (ADS)

Teoh, YJ; Bruka, MA; Idris, NM; Ismail, NA; Muztaza, NM

2018-04-01

Ground penetrating radar (GPR) are non-invasive geophysical techniques that enhance studies of the shallow subsurface. The purposes of this work are to study the subsurface composition of Balik Pulau area in Penang Island and to identify shallow subsurface geology features. Data acquisition for GPR is by using 250 MHz antenna to cover 200m survey line at Jalan Tun Sardon, Balik Pulau. GPR survey was divided into ten sections at 20 m each. Results from GPR shows that there is low EM reflection along the first 40 m of the survey line. Intense EM reflections were recorded along the distance 40 m to 100 m. Less noticeable radar reflections recorded along 100 m to 200 m distance of the survey line. As a conclusion, clear signal of radar wave reflection indicates dry region of the subsurface. Meanwhile, low signal of radar wave reflection indicates highly weathered granitic soil or clay of the subsurface.

Characterizing the In-Phase Reflection Bandwidth Theoretical Limit of Artificial Magnetic Conductors With a Transmission Line Model

NASA Technical Reports Server (NTRS)

Xie, Yunsong; Fan, Xin; Chen, Yunpeng; Wilson, Jeefrey D.; Simons, Rainee N.; Xiao, John Q.

2013-01-01

We validate through simulation and experiment that artificial magnetic conductors (AMC s) can be well characterized by a transmission line model. The theoretical bandwidth limit of the in-phase reflection can be expressed in terms of the effective RLC parameters from the surface patch and the properties of the substrate. It is found that the existence of effective inductive components will reduce the in-phase reflection bandwidth of the AMC. Furthermore, we propose design strategies to optimize AMC structures with an in-phase reflection bandwidth closer to the theoretical limit.

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

PubMed

Tsutsui, Takeki; Kumakura, Shin-Ichi; Tamura, Yukiko; Tsutsui, Takeo W; Sekiguchi, Mizuki; Higuchi, Tokihiro; Barrett, J Carl

2003-05-01

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

Systemic Therapy for Stage IV Non-Small-Cell Lung Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update.

PubMed

Hanna, Nasser; Johnson, David; Temin, Sarah; Baker, Sherman; Brahmer, Julie; Ellis, Peter M; Giaccone, Giuseppe; Hesketh, Paul J; Jaiyesimi, Ishmael; Leighl, Natasha B; Riely, Gregory J; Schiller, Joan H; Schneider, Bryan J; Smith, Thomas J; Tashbar, Joan; Biermann, William A; Masters, Gregory

2017-10-20

Purpose Provide evidence-based recommendations updating the 2015 ASCO guideline on systemic therapy for patients with stage IV non-small-cell lung cancer (NSCLC). Methods The ASCO NSCLC Expert Panel made recommendations based on a systematic review of randomized controlled trials from February 2014 to December 2016 plus the Cancer Care Ontario Program in Evidence-Based Care's update of a previous ASCO search. Results This guideline update reflects changes in evidence since the previous guideline update. Fourteen randomized controlled trials provide the evidence base; earlier phase trials also informed recommendation development. Recommendations New or revised recommendations include the following. Regarding first-line treatment for patients with non-squamous cell carcinoma or squamous cell carcinoma (without positive markers, eg, EGFR/ALK /ROS1), if the patient has high programmed death ligand 1 (PD-L1) expression, pembrolizumab should be used alone; if the patient has low PD-L1 expression, clinicians should offer standard chemotherapy. All other clinical scenarios follow 2015 recommendations. Regarding second-line treatment in patients who received first-line chemotherapy, without prior immune checkpoint therapy, if NSCLC tumor is positive for PD-L1 expression, clinicians should use single-agent nivolumab, pembrolizumab, or atezolizumab; if tumor has negative or unknown PD-L1 expression, clinicians should use nivolumab or atezolizumab. All immune checkpoint therapy is recommended alone plus in the absence of contraindications. For patients who received a prior first-line immune checkpoint inhibitor, clinicians should offer standard chemotherapy. For patients who cannot receive immune checkpoint inhibitor after chemotherapy, docetaxel is recommended; in patients with nonsquamous NSCLC, pemetrexed is recommended. In patients with a sensitizing EGFR mutation, disease progression after first-line epidermal growth factor receptor tyrosine kinase inhibitor therapy, and T790M mutation, osimertinib is recommended; if NSCLC lacks the T790M mutation, then chemotherapy is recommended. Patients with ROS1 gene rearrangement without prior crizotinib may be offered crizotinib, or if they previously received crizotinib, they may be offered chemotherapy.

Functional characterization of the 5'-flanking and the promoter region of the human UCP3 (hUCP3) gene.

PubMed

Tu, N; Chen, H; Winnikes, U; Reinert, I; Pirke, K M; Lentes, K U

2000-09-22

Uncoupling protein-3 (UCP3) is considered as an important regulator of energy expenditure and thermogenesis in humans. To get insight into the mechanisms regulating its expression we have cloned and characterized about 5 kb of the 5'-flanking region of the human UCP3 (hUCP3) gene. 5'-RACE analysis suggested a single transcription initiation site 187 bp upstream from the translational start site. The promoter region contains both TATA and CAAT boxes as well as consensus motifs for PPRE, TRE, CRE and muscle-specific factors like MyoD and MEF2 sites. Functional characterization of a 3 kb hUCP3 promoter fragment in multiple cell lines using a CAT-ELISA identified a cis-acting negative regulatory element between -2983 and -982 while the region between -982 and -284 showed greatly increased basal promoter activity suggesting the presence of a strong enhancer element. Promoter activity was particularly enhanced in the murine skeletal muscle cell line C2C12 reflecting the tissue-selective expression pattern of UCP3.

Non-contact thickness measurement using UTG

NASA Technical Reports Server (NTRS)

Bui, Hoa T. (Inventor)

1996-01-01

A measurement structure for determining the thickness of a specimen without mechanical contact but instead employing ultrasonic waves including an ultrasonic transducer and an ultrasonic delay line connected to the transducer by a retainer or collar. The specimen, whose thickness is to be measured, is positioned below the delay line. On the upper surface of the specimen a medium such as a drop of water is disposed which functions to couple the ultrasonic waves from the delay line to the specimen. A receiver device, which may be an ultrasonic thickness gauge, receives reflected ultrasonic waves reflected from the upper and lower surface of the specimen and determines the thickness of the specimen based on the time spacing of the reflected waves.

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

PubMed

Lee, Suk Kyoo; Lee, Gyun Min

2003-06-30

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.

The Nitric Oxide Prodrug JS-K Is Effective against Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo: Involvement of Reactive Oxygen SpeciesS⃞

PubMed Central

Chakrapani, Harinath; Saavedra, Joseph E.; Morris, Nicole L.; Holland, Ryan J.; Kosak, Ken M.; Shami, Paul J.; Anderson, Lucy M.; Keefer, Larry K.

2011-01-01

Non–small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non–small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy. PMID:20962031

The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species.

PubMed

Maciag, Anna E; Chakrapani, Harinath; Saavedra, Joseph E; Morris, Nicole L; Holland, Ryan J; Kosak, Ken M; Shami, Paul J; Anderson, Lucy M; Keefer, Larry K

2011-02-01

Non-small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O(2)-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non-small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy.

Relativistically Skewed Iron Emission and Disk Reflection in Galactic Microquasar XTE J1748-288

NASA Technical Reports Server (NTRS)

Miller, J. M.; Fox, D. W.; DiMatteo, T.; Wijnands, R.; Belloni, T.; Kouveliotou, C.; Lewin, W. H. G.

2000-01-01

We report evidence for an Fe K-alpha fluorescence line feature in the Very High, High, and Low state X-ray spectra of the galactic microquasar XTE JI748-288 during its June 1998 outburst. Spectral analyses were made on observations spread across the outburst, gathered with the Rossi X-ray Timing Explorer. Gaussian line. disk emission line, relativistic disk emission line, and disk reflection models are fit to the data. In the Very High State, the line profile is strongly redshifted and consistent with emission from the innermost radius of a maximally rotating Kerr black hole, 1.235 R(sub g). The line profile is less redshifted in the High State, but increasingly prominent. In the Low State, the line profile is very strong and centered af approx. 6.7 keV; disk line emission models constrain the inner edge of the disk to fluctuate between approx.20 and approx.59 R(sub g). We trace the disk reflection fraction across the full outburst of this source, and find well-constrained fractions below those observed in AGN in the Very High and High States, but consistent with other galactic sources in the Low State. We discuss the possible implications for black hole X-ray binary system dynamics and accretion flow geometry.

Expression of monoacylglycerol lipase as a marker of tumour invasion and progression in malignant melanoma.

PubMed

Baba, Yuko; Funakoshi, T; Mori, M; Emoto, K; Masugi, Y; Ekmekcioglu, S; Amagai, M; Tanese, K

2017-12-01

Accumulating evidence suggests that the lipid lytic enzyme monoacylglycerol lipase (MAGL) promotes tumour invasion and metastasis through up-regulation of pro-tumorigenic signalling lipids in several tumour cell lines. However, the expression status of MAGL in clinical melanoma tissues and its clinicopathological significance remain unclear. To correlate the tumour expression status of MAGL with the clinicopathological information of patients with malignant melanoma. Polymerase chain reaction (PCR) array screening was performed, and the results were validated using immunocytochemical analysis of tumour and non-tumour melanocytic cell lines. Immunohistochemical staining for MAGL was performed for 74 melanoma samples, including 48 primary and 26 metastatic tumours, in which the expression of MAGL was determined by evaluating the percentage of MAGL-positive tumour cells and the MAGL staining intensity. Finally, we analysed the association of MAGL expression status with tumour progression, tumour thickness and vascular invasion of the primary lesion. Immunocytochemical analysis revealed that MAGL was expressed in all 12 melanoma cell lines, but not in normal human epidermal melanocytes. In the immunohistochemical analysis, positive staining for MAGL was noted in 32 of 48 (64.5%) primary lesions, 14 of 17 (82.4%) lymph node metastatic lesions and 7 of 9 (77.8%) skin metastatic lesions. Metastatic tumours had a significantly higher staining intensity (P = 0.033 for lymph node, P = 0.010 for skin). In the analysis of primary lesions, higher MAGL expression correlated with greater tumour thickness (P = 0.015) and the presence of vascular invasion (P = 0.017). On further evaluation of MAGL-positive primary lesions, staining intensity of MAGL tended to be higher in deeper areas of the tumour mass. The expression of MAGL in tumour cells reflects the aggressiveness of melanoma cells and may serve as a marker of tumour progression. © 2017 European Academy of Dermatology and Venereology.

The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia.

PubMed

de Rooij, Martin F M; Kuil, Annemieke; Geest, Christian R; Eldering, Eric; Chang, Betty Y; Buggy, Joseph J; Pals, Steven T; Spaargaren, Marcel

2012-03-15

Small-molecule drugs that target the B-cell antigen receptor (BCR) signalosome show clinical efficacy in the treatment of B-cell non-Hodgkin lymphoma. These agents, including the Bruton tyrosine kinase (BTK) inhibitor PCI-32765, display an unexpected response in patients with chronic lymphocytic leukemia (CLL): a rapid and sustained reduction of lymphadenopathy accompanied by transient lymphocytosis, which is reversible upon temporary drug deprivation. We hypothesized that this clinical response reflects impaired integrin-mediated adhesion and/or migration. Here, we show that PCI-32765 strongly inhibits BCR-controlled signaling and integrin α(4)β(1)-mediated adhesion to fibronectin and VCAM-1 of lymphoma cell lines and primary CLL cells. Furthermore, PCI-32765 also inhibits CXCL12-, CXCL13-, and CCL19-induced signaling, adhesion, and migration of primary CLL cells. Our data indicate that inhibition of BTK by PCI-32765 overcomes BCR- and chemokine-controlled integrin-mediated retention and homing of malignant B cells in their growth- and survival-supporting lymph node and bone marrow microenvironment, which results in clinically evident CLL regression.

Single-molecule microscopy reveals membrane microdomain organization of cells in a living vertebrate.

PubMed

Schaaf, Marcel J M; Koopmans, Wiepke J A; Meckel, Tobias; van Noort, John; Snaar-Jagalska, B Ewa; Schmidt, Thomas S; Spaink, Herman P

2009-08-19

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.

Leukemia-lymphoma cell lines as model systems for hematopoietic research.

PubMed

Drexler, Hans G; MacLeod, Roderick A F

2003-01-01

Continuous human leukemia-lymphoma (LL) cell lines comprise a rich self-renewing resource of accessible and manipulable living cells which has illuminated the pathophysiology of hematopoietic tumors as well as basic cell biology. The major key advantages of continuous cell lines are the unlimited supply and worldwide availability of identical cell material and their cryopreservation. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro with 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 may be distinguished from Epstein-Barr virus (EBV)-immortalized normal cells, using various operational and conceptual parameters. The characterization and publication of new LL cell lines provides important and informative core data which, by opening new avenues for investigation, have become ubiquitous powerful research tools that are available to every investigator by reference cell repositories. There is a need in the scientific community for clean and authenticated LL cell lines to which every scientist has access as offered by these institutionalized public cell line banks. A list of the most useful, robust and freely available reference cell lines is proposed in this review. Clearly, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia.

High resolution measurements of solar induced chlorophyll fluorescence in the Fraunhofer oxigen bands

NASA Astrophysics Data System (ADS)

Mazzoni, M.; Agati, G.; Cecchi, G.; Toci, G.; Mazzinghi, P.

2017-11-01

Spectra of solar radiance reflected by leaves close to the Fraunhofer bands show the net contribution of chlorophyll fluorescence emission which adds to the reflected solar spectra. In a laboratory experiment, a low stray light, high resolution, 0.85 m double monochromator was used to filter radiation living leaves still attached to the plant in correspondence of the 687 nm and 760 nm O2 absorption bands. Reference spectra from a non fluorescent white reference were also acquired. Acquisition was performed by a Microchannel plate (MCP) intensified diode array with 512 elements. A fit of the spectral data outside the absorption lines allowed to retrieve the spectral base-line as a function of wavelength for the reference panel and the leaf. Reflectance functions were determined extending the Plascyck equation system to all the resolved lines of the oxygen absorption bands and using the base-lines for the continuum values. Fluorescence was deduced from the same equation system, using both the measured leaf and reference radiance spectra and the leaf reflectance fitting function.

Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines

PubMed Central

Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

2013-01-01

The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer. PMID:24149212

Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.

PubMed

Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

2013-12-01

The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

PubMed

Scharmach, E; Hessel, S; Niemann, B; Lampen, A

2009-11-30

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Heparanase expression is a prognostic indicator for postoperative survival in pancreatic adenocarcinoma

PubMed Central

Rohloff, J; Zinke, J; Schoppmeyer, K; Tannapfel, A; Witzigmann, H; Mössner, J; Wittekind, C; Caca, K

2002-01-01

Pancreatic ductal adenocarcinoma has a median survival of less than 6 months from diagnosis. This is due to the difficulty in early diagnosis, the aggressive biological behaviour of the tumour and a lack of effective therapies for advanced disease. Mammalian heparanase is a heparan-sulphate proteoglycan cleaving enzyme. It helps to degrade the extracellular matrix and basement membranes and is involved in angiogenesis. Degradation of extracellular matrix and basement membranes as well as angiogenesis are key conditions for tumour cell spreading. Therefore, we have analysed the expression of heparanase in human pancreatic cancer tissue and cell lines. Heparanase is expressed in cell lines derived from primary tumours as well as from metastatic sites. By immunohistochemical analysis, it is preferentially expressed at the invading edge of a tumour at both metastatic and primary tumour sites. There is a trend towards heparanase expression in metastasising tumours as compared to locally growing tumours. Postoperative survival correlates inversely with heparanase expression of the tumour reflected by a median survival of 34 and 17 month for heparanase negative and positive tumours, respectively. Our results suggest, that heparanase promotes cancer cell invasion in pancreatic carcinoma and could be used as a prognostic indicator for postoperative survival of patients. British Journal of Cancer (2002) 86, 1270–1275. DOI: 10.1038/sj/bjc/6600232 www.bjcancer.com © 2002 Cancer Research UK PMID:11953884

Melicope ptelefolia leaf extracts exhibit antioxidant activity and exert anti-proliferative effect with apoptosis induction on four different cancer cell lines.

PubMed

Kabir, Mohammad Faujul; Mohd Ali, Johari; Abolmaesoomi, Mitra; Hashim, Onn Haji

2017-05-05

Melicope ptelefolia is a well-known herb in a number of Asian countries. It is often used as vegetable salad and traditional medicine to address various ailments. However, not many studies have been currently done to evaluate the medicinal benefits of M. ptelefolia (MP). The present study reports antioxidant, anti-proliferative, and apoptosis induction activities of MP leaf extracts. Young MP leaves were dried, powdered and extracted sequentially using hexane (HX), ethyl acetate (EA), methanol (MeOH) and water (W). Antioxidant activity was evaluated using ferric reducing antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radicals scavenging and cellular antioxidant activity (CAA) assays. Anti-proliferative activity was evaluated through cell viability assay, using the following four human cancer cell lines: breast (HCC1937, MDA-MB-231), colorectal (HCT116) and liver (HepG2). The anti-proliferative activity was further confirmed through cell cycle and apoptosis assays, including annexin-V/7-aminoactinomycin D staining and measurements of caspase enzymes activation and inhibition. Overall, MP-HX extract exhibited the highest antioxidant potential, with IC 50 values of 267.73 ± 5.58 and 327.40 ± 3.80 μg/mL for ABTS and DPPH radical-scavenging assays, respectively. MP-HX demonstrated the highest CAA activity in Hs27 cells, with EC 50 of 11.30 ± 0.68 μg/mL, while MP-EA showed EC 50 value of 37.32 ± 0.68 μg/mL. MP-HX and MP-EA showed promising anti-proliferative activity towards the four cancer cell lines, with IC 50 values that were mostly below 100 μg/mL. MP-HX showed the most notable anti-proliferative activity against MDA-MB-231 (IC 50  = 57.81 ± 3.49 μg/mL) and HCT116 (IC 50  = 58.04 ± 0.96 μg/mL) while MP-EA showed strongest anti-proliferative activity in HCT116 (IC 50  = 64.69 ± 0.72 μg/mL). The anticancer potential of MP-HX and MP-EA were also demonstrated by their ability to induce caspase-dependent apoptotic cell death in all of the cancer cell lines tested. Cell cycle analysis suggested that both the MP-HX and MP-EA extracts were able to disrupt the cell cycle in most of the cancer cell lines. MP-HX and MP-EA extracts demonstrated notable antioxidant, anti-proliferative, apoptosis induction and cancer cell cycle inhibition activities. These findings reflect the promising potentials of MP to be a source of novel phytochemical(s) with health promoting benefits that are also valuable for nutraceutical industry and cancer therapy.

[Establishment of human embryonic stem cell lines and their therapeutic application].

PubMed

Suemori, Hirofumi

2004-03-01

Embryonic stem (ES) cell lines are pluripotent stem cell lines that can be propagated indefinitely in culture, retaining their potency to differentiate into every type of cell and tissue in the body. ES cell lines were first established from mouse blastocysts, and have been used for research in developmental biology. ES cells have been proven to be very valuable in the genetic modification of the mouse, especially in producing knockout mice. Since establishment of human ES cell lines was reported, their use in cell replacement therapies has been enthusiastically expected. There have been reports of the differentiation of several useful cell types from human ES cell lines, and clinical use of functional tissues and cells from human ES cells is anticipated. In Japan, there have also been many demands for the use of human ES cells in basic and pre-clinical research. We obtained governmental permission to establish human ES cell lines in April 2002 and started research using donated frozen embryos in January 2003. We successfully established three ES cell line from three blastocysts. These cell lines will be distributed at cost to researchers who have governmental permission to use human ES cells.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

PubMed

Jadi, R S; Sudeep, A B; Kumar, Satyendra; Arankalle, V A; Mishra, A C

2010-08-01

Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Coordinate Intracellular Expression of Salmonella Genes Induced during Infection

PubMed Central

Heithoff, Douglas M.; Conner, Christopher P.; Hentschel, Ute; Govantes, Fernando; Hanna, Philip C.; Mahan, Michael J.

1999-01-01

Salmonella typhimurium in vivo-induced (ivi) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg2+, Fe2+, and PhoPQ. All of the seven ivi fusions that are induced by both low pH and low Mg2+ (e.g., iviVI-A) are activated by the PhoPQ regulatory system. Iron-responsive ivi fusions include those induced under iron limitation (e.g., entF) as well as one induced by iron excess but only in the absence of PhoP (pdu). Intracellular expression studies showed that each of the pH- and Mg2+-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407. Each ivi fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed ivi genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types. Investigation of ivi expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments. PMID:9922242

Cell line: 2004-2014.

PubMed

2014-11-20

2014 marks Cell's 40th anniversary, and over the year we have looked back at how discoveries of the last four decades have molded our understanding of biology. The final decade of the Cell Line features a selection of the exceptional scientific work-both landmark papers and essential reviews. Select entries can be read as an "Annotated Classic," which includes the original paper and accompanying reflections of a leading scientist, considering the work from our current vantage point. Our last installment includes a harbinger of the interplay between microbiota and mammalian hosts in 2004, revolutionary papers in 2006 and 2007 unlocking cellular reprogramming, the discovery of beige adipocytes in 2012, and the first example of CRISPR-based genome editing in a nonhuman primate in 2014. In addition to landmark publications, there were innovative developments at the journal in this decade, with the complete redesign of the print journal and the creation of Leading Edge in late 2005 and the restructuring of the online display of the article in 2010. Keeping pace with the changing nature of biological research, over the decade Cell added new article types, introduced guidelines for the organization of supplementary material, and expanded the journal's web-based content to bring editors' and authors' excitement and perspective on individual papers to the readership. An interactive version of the timeline, with links to the papers, full author lists, and Annotated Classics, is available at http://dx.doi.org/10.1016/j.cell.2014.11.004.

STRONGER REFLECTION FROM BLACK HOLE ACCRETION DISKS IN SOFT X-RAY STATES

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

Steiner, James F.; Remillard, Ronald A.; García, Javier A.

We analyze 15,000 spectra of 29 stellar-mass black hole (BH) candidates collected over the 16 year mission lifetime of Rossi X-ray Timing Explorer using a simple phenomenological model. As these BHs vary widely in luminosity and progress through a sequence of spectral states, which we broadly refer to as hard and soft, we focus on two spectral components: the Compton power law and the reflection spectrum it generates by illuminating the accretion disk. Our proxy for the strength of reflection is the equivalent width of the Fe–K line as measured with respect to the power law. A key distinction ofmore » our work is that for all states we estimate the continuum under the line by excluding the thermal disk component and using only the component that is responsible for fluorescing the Fe–K line, namely, the Compton power law. We find that reflection is several times more pronounced (∼3) in soft compared to hard spectral states. This is most readily caused by the dilution of the Fe line amplitude from Compton scattering in the corona, which has a higher optical depth in hard states. Alternatively, this could be explained by a more compact corona in soft (compared to hard) states, which would result in a higher reflection fraction.« less

Crustal evolution of Eocene paleo arc around Ogasawara region obtained by seismic reflection survey

NASA Astrophysics Data System (ADS)

Yamashita, M.; Takahashi, N.; Kodaira, S.; Miura, S.; Ishizuka, O.; Tatsumi, Y.

2011-12-01

The Izu-Bonin (Ogasawara)-Mariana (IBM) arc is known to the typical oceanic island arc, and it is the most suitable area to understand the growth process of island arc. The existence of two paleo arc which consists of Oligocene and Eocene paleo age is known in IBM forearc region by geological and geophysical studies. The Ogasawara ridge is also known to locate the initial structure of arc evolution from geologic sampling of research submersible. In this region, IODP drilling site: IBM-2 is proposed in order to understand the temporal and spatial change in arc crust composition from 50 to 40Ma magmatism. Site IBM-2 consists of two offset drilling holes (BON-1, BON-2). BON-1 designed to first encounter forearc basalt and will reach the sheeted dykes. BON-2 will start in boninites and finish in fore arc basalts. The purpose of these drilling is sampling the full volcanic stratigraphy from gabbro to boninite. There is no seismic data around BON-1 and BON-2, therefore it is need to conduct the multi-channel seismic reflection survey. Japan Agency for Marine-Earth Science and Technology carried out multi-channel seismic reflection survey and wide-angle reflection survey using 7,800 cu.in. air gun, 5 km streamer with 444 ch hydrophones and 40 OBSs in March 2011. We obtained two seismic reflection profiles of lines KT06 and KT07 along the paleo arc around Ogasawara ridge. Line KT06 located the north side of Ogasawara ridge. Line KT07 located the trench side of Ogasawara ridge. Lines KT06 is also deployed the OBSs every 5 km interval. Thin sediments are covered with basement in both survey lines. There are some sediment filled in depression topography. The low-frequency reflection from the top of subducting Pacific plate is recognized in line KT06. The continuity of this reflection is not clear due to the complicated bathymetry. The displacement of basement in northern side of Ogasawara ridge is identified along the lineament of bathymetry in Line 06. This structure is estimated to relate the deformation in the Ogasawara Trough and lineament of paleo arc. We will discuss the relationship this lineament and deformation with regard to activity such as post volcanism.

Diffractive optical element-based glossmeter for the on-line measurement of normal reflectance on a printed porous coated paper

NASA Astrophysics Data System (ADS)

Oksman, Antti; Kuivalainen, Kalle; Ta˚G, Carl-Mikael; Juuti, Mikko; Mattila, Rauno; Hietala, Eero; Gane, Patrick A. C.; Peiponen, Kai-Erik

2011-04-01

Gloss of a product, such as print gloss, is mainly inspected with conventional white light glossmeters both at laboratory or production facilities. However, problems occur in conventional gloss measurement when the inspected surface is vertically moved in the plane of incidence and reflection or when the measurement area is small or curved. For a partial solution to these problems, we have previously introduced diffractive optical element-based glossmeters (DOGs) for the gloss inspection in laboratories and off-line use. We present a new construction of DOG, termed μDOG 1D, for the one-dimensional on-line print gloss measurement, in the form of the reflectance determination normal to the surface. The function of the glossmeter is demonstrated by laboratory tests and on-line measurements at a heat-set web offset printing machine. It is shown that gloss (i.e., normal reflectance) and minute gloss variation of papers and prints can be measured at the printing line using the glossmeter. This glossmeter is expected to be useful in real-time monitoring of the gloss and surface-specific absorption not only in the printing industry but also in inspection of products in other industrial sectors, such as metal finishing, laminating, paper, and construction materials manufacturing.

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

PubMed

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

No signatures of black hole spin in the X-ray spectrum of the Seyfert 1 galaxy Fairall 9

NASA Astrophysics Data System (ADS)

Yaqoob, T.; Turner, T. J.; Tatum, M. M.; Trevor, M.; Scholtes, A.

2016-11-01

Fairall 9 is one of several type 1 active galactic nuclei for which it has been claimed that the angular momentum (or spin) of the supermassive black hole can be robustly measured, using the Fe Kα emission line and Compton-reflection continuum in the X-ray spectrum. The method rests upon the interpretation of the Fe Kα line profile and associated Compton-reflection continuum in terms of relativistic broadening in the strong gravity regime in the innermost regions of an accretion disc, within a few gravitational radii of the black hole. Here, we re-examine a Suzaku X-ray spectrum of Fairall 9 and show that a face-on toroidal X-ray reprocessor model involving only non-relativistic and mundane physics provides an excellent fit to the data. The Fe Kα line emission and Compton-reflection continuum are calculated self-consistently, the iron abundance is solar, and an equatorial column density of ˜ 1024 cm- 2 is inferred. In this scenario, neither the Fe Kα line nor the Compton-reflection continuum provides any information on the black hole spin. Whereas previous analyses have assumed an infinite column density for the distant-matter reprocessor, the shape of the reflection spectrum from matter with a finite column density eliminates the need for a relativistically broadened Fe Kα line. We find a 90 per cent confidence range in the Fe Kα line full width at half-maximum of 1895-6205 km s- 1, corresponding to a distance of ˜3100 to 33 380 gravitational radii from the black hole, or 0.015-0.49 pc for a black hole mass of ˜1-3 × 108 M⊙.

3D printing of biomimetic microstructures for cancer cell migration.

PubMed

Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

2014-02-01

To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10 T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10 T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10 T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10 T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies.

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

77 FR 5489 - Identification of Human Cell Lines Project

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-03

...-01] Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology... cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding... cell lines accepted on the NIST Applied Genetics Group Web site at http://www.nist.gov/mml/biochemical...

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

NASA Astrophysics Data System (ADS)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

Butyrate-induced apoptotic cascade in colonic carcinoma cells: modulation of the beta-catenin-Tcf pathway and concordance with effects of sulindac and trichostatin A but not curcumin.

PubMed

Bordonaro, M; Mariadason, J M; Aslam, F; Heerdt, B G; Augenlicht, L H

1999-10-01

Short-chain fatty acids play a critical role in colonic homeostasis because they stimulate pathways of growth arrest, differentiation, and apoptosis. These effects have been well characterized in colonic cell lines in vitro. We investigated the role of beta-catenin-Tcf signaling in these responses to butyrate and other well-characterized inducers of apoptosis of colonic epithelial cells. Unlike wild-type APC, which down-regulates Tcf activity, butyrate, as well as sulindac and trichostatin A, all inducers of G0-G1 cell cycle arrest and apoptosis in the SW620 colonic carcinoma cell line, up-regulate Tcf activity. In contrast, structural analogues of butyrate that do not induce cell cycle arrest or apoptosis and curcumin, which stimulates G2-M arrest without inducing apoptosis, do not alter Tcf activity. Similar to the cell cycle arrest and apoptotic cascade induced by butyrate, the up-regulation of Tcf activity is dependent upon the presence of a mitochondrial membrane potential, unlike the APC-induced down-regulation, which is insensitive to collapse of the mitochondrial membrane potential. Moreover, the butyrate-induced increase in Tcf activity, which is reflected in an increase in beta-catenin-Tcf complex formation, is independent of the down-regulation caused by expression of wild-type APC. Thus, butyrate and wild-type APC have different and independent effects on beta-catenin-Tcf signaling. These data are consistent with other reports that suggest that the absence of wild-type APC, associated with the up-regulation of this signaling pathway, is linked to the probability of a colonic epithelial cell entering an apoptotic cascade.

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma.

PubMed

Narita, Tomoko; Ishida, Takashi; Ito, Asahi; Masaki, Ayako; Kinoshita, Shiori; Suzuki, Susumu; Takino, Hisashi; Yoshida, Takashi; Ri, Masaki; Kusumoto, Shigeru; Komatsu, Hirokazu; Imada, Kazunori; Tanaka, Yuetsu; Takaori-Kondo, Akifumi; Inagaki, Hiroshi; Scholz, Arne; Lienau, Philip; Kuroda, Taruho; Ueda, Ryuzo; Iida, Shinsuke

2017-08-31

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL. © 2017 by The American Society of Hematology.

Accumulation of N-Acetylglucosamine Oligomers in the Plant Cell Wall Affects Plant Architecture in a Dose-Dependent and Conditional Manner1[W][OPEN

PubMed Central

Vanholme, Bartel; Vanholme, Ruben; Turumtay, Halbay; Goeminne, Geert; Cesarino, Igor; Goubet, Florence; Morreel, Kris; Rencoret, Jorge; Bulone, Vincent; Hooijmaijers, Cortwa; De Rycke, Riet; Gheysen, Godelieve; Ralph, John; De Block, Marc; Meulewaeter, Frank; Boerjan, Wout

2014-01-01

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane. PMID:24664205

Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor

PubMed Central

Chaudhri, Virendra K.; Salzler, Gregory G.; Dick, Salihah A.; Buckman, Melanie S.; Sordella, Raffaella; Karoly, Edward D.; Mohney, Robert; Stiles, Brendon M.; Elemento, Olivier; Altorki, Nasser K.; McGraw, Timothy E.

2013-01-01

SUMMARY Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung tumor cancer-associated fibroblast lines (CAFs) to “normal” fibroblast lines (NFs) generated from adjacent non-neoplastic lung tissue. CAFs are pro-tumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor’s glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we demonstrate a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer. PMID:23475953

Circumnuclear Multi-phase Gas in the Circinus Galaxy. I. Non-LTE Calculations of CO Lines

NASA Astrophysics Data System (ADS)

Wada, Keiichi; Fukushige, Ryosuke; Izumi, Takuma; Tomisaka, Kohji

2018-01-01

In this study, we investigate the line emissions from cold molecular gas based on our previous “radiation-driven fountain model,” which reliably explains the spectral energy distribution of the nearest type 2 Seyfert galaxy, the Circinus galaxy. Using a snapshot of the best-fit radiation-hydrodynamic model for the central r≤slant 16 pc, in which non-equilibrium X-ray-dominated region chemistry is solved, we conduct post-processed non-local thermodynamic equilibrium radiation transfer simulations for the CO lines. We obtain a spectral line energy distribution with a peak around J≃ 6, and its distribution suggests that the lines are not thermalized. However, for a given line of sight, the optical depth distribution is highly non-uniform between {τ }ν \\ll 1 and {τ }ν \\gg 1. The CO-to-H2 conversion factor ({X}{CO}), which can be directly obtained from the results and is not a constant, depends strongly on the integrated intensity and differs from the fiducial value for local objects. {X}{CO} exhibits a large dispersion of more than one order of magnitude, reflecting the non-uniform internal structure of a “torus.” In addition, we found that the physical conditions differ between grid cells on a scale of a few parsecs along the observed lines of sight; therefore, a specific observed line ratio does not necessarily represent a single physical state of the interstellar medium.

The Onion Sign in neovascular age-related macular degeneration represents cholesterol crystals

PubMed Central

Pang, Claudine E.; Messinger, Jeffrey D.; Zanzottera, Emma C.; Freund, K. Bailey; Curcio, Christine A.

2015-01-01

Purpose To investigate the frequency, natural evolution and histological correlates of layered, hyperreflective, sub-retinal pigment epithelium (sub-RPE) lines, known as the Onion Sign, in neovascular age-related macular degeneration (nvAMD). Design Retrospective observational cohort study; an experimental laboratory study. Participants Two hundred thirty eyes of 150 consecutive patients with nvAMD; 40 human donor eyes with clinical and histopathologic diagnosis of nvAMD. Methods Spectral-domain optical coherence tomography (SD-OCT), near-infrared reflectance (nIR), color fundus images and medical charts were reviewed. Donor eyes underwent multimodal ex vivo imaging including SD-OCT before processing for high-resolution histology. Main Outcome Measures Presence of layered, hyperreflective sub-RPE lines, qualitative analysis of their change in appearance over time with SD-OCT, histological correlates of these lines, and associated findings within surrounding tissues. Results Sixteen of 230 eyes of patients (7.0%) and 2 of 40 donor eyes (5.0%) with nvAMD had layered, hyperreflective sub-RPE lines on SD-OCT imaging. These appeared as refractile, yellow-gray exudates on color imaging and hyperreflective lesions on nIR. In all 16 eyes, the Onion Sign persisted in follow-up for up to 5 years, with fluctuations in the abundance of lines and associated with intraretinal hyperreflective foci. Patients with the Onion Sign were disproportionately taking cholesterol-lowering medications (p = 0.025). Histology of 2 donor eyes revealed that hyperreflective lines correlated with clefts created by extraction of cholesterol crystals during tissue processing. Fluid surrounding crystals contained lipid yet was distinct from oily drusen. Intraretinal hyperreflective foci correlated with intraretinal RPE and lipid-filled cells of probable monocyte origin. Conclusion Persistent and dynamic, the Onion Sign represents sub-RPE cholesterol crystal precipitation in aqueous environment. The frequency of the Onion Sign in nvAMD in a referral practice and a pathology archive is 5–7%. Associations include use of cholesterol-lowering medication and intraretinal hyperreflective foci attributable to RPE cells and lipid-filled cells of monocyte origin. PMID:26298717

Leptin, immune responses and autoimmune disease. Perspectives on the use of leptin antagonists.

PubMed

Peelman, F; Iserentant, H; Eyckerman, S; Zabeau, L; Tavernier, J

2005-01-01

The pivotal role of leptin in regulating body weight and energy homeostasis is very well established. More recently, leptin also emerged as an important regulator of T-cell-dependent immunity. Reduced leptin levels, as observed during periods of starvation, correlate with an impaired cellular immune response, whereby especially the T(H)1 pro-inflammatory immune response appears to be affected. Physiologically, this could reflect the high energy demand of such processes, which are suppressed in animals or people with nutrient shortage. Several autoimmune diseases are T(H)1 T-cell dependent. In line with a pro-inflammatory role for leptin, animal models of leptin deficiency are markedly resistant to a variety of T-cell dependent autoimmune diseases. Here, we review the role of leptin in immune responses, with emphasis on autoimmune diseases. The design and potential use of leptin antagonists is also discussed.

The Production of lnterleukin-1 Receptor Antagonist by Human Bronchogenic Carcinoma

PubMed Central

Smith, Daniel R.; Kunkel, Steven L.; Standiford, Theodore J.; Chensue, Stephen W.; Rolfe, Mark W.; Orringer, Mark B.; Whyte, Richard I.; Burdick, Marie D.; Danforth, Jean M.; Gilbert, Andrew R.; Strieter, Robert M.

1993-01-01

Bronchogenic carcinoma displays an aggressive clinical course that may reflect a capacity to evade host defenses. We postulated that tumors may elaborate interleukin-1 receptor antagonist protein (IRAP) to escape host interleukin-1-dependent responses. Homogenates of human bronchogenic lung tumors demonstrated significant increases of IRAP compared with normal lung tissue controls (n = 48). There was no significant difference in interleukin-1 β levels between tumor and normal lung tissue. Immunohistochemical staining localized IRAP to tumor cells. Semiquantitative pathological analysis demonstrated a modest inflammatory cell infiltrate with qualitative differences between tumors of different histology. Western blot analysis of tumor homogenates demonstrated several molecular weight forms of IRAP. Finally, antigenic IRAP was detected in supernatants of the human bronchogenic carcinoma cell line (A549) maintained in vitro. These findings illustrate the capacity of bronchogenic tumors to produce and secrete IRAP that may be important in tumor evasion of host defenses. ImagesFigure 3Figure 4 PMID:8362978

Development and characterization of two new cell lines from milkfish (Chanos chanos) and grouper (Epinephelus coioides) for virus isolation.

PubMed

Parameswaran, V; Ishaq Ahmed, V P; Shukla, Ravi; Bhonde, R R; Sahul Hameed, A S

2007-01-01

Two new cell lines, SIMH and SIGE, were derived from the heart of milkfish (Chanos chanos), a euryhaline teleost, and from the eye of grouper (Epinephelus coioides), respectively. These cell lines were maintained in Leibovitz's L-15 supplemented with 20% fetal bovine serum (FBS). The SIMH cell line was subcultured more than 50 times over a period of 210 days and SIGE cell line has been subcultured 100 times over a period of 1 1/2 years. The SIMH cell line consists predominantly of fibroblastic-like cells. The SIGE cell line consists predominantly of epithelial cells. Both the cell lines were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 28 degrees C with optimum growth at the concentrations of 15% or 20% FBS. Seven marine fish viruses were tested to determine the susceptibility of these cell lines. The SIGE cell line was found to be susceptible to nodavirus, MABV NC-1 and Y6, and the infection was confirmed by cytopathic effect (CPE) and reverse transcriptase-polymerase chain reaction. When these cells were transfected with pEGFP-N1 vector DNA, significant fluorescent signals were observed, suggesting that these cell lines can be a useful tool for transgenic and genetic manipulation studies. Further, these cell lines are characterized by immunocytochemistry using confocal laser scanning microscopy (CFLSM).

[Morphological pathology of classic Kaposi's sarcoma. Ultrastructural studies and reflections on histogenesis].

PubMed

Holzhausen, H J; Stiller, D; Sachs, M

1988-01-01

Histological and electron-microscopic studies were conducted into biopsy material from cases of what is called the classical type of idiopathic Kaposi's sarcoma without acquired immunodeficiency syndrome. Ultrastructural analysis was conducted, with the view to characterizing a possible progenitor cell from which the angioblastic and fibroblastic elements were likely to originate. The biopsy material had been obtained from two males, aged 86 or 83 years, who had been afflicted with the disease for 18 or 8 years. The nodular lesions were typical of Kaposi's sarcoma and were, histologically, made up of variable mixtures of vascular and spindle cell elements. The angiomatous structures were a capillary meshwork or sinusoidal patterns lined by atypical endothelial cells. The spindle cell areas contained large numbers of slit-like spaces which were without endothelial lining but were stuffed with erythrocytes. Flattened endothelioid cells were recordable from semi-thin-sections of some clefts. Haemosiderin was, typically, deposited in places. Electron microscopically, the endothelial cells of vascular channels exhibited varying amounts of characteristic organelles, such as Weibel-Palade bodies, microfilaments and pinocytotic vesicles as well as basal membranes. Cells with typical endothelial markers, too, were detectable in solid sprouts or in capillary-like differentiations with narrow or small lumina. The spindle cell tumour areas consisted of fibroblastic cells with plenty of rough endoplasmic reticulum and surrounded by material of basal membrane nature. Also visible were solid, sprout-type multilayer cell complexes surrounded by basal membranes which exhibited undifferentiated or primitive cellular forms, endothelioid and pericytic. Transitional forms from these complexes to the above vascular tumours or the spindle-cell formations were detectable. These ultrastructural findings might be interpreted to the effect that an angioblastically determined mesenchymal cell, a so-called endothelioblast, was thinkable and was discussed as the precursor cell of atypical vascular and spindle cell proliferation in Kaposi's sarcoma.

[The distribution of B-lymphocytes in lymphoepithelial tissues as well as in tumors of the neck-, nose-, and throat region derived from lymphoreticular and lymphoepithelial tissues (author's transl)].

PubMed

Uhlmann, C; Krüger, G R; Sesterhenn, K; Wustrow, F; Fisher, R

1975-08-28

B-Lymphocytes carrying IgG-, IgM,- and IgA-surface receptors were estimated by fluorescence microscopy in the palatine tonsil of 50 patients aged 3 to 18 years as well as in 44 patients with various types of malignant lymphoms and lymphoepithelial carcinomas. Hyperplastic tonsillartissue contains large numbers of B-cells with a marked variability in concentration (4-30% IgG-cells, medium 12,9%;6-36 IgM-cells, medium 23.4%;3-38% IgA cells, medium 20.8%). There appears to exist an age-dependent increase in IgM-cells and an increase in IgG-and IgA-cells in patients with numerous recurrent infections of the upper respiratory tract. Malignant lymphomas can be grouped into three main categories: Such with a predominance of one B-cell line (above 75-80% of one immunological cell type); these include primarily malignant lymphomas of the well differentiated lymphocytic type (IgM and IgA receptors). Secondly, such with a significant decrease in B-cells (below 10%) which include primarily malignant lymphomas of the poorly differentiated lymphocytic type. Thirdly, such with an increased B-cell content but with more than one cell line participating in cell proliferation. The latter ones comprise certain cases of Hodkin's lymphomas. Lymphoepithial carcinomas are charactersized by a significant decrease in total B-cell content, except for IgE- and IgD-cells which were not investigated. The results show that the immunologic classification of malignant lymphomas correlates only to a certain degree with the morphologic classification; i.e. the same morphologic type of tumor may possess different immunologic characteristics. Since the immunologic characteristics may reflect a certain functional potential of these tumors as well as probably a certain kind of immunologic incompetence prior to tumor development, it is suggested, that future morphologic investigations of malignant lymphomas and lymphoepithelial carcinomas are combined with immunologic classifications.

Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

PubMed Central

Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

2017-01-01

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

PubMed

Povey, Jane F; O'Malley, Christopher J; Root, Tracy; Martin, Elaine B; Montague, Gary A; Feary, Marc; Trim, Carol; Lang, Dietmar A; Alldread, Richard; Racher, Andrew J; Smales, C Mark

2014-08-20

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

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

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Adventitious viruses in insect cell lines used for recombinant protein expression.

PubMed

Geisler, Christoph; Jarvis, Donald L

2018-04-01

Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines.

PubMed

Almanaa, Taghreed N; Geusz, Michael E; Jamasbi, Roudabeh J

2012-10-24

Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized. Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20-80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines. The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines. Our results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence.

Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

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

Gestl, Erin E., E-mail: egestl@wcupa.edu; Anne Boettger, S., E-mail: aboettger@wcupa.edu

2012-06-29

Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated withmore » p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53 gene expression levels compared to cell lines without p53 sequestration. Our data reveal the characteristic cytoplasmic sequestration of p53 by the heat shock protein mortalin in human colorectal adenocarcinoma cell lines, as is the case for other cancers, such as glioblastomas and hepatocellular carcinomas.« less

Seismic Reflectivity of the Crust in the Northern Salton Trough

NASA Astrophysics Data System (ADS)

Bauer, K.; Fuis, G. S.; Goldman, M.; Persaud, P.; Ryberg, T.; Langenheim, V. E.; Scheirer, D. S.; Rymer, M. J.; Hole, J. A.; Stock, J. M.; Catchings, R.

2015-12-01

The Salton Trough in southern California is a tectonically active pull-apart basin that was formed by migrating step-overs between strike-slip faults, of which the San Andreas Fault (SAF) and the Imperial Fault are the current, northernmost examples. The Salton Seismic Imaging Project (SSIP) was undertaken to improve our knowledge of fault geometry and seismic velocities within the sedimentary basins and underlying crystalline crust around the SAF. Such data are useful as input for modeling scenarios of strong ground shaking in the surrounding high-population areas. We used pre-stack depth migration of line segments from shot gathers in several seismic profiles that were acquired in the northern part of the SSIP study area (Lines 4 - 7). Our migration approach can be considered as an infinite-frequency approximation of the Fresnel volume pre-stack depth migration method. We use line segments instead of the original waveform data. We demonstrate the method using synthetic data and analyze real data from Lines 4 - 7 to illustrate the relationship between distinct phases in the time domain and their resulting image at depth. We show both normal-moveout reflections from sub-horizontal interfaces and reverse-moveout reflections from steep interfaces, such as faults. Migrated images of dipping faults, such as the SAF and the Pinto Mountain Fault, are presented in this way. The SAF is imaged along Line 4, through the Mecca Hills, as a number of steeply dipping fault segments that collectively form a flower structure, above 5 km depth, that sole into a moderately NE-dipping fault below that depth. The individual migrated reflection packages correlate with mapped surface fault traces in the Mecca Hills. A similar geometry is seen on Line 6, from Palm Springs through Yucca Valley, where fault splays sole or project into a moderately dipping SAF below 10-km depth. We also show and discuss the reflectivity pattern of the middle and lower crust for Lines 4 - 7.

Development of a new canine osteosarcoma cell line.

PubMed

Séguin, B; Zwerdling, T; McCallan, J L; DeCock, H E V; Dewe, L L; Naydan, D K; Young, A E; Bannasch, D L; Foreman, O; Kent, M S

2006-12-01

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1-deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

PubMed Central

Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D.; Ziller, Michael; Croft, Gist F.; Amoroso, Mackenzie W.; Oakley, Derek H.; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

2011-01-01

SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. PMID:21295703

New mounting improves solar-cell efficiency

NASA Technical Reports Server (NTRS)

Shepard, N. F., Jr.

1980-01-01

Method boosts output by about 20 percent by trapping and redirecting solar radiation without increasing module depth. Mounted solar-cell array is covered with internally reflecting plate. Plate is attached to each cell by transparent adhesive, and space between cells is covered with layer of diffusely reflecting material. Solar energy falling on space between cells is diffused and reflected internally by plate until it is reflected onto solar cell.

A time- and matrix-dependent TGFBR3–JUND–KRT5 regulatory circuit in single breast epithelial cells and basal-like premalignancies

PubMed Central

Wang, Chun-Chao; Bajikar, Sameer S.; Jamal, Leen; Atkins, Kristen A.; Janes, Kevin A.

2014-01-01

Basal-like breast carcinoma is characterized by poor prognosis and high intratumor heterogeneity. In an immortalized basal-like breast epithelial cell line, we identified two anti-correlated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first contains multiple TGFβ-related genes including TGFBR3, whereas the second contains JUND and the basal-like marker, KRT5. TGFBR3 and JUND interconnect through four negative-feedback loops to form a circuit that exhibits spontaneous damped oscillations in 3D culture. The TGFBR3–JUND circuit appears conserved in some premalignant lesions that heterogeneously express KRT5. The circuit depends on ECM engagement, as detachment causes a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine tenascin C, which is critical for intraductal colonization of basal-like breast cancer cells in vivo. Intratumor heterogeneity need not stem from partial differentiation and could instead reflect dynamic toggling of cells between expression states that are not cell autonomous. PMID:24658685

Luminance, Colour, Viewpoint and Border Enhanced Disparity Energy Model

PubMed Central

Martins, Jaime A.; Rodrigues, João M. F.; du Buf, Hans

2015-01-01

The visual cortex is able to extract disparity information through the use of binocular cells. This process is reflected by the Disparity Energy Model, which describes the role and functioning of simple and complex binocular neuron populations, and how they are able to extract disparity. This model uses explicit cell parameters to mathematically determine preferred cell disparities, like spatial frequencies, orientations, binocular phases and receptive field positions. However, the brain cannot access such explicit cell parameters; it must rely on cell responses. In this article, we implemented a trained binocular neuronal population, which encodes disparity information implicitly. This allows the population to learn how to decode disparities, in a similar way to how our visual system could have developed this ability during evolution. At the same time, responses of monocular simple and complex cells can also encode line and edge information, which is useful for refining disparities at object borders. The brain should then be able, starting from a low-level disparity draft, to integrate all information, including colour and viewpoint perspective, in order to propagate better estimates to higher cortical areas. PMID:26107954

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

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

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

PubMed Central

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

PubMed

Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

2017-06-01

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Measuring contact angle and meniscus shape with a reflected laser beam.

PubMed

Eibach, T F; Fell, D; Nguyen, H; Butt, H J; Auernhammer, G K

2014-01-01

Side-view imaging of the contact angle between an extended planar solid surface and a liquid is problematic. Even when aligning the view perfectly parallel to the contact line, focusing one point of the contact line is not possible. We describe a new measurement technique for determining contact angles with the reflection of a widened laser sheet on a moving contact line. We verified this new technique measuring the contact angle on a cylinder, rotating partially immersed in a liquid. A laser sheet is inclined under an angle φ to the unperturbed liquid surface and is reflected off the meniscus. Collected on a screen, the reflection image contains information to determine the contact angle. When dividing the laser sheet into an array of laser rays by placing a mesh into the beam path, the shape of the meniscus can be reconstructed from the reflection image. We verified the method by measuring the receding contact angle versus speed for aqueous cetyltrimethyl ammonium bromide solutions on a smooth hydrophobized as well as on a rough polystyrene surface.

Measuring contact angle and meniscus shape with a reflected laser beam

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

Eibach, T. F.; Nguyen, H.; Butt, H. J.

2014-01-15

Side-view imaging of the contact angle between an extended planar solid surface and a liquid is problematic. Even when aligning the view perfectly parallel to the contact line, focusing one point of the contact line is not possible. We describe a new measurement technique for determining contact angles with the reflection of a widened laser sheet on a moving contact line. We verified this new technique measuring the contact angle on a cylinder, rotating partially immersed in a liquid. A laser sheet is inclined under an angle φ to the unperturbed liquid surface and is reflected off the meniscus. Collectedmore » on a screen, the reflection image contains information to determine the contact angle. When dividing the laser sheet into an array of laser rays by placing a mesh into the beam path, the shape of the meniscus can be reconstructed from the reflection image. We verified the method by measuring the receding contact angle versus speed for aqueous cetyltrimethyl ammonium bromide solutions on a smooth hydrophobized as well as on a rough polystyrene surface.« less

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

NASA Astrophysics Data System (ADS)

Narciso, Cody; Wu, Qinfeng; Brodskiy, Pavel; Garston, George; Baker, Ruth; Fletcher, Alexander; Zartman, Jeremiah

2015-10-01

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ’s final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal-distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue.

Tunneling nanotube (TNT) formation is downregulated by cytarabine and NF-κB inhibition in acute myeloid leukemia (AML)

PubMed Central

Omsland, Maria; Bruserud, Øystein; Gjertsen, Bjørn T; Andresen, Vibeke

2017-01-01

Acute myeloid leukemia (AML) is a bone marrow derived blood cancer where intercellular communication in the leukemic bone marrow participates in disease development, progression and chemoresistance. Tunneling nanotubes (TNTs) are intercellular communication structures involved in transport of cellular contents and pathogens, also demonstrated to play a role in both cell death modulation and chemoresistance. Here we investigated the presence of TNTs by live fluorescent microscopy and identified TNT formation between primary AML cells and in AML cell lines. We found that NF-κB activity was involved in TNT regulation and formation. Cytarabine downregulated TNTs and inhibited NF-κB alone and in combination with daunorubicin, providing additional support for involvement of the NF-κB pathway in TNT formation. Interestingly, daunorubicin was found to localize to lysosomes in TNTs connecting AML cells indicating a novel function of TNTs as drug transporting devices. We conclude that TNT communication could reflect important biological features of AML that may be explored in future therapy development. PMID:27974700

Electromagnetically induced reflectance and Fano resonance in one dimensional superconducting photonic crystal

NASA Astrophysics Data System (ADS)

Athe, Pratik; Srivastava, Sanjay; Thapa, Khem B.

2018-04-01

In the present work, we demonstrate the generation of optical Fano resonance and electromagnetically induced reflectance (EIR) in one-dimensional superconducting photonic crystal (1D SPC) by numerical simulation using transfer matrix method as analysis tool. We investigated the optical response of 1D SPC structure consisting of alternate layer of two different superconductors and observed that the optical spectra of this structure exhibit two narrow reflectance peaks with zero reflectivity of sidebands. Further, we added a dielectric cap layer to this 1D SPC structure and found that addition of dielectric cap layer transforms the line shape of sidebands around the narrow reflectance peaks which leads to the formation of Fano resonance and EIR line shape in reflectance spectra. We also studied the effects of the number of periods, refractive index and thickness of dielectric cap layer on the lineshape of EIR and Fano resonances. It was observed that the amplitude of peak reflectance of EIR achieves 100% reflectance by increasing the number of periods.

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research

PubMed Central

Girard, Luc; Lockwood, William W.; Lam, Wan L.; Minna, John D.

2010-01-01

Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non–small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel. PMID:20679594

Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

PubMed

von Erichsen, J; Hecht, W; Löhberg-Gruene, C; Reinacher, M

2012-07-01

The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

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

Chiang, Chia-Ying; Cackett, Edward M.; Miller, Jon M.

Broad Fe K emission lines have been widely observed in the X-ray spectra of black hole systems as well as in neutron star systems. The intrinsically narrow Fe K fluorescent line is generally believed to be part of the reflection spectrum originating in an illuminated accretion disk which is broadened by strong relativistic effects. However, the nature of the lines in neutron star low-mass X-ray binaries (LMXBs) has been a matter of debate. We therefore obtained the longest, high-resolution X-ray spectrum of a neutron star LMXB to date with a 300 ks Chandra High Energy Transmission Grating Spectrometer (HETGS) observationmore » of Serpens X-1. The observation was taken under the “continuous clocking” mode, and thus was free of photon pile-up effects. We carry out a systematic analysis and find that the blurred reflection model fits the Fe line of Serpens X-1 significantly better than a broad Gaussian component does, implying that the relativistic reflection scenario is much preferred. Chandra HETGS also provides a highest spectral resolution view of the Fe K region and we find no strong evidence for additional narrow lines.« less

Analysis of folded pulse forming line operation.

PubMed

Domonkos, M T; Watrous, J; Parker, J V; Cavazos, T; Slenes, K; Heidger, S; Brown, D; Wilson, D

2014-09-01

A compact pulse forming line (CPFL) concept based on a folded transmission line and high-breakdown strength dielectric was explored through an effort combining proof-of-principle experiments with electromagnetic modeling. A small-scale folded CPFL was fabricated using surface-mount ceramic multilayer capacitors. The line consisted of 150 capacitors close-packed in parallel and delivered a 300 ns flat-top pulse. The concept was carried to a 10 kV class device using a polymer-ceramic nanocomposite dielectric with a permittivity of 37.6. The line was designed for a 161 ns FWHM length pulse into a matched load. The line delivered a 110 ns FWHM pulse, and the pulse peak amplitude exceeded the matched load ideal. Transient electromagnetic analysis using the particle-in-cell code ICEPIC was conducted to examine the nature of the unexpected pulse shortening and distortion. Two-dimensional analysis failed to capture the anomalous behavior. Three-dimensional analysis replicated the pulse shape and revealed that the bends were largely responsible for the pulse shortening. The bends not only create the expected reflection of the incident TEM wave but also produce a non-zero component of the Poynting vector perpendicular to the propagation direction of the dominant electromagnetic wave, resulting in power flow largely external to the PFL. This analysis explains both the pulse shortening and the amplitude of the pulse.

Analysis of folded pulse forming line operation

NASA Astrophysics Data System (ADS)

Domonkos, M. T.; Watrous, J.; Parker, J. V.; Cavazos, T.; Slenes, K.; Heidger, S.; Brown, D.; Wilson, D.

2014-09-01

A compact pulse forming line (CPFL) concept based on a folded transmission line and high-breakdown strength dielectric was explored through an effort combining proof-of-principle experiments with electromagnetic modeling. A small-scale folded CPFL was fabricated using surface-mount ceramic multilayer capacitors. The line consisted of 150 capacitors close-packed in parallel and delivered a 300 ns flat-top pulse. The concept was carried to a 10 kV class device using a polymer-ceramic nanocomposite dielectric with a permittivity of 37.6. The line was designed for a 161 ns FWHM length pulse into a matched load. The line delivered a 110 ns FWHM pulse, and the pulse peak amplitude exceeded the matched load ideal. Transient electromagnetic analysis using the particle-in-cell code ICEPIC was conducted to examine the nature of the unexpected pulse shortening and distortion. Two-dimensional analysis failed to capture the anomalous behavior. Three-dimensional analysis replicated the pulse shape and revealed that the bends were largely responsible for the pulse shortening. The bends not only create the expected reflection of the incident TEM wave but also produce a non-zero component of the Poynting vector perpendicular to the propagation direction of the dominant electromagnetic wave, resulting in power flow largely external to the PFL. This analysis explains both the pulse shortening and the amplitude of the pulse.

Attempt to develop taste bud models in three-dimensional culture.

PubMed

Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

2011-09-01

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

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

Blattmann, Claudia, E-mail: claudia.blattmann@med.uni-heidelberg.d; Oertel, Susanne; Ehemann, Volker

2010-09-01

Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced anmore » inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.« less

Effect of small-scale biomass gasification at the state of refractory lining the fixed bed reactor

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

Janša, Jan, E-mail: jan.jansa@vsb.cz; Peer, Vaclav, E-mail: vaclav.peer@vsb.cz; Pavloková, Petra, E-mail: petra.pavlokova@vsb.cz

The article deals with the influence of biomass gasification on the condition of the refractory lining of a fixed bed reactor. The refractory lining of the gasifier is one part of the device, which significantly affects the operational reliability and durability. After removing the refractory lining of the gasifier from the experimental reactor, there was done an assessment how gasification of different kinds of biomass reflected on its condition in terms of the main factors affecting its life. Gasification of biomass is reflected on the lining, especially through sticking at the bottom of the reactor. Measures for prolonging the lifemore » of lining consist in the reduction of temperature in the reactor, in this case, in order to avoid ash fusion biomass which it is difficult for this type of gasifier.« less

Generation of genome-modified Drosophila cell lines using SwAP.

PubMed

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

PubMed

Santoso, D; Thornburg, R

2000-08-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Fluoroorotic Acid-Selected Nicotiana plumbaginifolia Cell Lines with a Stable Thymine Starvation Phenotype Have Lost the Thymine-Regulated Transcriptional Program1

PubMed Central

Santoso, Djoko; Thornburg, Robert

2000-01-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. PMID:10938367

The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer.

PubMed

Yu, Sungryul; Kim, Taemook; Yoo, Kyung Hyun; Kang, Keunsoo

2017-05-06

Cell lines are often used as in vitro tools to mimic certain types of in vivo system; several cell lines, including MCF-7 and T47D, have been widely used in breast cancer studies without investigating the cell lines' characteristics. In this study, we compared the genome-wide binding profiles of ERα, PR, and P300, and the gene expression changes between MCF-7 and T47D cell lines that represent the luminal A subtype of breast cancer. Surprisingly, several thousand genes were differentially expressed under estrogenic condition. In addition, ERα, PR, and P300 binding to regulatory elements showed distinct genomic landscapes between MCF-7 and T47D cell lines in the same hormonal states. In particular, the T47D cell line was markedly susceptible to progesterone, whereas the MCF-7 cell line did not respond to progesterone in the presence of estrogen. Consistently, changes in the expression level of the PR-target gene, STAT5A, were only observed in the T47D cell line, not the MCF-7 cell line, when treated with progesterone. Overall, the results highlight the importance of selecting appropriate cell lines for breast cancer studies and suggest that T47D cell lines can be an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

PubMed

Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines.

PubMed

Madhusudana, Shampur Narayan; Sundaramoorthy, Subha; Ullas, Padinjaremattatthil Thankappan

2010-12-01

A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research. Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-10-01

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior

PubMed Central

Tomiyasu, Hirotaka; Garbe, John R.; Cornax, Ingrid; Amaya, Clarissa; O'Sullivan, M. Gerard; Subramanian, Subbaya

2016-01-01

ABSTRACT Osteosarcoma (OS) is a heterogeneous and rare disease with a disproportionate impact because it mainly affects children and adolescents. Lamentably, more than half of patients with OS succumb to metastatic disease. Clarification of the etiology of the disease, development of better strategies to manage progression, and methods to guide personalized treatments are among the unmet health needs for OS patients. Progress in managing the disease has been hindered by the extreme heterogeneity of OS; thus, better models that accurately recapitulate the natural heterogeneity of the disease are needed. For this study, we used cell lines derived from two spontaneous canine OS tumors with distinctly different biological behavior (OS-1 and OS-2) for heterotypic in vivo modeling that recapitulates the heterogeneous biology and behavior of this disease. Both cell lines demonstrated stability of the transcriptome when grown as orthotopic xenografts in athymic nude mice. Consistent with the behavior of the original tumors, OS-2 xenografts grew more rapidly at the primary site and had greater propensity to disseminate to lung and establish microscopic metastasis. Moreover, OS-2 promoted formation of a different tumor-associated stromal environment than OS-1 xenografts. OS-2-derived tumors comprised a larger percentage of the xenograft tumors than OS-1-derived tumors. In addition, a robust pro-inflammatory population dominated the stromal cell infiltrates in OS-2 xenografts, whereas a mesenchymal population with a gene signature reflecting myogenic signaling dominated those in the OS-1 xenografts. Our studies show that canine OS cell lines maintain intrinsic features of the tumors from which they were derived and recapitulate the heterogeneous biology and behavior of bone cancer in mouse models. This system provides a resource to understand essential interactions between tumor cells and the stromal environment that drive the progression and metastatic propensity of OS. PMID:27874835

Heterotypic mouse models of canine osteosarcoma recapitulate tumor heterogeneity and biological behavior.

PubMed

Scott, Milcah C; Tomiyasu, Hirotaka; Garbe, John R; Cornax, Ingrid; Amaya, Clarissa; O'Sullivan, M Gerard; Subramanian, Subbaya; Bryan, Brad A; Modiano, Jaime F

2016-12-01

Osteosarcoma (OS) is a heterogeneous and rare disease with a disproportionate impact because it mainly affects children and adolescents. Lamentably, more than half of patients with OS succumb to metastatic disease. Clarification of the etiology of the disease, development of better strategies to manage progression, and methods to guide personalized treatments are among the unmet health needs for OS patients. Progress in managing the disease has been hindered by the extreme heterogeneity of OS; thus, better models that accurately recapitulate the natural heterogeneity of the disease are needed. For this study, we used cell lines derived from two spontaneous canine OS tumors with distinctly different biological behavior (OS-1 and OS-2) for heterotypic in vivo modeling that recapitulates the heterogeneous biology and behavior of this disease. Both cell lines demonstrated stability of the transcriptome when grown as orthotopic xenografts in athymic nude mice. Consistent with the behavior of the original tumors, OS-2 xenografts grew more rapidly at the primary site and had greater propensity to disseminate to lung and establish microscopic metastasis. Moreover, OS-2 promoted formation of a different tumor-associated stromal environment than OS-1 xenografts. OS-2-derived tumors comprised a larger percentage of the xenograft tumors than OS-1-derived tumors. In addition, a robust pro-inflammatory population dominated the stromal cell infiltrates in OS-2 xenografts, whereas a mesenchymal population with a gene signature reflecting myogenic signaling dominated those in the OS-1 xenografts. Our studies show that canine OS cell lines maintain intrinsic features of the tumors from which they were derived and recapitulate the heterogeneous biology and behavior of bone cancer in mouse models. This system provides a resource to understand essential interactions between tumor cells and the stromal environment that drive the progression and metastatic propensity of OS. © 2016. Published by The Company of Biologists Ltd.

Collective Lamb Shift of a Nanoscale Atomic Vapor Layer within a Sapphire Cavity

NASA Astrophysics Data System (ADS)

Peyrot, T.; Sortais, Y. R. P.; Browaeys, A.; Sargsyan, A.; Sarkisyan, D.; Keaveney, J.; Hughes, I. G.; Adams, C. S.

2018-06-01

We measure the near-resonant transmission of light through a dense medium of potassium vapor confined in a cell with nanometer thickness in order to investigate the origin and validity of the collective Lamb shift. A complete model including the multiple reflections in the nanocell reproduces accurately the observed line shape. It allows the extraction of a density-dependent shift and width of the bulk atomic medium resonance, deconvolved from the cavity effect. We observe an additional, unexpected dependence of the shift with the thickness of the medium. This extra dependence demands further experimental and theoretical investigations.

[Gas pipeline leak detection based on tunable diode laser absorption spectroscopy].

PubMed

Zhang, Qi-Xing; Wang, Jin-Jun; Liu, Bing-Hai; Cai, Ting-Li; Qiao, Li-Feng; Zhang, Yong-Ming

2009-08-01

The principle of tunable diode laser absorption spectroscopy and harmonic detection technique was introduced. An experimental device was developed by point sampling through small multi-reflection gas cell. A specific line near 1 653. 7 nm was targeted for methane measurement using a distributed feedback diode laser as tunable light source. The linearity between the intensity of second harmonic signal and the concentration of methane was determined. The background content of methane in air was measured. The results show that gas sensors using tunable diode lasers provide a high sensitivity and high selectivity method for city gas pipeline leak detection.

Bifidobacterium breve - HT-29 cell line interaction: modulation of TNF-α induced gene expression.

PubMed

Boesten, R J; Schuren, F H J; Willemsen, L E M; Vriesema, A; Knol, J; De Vos, W M

2011-06-01

To provide insight in the molecular basis for intestinal host-microbe interactions, we determined the genome-wide transcriptional response of human intestinal epithelial cells following exposure to cells of Bifidobacterium breve. To select an appropriate test system reflecting inflammatory conditions, the responsiveness to TNF-α was compared in T84, Caco-2 and HT-29 cells. The highest TNF-α response was observed in HT-29 cells and this cell line was selected for exposure to the B. breve strains M-16V, NR246 and UCC2003. After one hour of bacterial pre-incubation followed by two hours of additional TNF-α stimulation, B. breve M-16V (86%), but to a much lesser extent strains NR246 (50%) or UCC2003 (32%), showed a strain-specific reduction of the HT-29 transcriptional response to the inflammatory treatment. The most important functional groups of genes that were transcriptionally suppressed by the presence of B. breve M-16V, were found to be involved in immune regulation and apoptotic processes. About 54% of the TNF-α induced genes were solely suppressed by the presence of B. breve M-16V. These included apoptosis-related cysteine protease caspase 7 (CASP7), interferon regulatory factor 3 (IRF3), amyloid beta (A4) precursor proteinbinding family A member 1 (APBA1), NADPH oxidase (NOX5), and leukemia inhibitory factor receptor (LIFR). The extracellular IL-8 concentration was determined by an immunological assay but did not change significantly, indicating that B. breve M-16V only partially modulates the TNF-α pathway. In conclusion, this study shows that B. breve strains modulate gene expression in HT-29 cells under inflammatory conditions in a strain-specific way.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-12

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape.

Defining the Enterovirus Diversity Landscape of a Fecal Sample: A Methodological Challenge?

PubMed Central

Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Adeniji, Johnson Adekunle

2016-01-01

Enteroviruses are a group of over 250 naked icosahedral virus serotypes that have been associated with clinical conditions that range from intrauterine enterovirus transmission withfataloutcome through encephalitis and meningitis, to paralysis. Classically, enterovirus detection was done by assaying for the development of the classic enterovirus-specific cytopathic effect in cell culture. Subsequently, the isolates were historically identified by a neutralization assay. More recently, identification has been done by reverse transcriptase-polymerase chain reaction (RT-PCR). However, in recent times, there is a move towards direct detection and identification of enteroviruses from clinical samples using the cell culture-independent RT semi-nested PCR (RT-snPCR) assay. This RT-snPCR procedure amplifies the VP1 gene, which is then sequenced and used for identification. However, while cell culture-based strategies tend to show a preponderance of certain enterovirus species depending on the cell lines included in the isolation protocol, the RT-snPCR strategies tilt in a different direction. Consequently, it is becoming apparent that the diversity observed in certain enterovirus species, e.g., enterovirus species B(EV-B), might not be because they are the most evolutionarily successful. Rather, it might stem from cell line-specific bias accumulated over several years of use of the cell culture-dependent isolation protocols. Furthermore, it might also be a reflection of the impact of the relative genome concentration on the result of pan-enterovirus VP1 RT-snPCR screens used during the identification of cell culture isolates. This review highlights the impact of these two processes on the current diversity landscape of enteroviruses and the need to re-assess enterovirus detection and identification algorithms in a bid to better balance our understanding of the enterovirus diversity landscape. PMID:26771630

[Establishment of Z-HL16C cell line.].

PubMed

Chen, J P; Li, J; Zhao, S L; Tian, J Y; Ye, F

2006-09-01

To establish and study the nature and the application of Z-HL16C cell line. The cell line was continuously passed, frozen stored and recovered. Its application was expanded and the cell type was identified. The cell line had an epithelial-cell-like shape, the size appeared uniform, the cell boundary was distinct. It has been continuously passed, frozen stored and recovered for ten years. Its recovery rate was about 90%. It has been proved to be sensitive to the tested viruses which were enteroviruses (Polio, Cox, Echo), influenza viruses, parainfluenzaviruses, adenoviruses, measles virus. This cell line has been identified as a cancerization cell. The cell line Z-HL16C has been stably established, it has a broad spectrum in sensitivity for culturing viruses.

Anti-cancer activity of withaferin A in B-cell lymphoma

PubMed Central

McKenna, MK; Gachuki, BW; Alhakeem, SS; Oben, KN; Rangnekar, VM; Gupta, RC; Bondada, S

2015-01-01

Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90. PMID:26020511

Anti-cancer activity of withaferin A in B-cell lymphoma.

PubMed

McKenna, M K; Gachuki, B W; Alhakeem, S S; Oben, K N; Rangnekar, V M; Gupta, R C; Bondada, S

2015-01-01

Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90.

Inhibition of Excessive Monoamine Oxidase A/B Activity Protects Against Stress-induced Neuronal Death in Huntington Disease.

PubMed

Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A

2015-12-01

Monoamine oxidases (MAO) are important components of the homeostatic machinery that maintains the levels of monoamine neurotransmitters, including dopamine, in balance. Given the imbalance in dopamine levels observed in Huntington disease (HD), the aim of this study was to examine MAO activity in a mouse striatal cell model of HD and in human neural cells differentiated from control and HD patient-derived induced pluripotent stem cell (hiPSC) lines. We show that mouse striatal neural cells expressing mutant huntingtin (HTT) exhibit increased MAO expression and activity. We demonstrate using luciferase promoter assays that the increased MAO expression reflects enhanced epigenetic activation in striatal neural cells expressing mutant HTT. Using cellular stress paradigms, we further demonstrate that the increase in MAO activity in mutant striatal neural cells is accompanied by enhanced susceptibility to oxidative stress and impaired viability. Treatment of mutant striatal neural cells with MAO inhibitors ameliorated oxidative stress and improved cellular viability. Finally, we demonstrate that human HD neural cells exhibit increased MAO-A and MAO-B expression and activity. Altogether, this study demonstrates abnormal MAO expression and activity and suggests a potential use for MAO inhibitors in HD.

Development and characterization of a cell line WAF from freshwater shark Wallago attu.

PubMed

Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Sharma, Bhagwati S

2014-02-01

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

PubMed

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

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

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Acinar cell cystadenoma of the pancreas: a benign neoplasm or non-neoplastic ballooning of acinar and ductal epithelium?

PubMed

Singhi, Aatur D; Norwood, Stephanie; Liu, Ta-Chiang; Sharma, Rajni; Wolfgang, Christopher L; Schulick, Richard D; Zeh, Herbert J; Hruban, Ralph H

2013-09-01

Acinar cell cystadenoma (ACA) of the pancreas was initially described as a non-neoplastic cyst of the pancreas and, at that time, referred to as "acinar cystic transformation." In subsequent studies, these lesions were given the designation of "-oma," despite the relative lack of evidence supporting a neoplastic process. To characterize these lesions further, we examined the clinical, pathologic, and immunohistochemical features of 8 ACAs. The majority of patients were female (7 of 8, 88%) and ranged in age from 18 to 57 years (mean, 43 y). Grossly, the cysts involved the head (n=5), body (n=1), or the entire pancreas (n=2). ACAs were either multilocular (n=4) or unilocular (n=4) and ranged in size from 1.8 to 15 cm (mean, 6.8 cm). Histologically, multilocular ACAs were lined by patches of acinar and ductal epithelium. Immunolabeling, including double-labeling for cytokeratin 19 and chymotrypsin, highlighted the patchy pattern of the ductal and acinar cells lining the cysts. In some areas, the cysts with patches of acinar and ductal differentiation formed larger locules with incomplete septa as they appeared to fuse with other cysts. In contrast, the unilocular cases were lined by 1 to 2 cell layers of acinar cells with little intervening ductal epithelium. Nuclear atypia, mitotic figures, necrosis, infiltrative growth, and associated invasive carcinoma were absent in all cases. In addition, we assessed the clonal versus polyclonal nature of ACAs, occurring in women, using X-chromosome inactivation analysis of the human androgen receptor (AR) gene. Five of 7 cases were informative and demonstrated a random X-chromosome inactivation pattern. Clinical follow-up information was available for all patients, and follow-up ranged from 10 months to 7.8 years (mean, 3.6 y), with no evidence of recurrence or malignant transformation. We hypothesize that early lesions are marked by acinar dilatation that expands into and incorporates smaller ductules and later larger ducts. As the cysts increase in size, they fuse forming larger cysts. Later lesions demonstrate a unilocular cyst lined by predominantly acinar epithelium with scattered ductal cells. The term cystadenoma, with its neoplastic connotation, does not seem to accurately reflect the histologic, immunohistochemical, or molecular features of these lesions. We suggest readopting the term "acinar cystic transformation" until the non-neoplastic versus neoplastic origin of these lesions can be resolved.

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

USDA-ARS?s Scientific Manuscript database

The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Reflector system for a lighting fixture

DOEpatents

Siminovitch, Michael J.; Page, Erik; Gould, Carl T.

1998-01-01

Disclosed herein is a reflector system for a lighting fixture having a illumination source surrounded by an envelope. The reflector system includes a first reflector surrounding the illumination source. The reflector system also includes a second reflector which is non-contiguous with the first reflector and which surrounds the illumination source. The illumination source creates light rays which are reflected by the first and second reflectors. The first reflector directs light rays toward the center line of the fixture. However, the reflected rays despite being so reflected do not substantially intersect the envelope. The reflected light rays from the second reflector being directed so that they diverge from the center line of the fixture avoiding intersection with the semi-transparent envelope.

Reflector system for a lighting fixture

DOEpatents

Siminovitch, Michael J.; Page, Erik; Gould, Carl T.

2001-01-01

Disclosed herein is a reflector system for a lighting fixture having a illumination source surrounded by an envelope. The reflector system includes a first reflector surrounding the illumination source. The reflector system also includes a second reflector which is non-contiguous with the first reflector and which surrounds the illumination source. The illumination source creates light rays which are reflected by the first and second reflectors. The first reflector directs light rays toward the center line of the fixture. However, the reflected rays despite being so reflected do not substantially intersect the envelope. The reflected light rays from the second reflector being directed so that they diverge from the center line of the fixture avoiding intersection with the semi-transparent envelope.

An Automated Method to Monitor Cell Migration.

NASA Astrophysics Data System (ADS)

Giaever, Ivar; Keese, Charles R.

2002-03-01

Electric cell-substrate impedance sensing (ECIS) has been developed as a non-invasive means to follow cell behavior in culture. In this method cells are cultured on small (250 micrometer diameter) gold film electrodes. The impedance of the electrode is measured by an AC current about 1 microampere. When challenged by biochemical or physical stimuli the cells will respond by changing their morphology and motion. These changes are reflected in the measured impedance values. In this study, the basic ECIS system was used in both a non-invasive and invasive mode to carry out an automated wound-healing assay for quantifying cell migration activity. BSC-1, MDCK, and NRK cell lines were grown to confluence in ECIS wells before data was collected. An AC current of approximately 1 milliampere at 40,000 Hz was applied for several seconds, killing the cells in contact with the ECIS electrode and dropping the impedance to that of a cell-free electrode. For the next few hours following this incursion, the neighboring cells migrate into the wounded area replacing the dead cells, and the electrodes return to impedance values of unwounded controls. Data shows that the time required for the completion of this activity is strongly dependent upon cell type, medium composition, and the type of protein adsorbed to the substrate.

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

PubMed Central

ASADA, Hajime; TOMIYASU, Hirotaka; GOTO-KOSHINO, Yuko; FUJINO, Yasuhito; OHNO, Koichi; TSUJIMOTO, Hajime

2015-01-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS. PMID:25715778

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.

PubMed

Asada, Hajime; Tomiyasu, Hirotaka; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

2015-06-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.

Genetic Profiling Reveals Cross-Contamination and Misidentification of 6 Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS and CAC2

PubMed Central

Phuchareon, Janyaporn; Ohta, Yoshihito; Woo, Jonathan M.; Eisele, David W.; Tetsu, Osamu

2009-01-01

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells. PMID:19557180

Directional Reflective Surface Formed via Gradient-Impeding Acoustic Meta-Surfaces

PubMed Central

Song, Kyungjun; Kim, Jedo; Hur, Shin; Kwak, Jun-Hyuk; Lee, Seong-Hyun; Kim, Taesung

2016-01-01

Artificially designed acoustic meta-surfaces have the ability to manipulate sound energy to an extraordinary extent. Here, we report on a new type of directional reflective surface consisting of an array of sub-wavelength Helmholtz resonators with varying internal coiled path lengths, which induce a reflection phase gradient along a planar acoustic meta-surface. The acoustically reshaped reflective surface created by the gradient-impeding meta-surface yields a distinct focal line similar to a parabolic cylinder antenna, and is used for directive sound beamforming. Focused beam steering can be also obtained by repositioning the source (or receiver) off axis, i.e., displaced from the focal line. Besides flat reflective surfaces, complex surfaces such as convex or conformal shapes may be used for sound beamforming, thus facilitating easy application in sound reinforcement systems. Therefore, directional reflective surfaces have promising applications in fields such as acoustic imaging, sonic weaponry, and underwater communication. PMID:27562634

Continuous human cell lines and method of making same

DOEpatents

Stampfer, Martha R.

1989-01-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

PubMed

Zhu, Bing; Guo, Zhili; Jin, Muzi; Bai, Yujuan; Yang, Wenliang; Hui, Lihua

2018-05-01

To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA) + /cytokeratin 13 + , while the dermal papilla cells were α-SMA + /cytokeratin 13 - and the outer root sheath cells were α-SMA - /cytokeratin 13 + . Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed

Laferté, S; Loh, L C

1992-04-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours.

Dual-Color Fluorescence Imaging to Monitor CYP3A4 and CYP3A7 Expression in Human Hepatic Carcinoma HepG2 and HepaRG Cells

PubMed Central

Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps. PMID:25101946

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).

Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

PubMed

Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

2015-01-01

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

Synergistic anticancer effects of cisplatin and histone deacetylase inhibitors (SAHA and TSA) on cholangiocarcinoma cell lines.

PubMed

Asgar, Md Ali; Senawong, Gulsiri; Sripa, Banchob; Senawong, Thanaset

2016-01-01

Clinical application of cisplatin against cholangiocarcinoma is often associated with resistance and toxicity posing urgent demand for combination therapy. In this study, we evaluated the combined anticancer effect of cisplatin and histone deacetylase inhibitors (HDACIs), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on the cholangiocarcinoma KKU-100 and KKU-M214 cell lines. Antiproliferative activity was evaluated using MTT assay. Apoptosis induction and cell cycle arrest were analyzed by flow cytometry. Cell cycle and apoptosis regulating proteins were evaluated by western blot analysis. MTT assay showed that cisplatin, SAHA and TSA dose-dependently reduced the viability of KKU-100 and KKU-M214 cells. The combination of cisplatin and HDACIs exerted significantly more cytotoxicity than the single drugs. Combination indices below 1.0 reflect synergism between cisplatin and HDACIs, leading to positive dose reductions of cisplatin and HDACIs. Cisplatin and HDACIs alone induced G0/G1 phase arrest in KKU-100 cells, but the drug combinations increased sub-G1 percent more than either drug. However, cisplatin and HDACIs alone or in combination increased only the sub-G1 percent in KKU-M214 cells. Annexin V-FITC staining revealed that cisplatin and HDACIs combinations induced more apoptotic cell death of both KKU-100 and KKU-M214 cells than the single drug. In KKU-100 cells, growth inhibition was accompanied by upregulation of p53 and p21 and downregulation of CDK4 and Bcl-2 due to exposure to cisplatin, SAHA and TSA alone or in combination. Moreover, combination of agents exerted higher impacts on protein expression. Single agents or combination did not affect p53 expression, however, combination of cisplatin and HDACIs increased the expression of p21 in KKU-M214 cells. Taken together, cisplatin and HDACIs combination may improve the therapeutic outcome in cholangiocarcinoma patients.

Notch-1 regulates pulmonary neuroendocrine cell differentiation in cell lines and in transgenic mice.

PubMed

Shan, Lin; Aster, Jon C; Sklar, Jeffrey; Sunday, Mary E

2007-02-01

The notch gene family encodes transmembrane receptors that regulate cell differentiation by interacting with surface ligands on adjacent cells. Previously, we demonstrated that tumor necrosis factor-alpha (TNF) induces neuroendocrine (NE) cell differentiation in H82, but not H526, undifferentiated small cell lung carcinoma lines. We now test the hypothesis that TNF mediates NE cell differentiation in part by altering Notch gene expression. First, using RT-PCR, we determined that TNF treatment of H82, but not H526, transiently decreases notch-1 mRNA in parallel with induction of gene expression for the NE-specific marker DOPA decarboxylase (DDC). Second, we treated H82 and H526 with notch-1 antisense vs. sense oligodeoxynucleotides. Using quantitative RT-PCR and Western analyses we demonstrate that DDC mRNA and protein are increased in H82 by notch-1 antisense, whereas notch-1 mRNA and activated Notch-1 protein are decreased. mRNA for Hes1, a transcription factor downstream from activated Notch, is also decreased by Notch-1 antisense in H82 but not H526. After 7 days of Notch-1 antisense treatment, neural cell adhesion molecule (NCAM) immunoreactivity is induced in H82 but not H526. Third, we generated transgenic mice bearing notch-1 driven by the neural/NE-specific calcitonin promoter, which express activated Notch-1 in developing lung epithelium. Newborn NotchCal mouse lungs have high levels of hes1 mRNA, reflecting increased activated Notch, compared with wild-type. NotchCal lungs have decreased CGRP-positive NE cells, decreased protein gene product 9.5 (PGP9.5)-positive NE cells, and decreased gastrin-releasing peptide (GRP), CGRP, and DDC mRNA levels compared with normal littermates. Cumulatively, these observations provide further support for a role for Notch-1 signaling in regulating pulmonary NE cell differentiation.

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

PubMed

Tsuji, Saori; Kawamura, Fumihiko; Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

PubMed Central

2010-01-01

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486

Molecular characterization of breast cancer cell lines through multiple omic approaches.

PubMed

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways. These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results.

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

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

PubMed Central

Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Becet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub O.; Ladewig, Erik; Dai, Qi; Okamura, Katsutomo; Flynt, Alex S.; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C.; Cherbas, Peter; Siepel, Adam; Lai, Eric C.

2014-01-01

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage. PMID:24985917

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

The use of human tumour cell lines in the discovery of new cancer chemotherapeutic drugs.

PubMed

Baguley, Bruce C; Marshall, Elaine S

2008-02-01

Human tumour cell lines have played a major role in anticancer drug discovery, but cell lines may model only some aspects of tumour behaviour in cancer patients. Growing evidence supports a theory that stem cells with self-renewing properties sustain tumours. This review considers the extent to which a deeper understanding of the origin and properties of tumour cell lines might lead to new strategies for anticancer drug discovery. Recent literature on normal and tumour stem cells is reviewed and placed in the context of a discussion on the derivation and properties of tumour cell lines. Early-passage cell lines may model the more rapidly proliferating cells in human tumours and, thus, retain some of the properties of tumour stem cells. The effects of anticancer drugs on cell lines should be considered not only with regards to the induction of apoptosis, but also to the induction of senescence or other pathways that lead to host immune and inflammatory responses.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

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

Belakavadi, Madesh; Prabhakar, B.T.; Salimath, Bharathi P.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptoticmore » effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.« less

Replication of Heliothis virescens ascovirus in insect cell lines.

PubMed

Asgari, S

2006-09-01

Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

Gamma-ray mirror technology for NDA of spent fuel

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

Descalle, M. A.; Ruz-Armendariz, J.; Decker, T.

Direct measurements of gamma rays emitted by fissile material have been proposed as an alternative to measurements of the gamma rays from fission products. From a safeguards applications perspective, direct detection of uranium (U) and plutonium (Pu) K-shell fluorescence emission lines and specific lines from some of their isotopes could lead to improved shipper-receiver difference or input accountability at the start of Pu reprocessing. However, these measurements are difficult to implement when the spent fuel is in the line-of-sight of the detector, as the detector is exposed to high rates dominated by fission product emissions. To overcome the combination ofmore » high rates and high background, grazing incidence multilayer mirrors have been proposed as a solution to selectively reflect U and Pu hard X-ray and soft gamma rays in the 90 to 420 keV energy into a high-purity germanium (HPGe) detector shielded from the direct line-of-sight of spent fuel. Several groups demonstrated that K-shell fluorescence lines of U and Pu in spent fuel could be detected with Ge detectors. In the field of hard X-ray optics the performance of reflective multilayer coated reflective optics was demonstrated up to 645 keV at the European Synchrotron Radiation Facility. Initial measurements conducted at Oak Ridge National Laboratory with sealed sources and scoping experiments conducted at the ORNL Irradiated Fuels Examination Laboratory (IFEL) with spent nuclear fuel further demonstrated the pass-band properties of multilayer mirrors for reflecting specific emission lines into 1D and 2D HPGe detectors, respectively.« less

Transcriptional Inducers of Acetylcholinesterase Expression as Novel Antidotes for Protection Against Chemical Warfare Agents

DTIC Science & Technology

2005-10-01

neuroblastoma cell line , P19 and a human neuroblastoma cell line SH - SY5Y (data not shown). Effect of trichostatin A on...mouse neuroblastoma P19 cell line and a human neuroblastoma cell line SH - SY5Y . More experiments are needed to prove the potential of AChE expression in...treatment of nerve agent exposure. MATERIALS AND METHODS Neuronal cell lines and

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines

PubMed Central

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines. PMID:18927105

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

PubMed

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

DTIC Science & Technology

2007-11-01

cells ( gray fill), cells preincubated with PBS and infected with virus (solid line), and cells preincubated with recombinant knob protein and incubated...U118-hCAR-tailless cells (b). Gray line¼ cells alone, solid line¼ cells+Ad5Luc1-CK1, dashed line¼ cells+Ad5Luc1. Figure 5 Ad5Luc1-CK1CAR-independent...line, whereas U118MG-hCAR-tailless stably expresses the extracellular domain of human CAR. Cells were infected with Ad5Luc1 ( gray bar) and Ad5-r1 (black

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

PubMed Central

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Assembly And Initial Characterization Of A Panel Of 85 Genomically Validated Cell Lines From Diverse Head And Neck Tumor Sites

PubMed Central

Zhao, Mei; Sano, Daisuke; Pickering, Curtis R.; Jasser, Samar A.; Henderson, Ying C.; Clayman, Gary L.; Sturgis, Erich M.; Ow, Thomas J.; Lotan, Reuben; Carey, Thomas E.; Sacks, Peter G.; Grandis, Jennifer R.; Sidransky, David; Heldin, Nils Erik; Myers, Jeffrey N.

2011-01-01

Purpose Human cell lines are useful for studying cancer biology and pre-clinically modeling cancer therapy, but can be misidentified and cross contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma (HNSCC), thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium, was assembled from the collections of several individuals and institutions. Authenticity was verified by performing short tandem repeat (STR) analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination demonstrates a wide range of in vitro phenotypes. Conclusion This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be utilized for biological as well as preclinical studies. PMID:21868764

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

PubMed

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

PubMed Central

Yu, Channing; Mannan, Aristotle M.; Yvone, Griselda Metta; Ross, Kenneth N.; Zhang, Yan-Ling; Marton, Melissa A.; Taylor, Bradley R.; Crenshaw, Andrew; Gould, Joshua Z.; Tamayo, Pablo; Weir, Barbara A.; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A.; Shamji, Alykhan F.; Palmer, Michelle A.; Foley, Michael A.; Winckler, Wendy; Schreiber, Stuart L.; Kung, Andrew L.; Golub, Todd R.

2016-01-01

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control1-4. Here, we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM displayed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo. PMID:26928769

Inhibition of melanoma development in the Nras((Q61K)) ::Ink4a(-/-) mouse model by the small molecule BI-69A11.

PubMed

Feng, Yongmei; Lau, Eric; Scortegagna, Marzia; Ruller, Chelsea; De, Surya K; Barile, Elisa; Krajewski, Stan; Aza-Blanc, Pedro; Williams, Roy; Pinkerton, Anthony B; Jackson, Michael; Chin, Lynda; Pellecchia, Maurizio; Bosenberg, Marcus; Ronai, Ze'ev A

2013-01-01

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15-20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI-69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon- and cell death-related genes that were associated with responsiveness of melanoma cell lines to BI-69A11. Strikingly, the administration of BI-69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras((Q61K)) ::Ink4a(-/-) ). Biweekly administration of BI-69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI-69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI-69A11-resistant Nras((Q61K)) ::Ink4a(-/-) tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI-69A11 for clinical assessment. © 2012 John Wiley & Sons A/S.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

USGS Publications Warehouse

Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

2014-01-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

PubMed

Soni, Pankaj; Pradhan, Pravata K; Swaminathan, T R; Sood, Neeraj

2018-06-01

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32° C, with an optimum temperature of 28° C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

PubMed

de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

2017-03-01

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. © 2015 John Wiley & Sons Ltd.

The transcriptional diversity of 25 Drosophila cell lines

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

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signalingmore » pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. In addition, we offer an initial analysis of previously unannotated transcripts in the cell lines.« less

BHD Tumor Cell Line and Renal Cell Carcinoma Line | NCI Technology Transfer Center | TTC

Cancer.gov

Scientists at the National Cancer Institute  have developed a novel renal cell carcinoma (RCC) cell line designated UOK257, which was derived from the surgical kidney tissue of a patient with hereditary Birt-Hogg-Dube''''(BHD) syndrome and companion cell line UOK257-2 in which FLCN expression has been restored by lentivirus infection. The NCI Urologic Oncology Branch seeks parties interested in licensing or collaborative research to co-develop, evaluate, or commercialize kidney cancer tumor cell lines.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1998-01-01

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

Immortalization capacity of HPV types is inversely related to chromosomal instability.

PubMed

Schütze, Denise M; Krijgsman, Oscar; Snijders, Peter J F; Ylstra, Bauke; Weischenfeldt, Joachim; Mardin, Balca R; Stütz, Adrian M; Korbel, Jan O; de Winter, Johan P; Meijer, Chris J L M; Quint, Wim G V; Bosch, Leontien; Wilting, Saskia M; Steenbergen, Renske D M

2016-06-21

High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC.Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis.

Immortalization capacity of HPV types is inversely related to chromosomal instability

PubMed Central

Schütze, Denise M.; Krijgsman, Oscar; Snijders, Peter J.F.; Ylstra, Bauke; Weischenfeldt, Joachim; Mardin, Balca R.; Stütz, Adrian M.; Korbel, Jan O.; Meijer, Chris J.L.M.; Quint, Wim G.V.; Bosch, Leontien; Wilting, Saskia M.; Steenbergen, Renske D.M.

2016-01-01

High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs. Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied. All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC. Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis. PMID:26993771

Discovery of Potent Antiproliferative Agents Targeting EGFR Tyrosine Kinase Based on the Pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine Scaffold.

PubMed

Aziz, Yasmine Mohamed Abdel; Said, Mohamed Mokhtar; El Shihawy, Hosam Ahmed; Tolba, Mai Fathy; Abouzid, Khaled Abouzid Mohamed

2015-01-01

A series of pyridothieno[3,2-d]pyrimidin-4-amines was designed and synthesized as congeners to the classical 4-anilinoquinazolines as ATP-competitive epidermal growth factor receptor (EGFR) inhibitors. Compound 5a exhibited the most potent and selective inhibitory activity against EGFR with an IC50 value of 36.7 nM. Moreover, compounds 4b and 5a showed remarkable cell growth inhibition against leukemia, central nervous system cancer, and non-small cell lung cancer cell lines that overexpress EGFR, with growth inhibition of 50% (GI50) values of around 10 nM in the full U.S. National Cancer Institute 60 cell panel assay. Cell cycle studies indicated that compounds 4b and 5a induced significant cell cycle arrest in the S-phase and G0/G1, respectively, in addition to boosting P27(kip) expression. Compound 5a did not alter the viability of placental trophoblasts, which reflects its safety for normal cells. The standard COMPARE analyses demonstrated considerable correlation levels between compounds 4b and 5a and erlotinib, with pyridinium chlorochromate (PCC) values of 0.707 and 0.727, respectively.

Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells.

PubMed

Alais, Sandrine; Dutartre, Hélène; Mahieux, Renaud

2017-01-01

Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.

Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model

PubMed Central

Borgna, Vincenzo; Villegas, Jaime; Burzio, Verónica A.; Belmar, Sebastián; Araya, Mariela; Jeldes, Emanuel; Lobos-González, Lorena; Silva, Verónica; Villota, Claudio; Oliveira-Cruz, Luciana; Lopez, Constanza; Socias, Teresa; Castillo, Octavio; Burzio, Luis O.

2017-01-01

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma. PMID:28620146

Mitochondrial ASncmtRNA-1 and ASncmtRNA-2 as potent targets to inhibit tumor growth and metastasis in the RenCa murine renal adenocarcinoma model.

PubMed

Borgna, Vincenzo; Villegas, Jaime; Burzio, Verónica A; Belmar, Sebastián; Araya, Mariela; Jeldes, Emanuel; Lobos-González, Lorena; Silva, Verónica; Villota, Claudio; Oliveira-Cruz, Luciana; Lopez, Constanza; Socias, Teresa; Castillo, Octavio; Burzio, Luis O

2017-07-04

Knockdown of antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptosis in several human and mouse tumor cell lines, but not normal cells, suggesting this approach for a selective therapy against different types of cancer. Here we show that in vitro knockdown of murine ASncmtRNAs induces apoptotic death of mouse renal adenocarcinoma RenCa cells, but not normal murine kidney epithelial cells. In a syngeneic subcutaneous RenCa model, treatment delayed and even reversed tumor growth. Since the subcutaneous model does not reflect the natural microenviroment of renal cancer, we used an orthotopic model of RenCa cells inoculated under the renal capsule. These studies showed inhibition of tumor growth and metastasis. Direct metastasis assessment by tail vein injection of RenCa cells also showed a drastic reduction in lung metastatic nodules. In vivo treatment reduces survivin, N-cadherin and P-cadherin levels, providing a molecular basis for metastasis inhibition. In consequence, the treatment significantly enhanced mouse survival in these models. Our results suggest that the ASncmtRNAs could be potent and selective targets for therapy against human renal cell carcinoma.

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

PubMed

Franco, Marco E; Sutherland, Grace E; Lavado, Ramon

2018-04-01

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies. Copyright © 2018 Elsevier Inc. All rights reserved.

An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

PubMed

Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

2018-05-15

Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, M.R.

1985-07-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

Synthesis and biological activity of chimeric structures derived from the cytotoxic natural compounds dolastatin 10 and dolastatin 15.

PubMed

Poncet, J; Busquet, M; Roux, F; Pierré, A; Atassi, G; Jouin, P

1998-04-23

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were synthesized in order to evaluate the possible equivalence of these units. These compounds can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the former and the C-terminal part of the latter and vice versa. Both analogues exhibited a marked decrease in their cytotoxic activity but showed similar differential cytotoxicity with regard to the cell lines assayed compared with the parent compounds. HT-29 cell line was the least sensitive one. However, this activity was in the nanomolar level and close to that of vincristine. The differences in their effect on tubulin polymerization were less pronounced. We confirmed the already known crucial role of the Dil residue in this assay. The nonequivalence of the Dil unit and the MeVal-Pro dipeptide probably reflects modification in the relative positions of the N-dimethylamino and the phenyl moieties.

Evolution of the Cerro Prieto geothermal system as interpreted from vitrinite reflectance under isothermal conditions

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

Barker, C.E.; Pawlewicz, M.J.; Bostick, N.H.

1981-01-01

Temperature estimates from reflectance data in the Cerro Prieto system correlate with modern temperature logs and temperature estimates from fluid inclusion and oxygen isotope geothermometry indicating that the temperature in the central portion of the Cerro Prieto System is now at its historical maximum. Isoreflectance lines formed by contouring vitrinite reflectance data for a given isothermal surface define an imaginary surface that indicates an apparent duration of heating in the system. The 250/sup 0/C isothermal surface has a complex dome-like form suggesting a localized heat source that has caused shallow heating in the central portion of this system. Isoreflectance linesmore » relative to this 250/sup 0/C isothermal surface define a zone of low reflectance roughly corresponding to the crest of the isothermal surface. Comparison of these two surfaces suggest that the shallow heating in the central portion of Cerro Prieto is young relative to the heating (to 250/sup 0/C) on the system margins. Laboratory and theoretical models of hydrothermal convection cells suggest that the form of the observed 250/sup 0/C isothermal surface and the reflectance surface derived relative to it results from the convective rise of thermal fluids under the influence of a regional hydrodynamic gradient that induces a shift of the hydrothermal heating effects to the southwest.« less

Effects of texturization due to chemical etching and laser on the optical properties of multicrystalline silicon for applications in solar cells

NASA Astrophysics Data System (ADS)

Vera, D.; Mass, J.; Manotas, M.; Cabanzo, R.; Mejia, E.

2016-02-01

In this work we carried out the texturization of surfaces of multicrystalline silicon type-p in order to decrease the reflection of light on the surface, using the chemical etching method and then a treatment with laser. In the first method, it was immersed in solutions of HF:HNO3:H2O, HF:HNO3:CH3COOH, HF:HNO3:H3PO4, in the proportion 14:01:05, during 30 seconds, 1, 2 and 3 minutes. Subsequently with a laser (ND:YAG) grids were generated beginning with parallel lines separated 50μm. The samples were analyzed by means of diffuse spectroscopy (UV-VIS) and scanning electron micrograph (SEM) before and after the laser treatment. The lowest result of reflectance obtained by HF:HNO3:H2O during 30 seconds, was of 15.5%. However, after applying the treatment with laser the reflectance increased to 17.27%. On the other hand, the samples treated (30 seconds) with acetic acid and phosphoric acid as diluents gives as a result a decrease in the reflectance values after applying the laser treatment from 21.97% to 17.79% and from 27.73% to 20.03% respectively. The above indicates that in some cases it is possible to decrease the reflectance using jointly the method of chemical etching and then a laser treatment.

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile 1

PubMed Central

Shedletzky, Esther; Shmuel, Miri; Trainin, Tali; Kalman, Sara; Delmer, Deborah

1992-01-01

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca2+-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16652933

Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

PubMed

Ostrowski, K; Dziedzic-Goclawska, A; Strojny, P; Grzesik, W; Kieler, J; Christensen, B; Mareel, M

1986-01-01

The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppressing lossy-film-induced angular mismatches between reflectance and transmittance extrema: optimum optical designs of interlayers and AR coating for maximum transmittance into active layers of CIGS solar cells.

PubMed

Chang, Yin-Jung

2014-01-13

The investigation of optimum optical designs of interlayers and antireflection (AR) coating for achieving maximum average transmittance (T(ave)) into the CuIn(1-x)Ga(x)Se2 (CIGS) absorber of a typical CIGS solar cell through the suppression of lossy-film-induced angular mismatches is described. Simulated-annealing algorithm incorporated with rigorous electromagnetic transmission-line network approach is applied with criteria of minimum average reflectance (R(ave)) from the cell surface or maximum T(ave) into the CIGS absorber. In the presence of one MgF2 coating, difference in R(ave) associated with optimum designs based upon the two distinct criteria is only 0.3% under broadband and nearly omnidirectional incidence; however, their corresponding T(ave) values could be up to 14.34% apart. Significant T(ave) improvements associated with the maximum-T(ave)-based design are found mainly in the mid to longer wavelengths and are attributed to the largest suppression of lossy-film-induced angular mismatches over the entire CIGS absorption spectrum. Maximum-T(ave)-based designs with a MgF2 coating optimized under extreme deficiency of angular information is shown, as opposed to their minimum-R(ave)-based counterparts, to be highly robust to omnidirectional incidence.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro.

PubMed

Gedye, Craig; Cardwell, Tracy; Dimopoulos, Nektaria; Tan, Bee Shin; Jackson, Heather; Svobodová, Suzanne; Anaka, Matthew; Behren, Andreas; Maher, Christopher; Hofmann, Oliver; Hide, Winston; Caballero, Otavia; Davis, Ian D; Cebon, Jonathan

2016-02-01

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Observations from ERTS of overgrazing and cultivation impact on the earth's surface

NASA Technical Reports Server (NTRS)

Otterman, J.; Walter, L. S.; Schmugge, T. J.

1976-01-01

ERTS-1 imagery was surveyed for cases of anthropogenic impact, similar to that in the Sinai, evidenced by the Sinai/Negev demarcation line. Reflectivities in several such cases were analyzed using ERTS computer compatible tapes. Sharp increases of surface reflectivity show the severe impact of overgrazing found in several places around the world. Agricultural cultivation can also result in pronounced change of reflectivities, as evidenced by the Montana, USA/Canada demarcation line. Such processes have probably been going on since prehistoric times, increasing with the human population and with the population of the grazing herds.

The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies.

PubMed

Marion, Marie-Jeanne; Hantz, Olivier; Durantel, David

2010-01-01

Liver progenitor cells may play an important role in carcinogenesis in vivo and represent therefore useful cellular materials for in vitro studies. The HepaRG cell line, which is a human bipotent progenitor cell line capable to differentiate toward two different cell phenotypes (i.e., biliary-like and hepatocyte-like cells), has been established from a liver tumor associated with chronic hepatitis C. This cell line represents a valuable alternative to ex vivo cultivated primary human hepatocytes (PHH), as HepaRG cells share some features and properties with adult hepatocytes. The cell line is particularly useful to evaluate drugs and perform drug metabolism studies, as many detoxifying enzymes are expressed and functional. It is also an interesting tool to study some aspect of progenitor biology (e.g., differentiation process), carcinogenesis, and the infection by some pathogens for which the cell line is permissive (e.g., HBV infection). Overall, this chapter gives a concise overview of the biological properties and potential applications of this cell line.

[Expression of Chemokine receptor CXCR6 and its significance in breast cancer cell lines].

PubMed

Cheng, Hao; Chen, Nian-yong

2014-05-01

To detect the expression of Chemokine receptor CXCR6 in invasive breast cancer cell lines and normal mammary epithelial cell line, and assess the relationship between CXCR6 expression and malignant behavior of breast cancer cells. Expression level of CXCR6 in different invasive breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-231) and normal mammary epithelial cell line (MCF-10A)was detected by real time reverse transcription-polymerase chain reaction (real time-PCR) and Western blot. Lentivirus was employed to interfere CXCR6 expression in MDA-MB-231. MTT assay and transwell chamber were used to study proliferative and invasive ability of those cells respectively. Vascular enothelial growth factor (VEGF) expression was detected to study the role of CXCR6 in angiogenesis. At both mRNA level and protein level, normal mammary epithelial cell line MCF-10A showed the weakest CXCR6 expression. The breast cancer cell lines expressed CXCR6 in different levels, the expression level of CXCR6 in highly invasive cell line MDA-MB-231 was significantly higher than that in two low-invasive cell lines SK-BR-3 and MCF-7 (P < 0.05). Silencing CXCR6 gene by Lentivirus-mediated RNA interference in MDA-MB-231 inhibited its proliferation ability, invasion ability and angiogenesis ability in vitro (P < 0.05). Different invasive breast cancer cell lines express CXCR6 at different levels, positively correlated with its invasive ability.

Cytogenetics of small cell carcinoma of the lung.

PubMed

Wurster-Hill, D H; Cannizzaro, L A; Pettengill, O S; Sorenson, G D; Cate, C C; Maurer, L H

1984-12-01

Nineteen cell lines derived from various malignant tissues of 15 patients with small cell carcinoma of the lung (SCCL) have been studied. The results showed heterogeneity in all cell lines, with no one consistent abnormality among them. Cell lines from 11 of the patients had minute and double minute chromosomes, and cell lines from 2 patients had abnormally banding regions, designated as ABRs, as distinguished from homogeneously staining regions (HSRs). The latter 2 and several of the former cell lines were derived from specimens taken before the patients were placed on therapy. All but 2 of the cell lines had a constant marker load, consisting of 24%-35% of the complement. Some markers remained stable through months and years of culture life, while other markers came and went. Chromosomes #1, #6 and #11 were most frequently involved in marker formation in the cell lines, and these were compared to similar markers in direct bone marrow preparations. Chromosome #1 markers were of variable structure, whereas #6 and #11 most often took the form of 6q- and 11p+ markers, with breakpoints most frequently at 6q23-25 and 11p11-12. A 3p- marker was found in a minority of cell lines. All of these markers were also found in direct marrow preparations from some patients with SCCL. Nonmonoclonal tumors arose from inoculation of bimodal cell lines into nude mice, but population selection by undetermined mechanism was evident. Cytogenetic parameters showed no positive correlation with hormone production by these cell lines.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

PubMed

Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

2015-01-01

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout. Copyright © 2014 Elsevier Ltd. All rights reserved.

Topological nodal-line fermions in spin-orbit metal PbTaSe2

PubMed Central

Bian, Guang; Chang, Tay-Rong; Sankar, Raman; Xu, Su-Yang; Zheng, Hao; Neupert, Titus; Chiu, Ching-Kai; Huang, Shin-Ming; Chang, Guoqing; Belopolski, Ilya; Sanchez, Daniel S.; Neupane, Madhab; Alidoust, Nasser; Liu, Chang; Wang, BaoKai; Lee, Chi-Cheng; Jeng, Horng-Tay; Zhang, Chenglong; Yuan, Zhujun; Jia, Shuang; Bansil, Arun; Chou, Fangcheng; Lin, Hsin; Hasan, M. Zahid

2016-01-01

Topological semimetals can support one-dimensional Fermi lines or zero-dimensional Weyl points in momentum space, where the valence and conduction bands touch. While the degeneracy points in Weyl semimetals are robust against any perturbation that preserves translational symmetry, nodal lines require protection by additional crystalline symmetries such as mirror reflection. Here we report, based on a systematic theoretical study and a detailed experimental characterization, the existence of topological nodal-line states in the non-centrosymmetric compound PbTaSe2 with strong spin-orbit coupling. Remarkably, the spin-orbit nodal lines in PbTaSe2 are not only protected by the reflection symmetry but also characterized by an integer topological invariant. Our detailed angle-resolved photoemission measurements, first-principles simulations and theoretical topological analysis illustrate the physical mechanism underlying the formation of the topological nodal-line states and associated surface states for the first time, thus paving the way towards exploring the exotic properties of the topological nodal-line fermions in condensed matter systems. PMID:26829889

Transmission-line model to design matching stage for light coupling into two-dimensional photonic crystals.

PubMed

Miri, Mehdi; Khavasi, Amin; Mehrany, Khashayar; Rashidian, Bizhan

2010-01-15

The transmission-line analogy of the planar electromagnetic reflection problem is exploited to obtain a transmission-line model that can be used to design effective, robust, and wideband interference-based matching stages. The proposed model based on a new definition for a scalar impedance is obtained by using the reflection coefficient of the zeroth-order diffracted plane wave outside the photonic crystal. It is shown to be accurate for in-band applications, where the normalized frequency is low enough to ensure that the zeroth-order diffracted plane wave is the most important factor in determining the overall reflection. The frequency limitation of employing the proposed approach is explored, highly dispersive photonic crystals are considered, and wideband matching stages based on binomial impedance transformers are designed to work at the first two photonic bands.

[Optimization of Glycyrrhiza flavonoid and ferulic acid cream by reflect-line orthogonal simplex method].

PubMed

Liu, Sheng; Xie, Jun; Chen, Xiangqing; Yang, Liqiang; Su, Dan; Fang, Yan; Yu, Na; Fang, Wei

2010-02-01

To optimize the formula of Glycyrrhiza flavonoid and ferulic acid cream and set up its quality control parameters. Reflect-line orthogonal simplex method was used to optimize the main factors such as amount of Myrj52-glyceryl monostearate and dimethicone, based on the appearance, spreadability and stability of the cream. 9.0% Myrj52-glyceryl monostearate (3:2) and 2.5% dimethicone were chosen in prescription. The prepared cream presented a good stability after being placed 24 h at 5 degrees C, 25 degrees C and 37 degrees C respectively,and its spreadability suited with the property of semi-fluid cream. [corrected] The formula of Glycyrrhiza flavonoid and ferulic acid cream is suitable, and its quality is stable. The reflect-line orthogonal simplex method is suitable for the formula optimization of cream.

Chitosan Stabilized Gold-Folate-Poly(lactide-co-glycolide) Nanoplexes Facilitate Efficient Gene Delivery in Hepatic and Breast Cancer Cells.

PubMed

Akinyelu, Jude; Singh, Moganavelli

2018-07-01

The biodegradable polymer, poly(lactide-co-glycolide) is a popular polymer of choice in many nanotherapeutic studies. Herein, we report on the synthesis and evaluation of four chitosan stabilized poly(lactide-co-glycolide) nanoparticles with and without coating with gold, and the targeting ligand, folic acid, as potential non-viral gene delivery vectors. The poly(lactide-co-glycolide) nanoparticles were synthesized via nanoprecipitation/solvent evaporation method in conjunction with the surface functionalizing folic acid and chitosan. The physiochemical properties (morphology, particle size, zeta potential, folic acid/chitosan presence, DNA binding), and biological properties (nuclease protection, in vitro cytotoxicity and transfection potential in human kidney, hepatocellular carcinoma and breast adenocarcinoma cells), of all four gene bound nanoparticles were evaluated. Gel retardation assays confirmed that all the nanoparticles were able to successfully bind the reporter plasmid, pCMV-luc DNA at varying weight ratios. The gold-folate-poly(lactide-co-glycolide) nanoplexes with the highest binding efficiency (w/w ratio 4:1), best protected the plasmid DNA as evidenced from the nuclease protection assays. Furthermore, these nanoplexes presented as spherical particles with an average particle size of 199.4 nm and zeta potential of 35.7 mV. Folic acid and chitosan functionalization of the nanoparticles was confirmed by attenuated total reflection-Fourier transform infrared spectroscopy. All nanoplexes maintained over 90% cell viability in all cell lines investigated. Interestingly, the gold-folate-poly(lactide-co-glycolide) nanoplexes showed a greater transgene activity in the hepatic and breast cancer cells compared to the other nanocomplexes in the same cell lines. The favorable size, colloidal stability, low cytotoxicity, significant transgene expression, and nuclease protection ability in vitro, all provide support for the use of gold-folate-poly(lactide-co-glycolide) nanoplexes in future gene therapy applications.

Combination of systemic chemotherapy with local stem cell delivered S-TRAIL in resected brain tumors.

PubMed

Redjal, Navid; Zhu, Yanni; Shah, Khalid

2015-01-01

Despite advances in standard therapies, the survival of glioblastoma multiforme (GBM) patients has not improved. Limitations to successful translation of new therapies include poor delivery of systemic therapies and use of simplified preclinical models which fail to reflect the clinical complexity of GBMs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically in tumor cells and we have tested its efficacy by on-site delivery via engineered stem cells (SC) in mouse models of GBM that mimic the clinical scenario of tumor aggressiveness and resection. However, about half of tumor lines are resistant to TRAIL and overcoming TRAIL-resistance in GBM by combining therapeutic agents that are currently in clinical trials with SC-TRAIL and understanding the molecular dynamics of these combination therapies are critical to the broad use of TRAIL as a therapeutic agent in clinics. In this study, we screened clinically relevant chemotherapeutic agents for their ability to sensitize resistant GBM cell lines to TRAIL induced apoptosis. We show that low dose cisplatin increases surface receptor expression of death receptor 4/5 post G2 cycle arrest and sensitizes GBM cells to TRAIL induced apoptosis. In vivo, using an intracranial resection model of resistant primary human-derived GBM and real-time optical imaging, we show that a low dose of cisplatin in combination with synthetic extracellular matrix encapsulated SC-TRAIL significantly decreases tumor regrowth and increases survival in mice bearing GBM. This study has the potential to help expedite effective translation of local stem cell-based delivery of TRAIL into the clinical setting to target a broad spectrum of GBMs. © 2014 AlphaMed Press.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma.

PubMed

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E

2017-10-17

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma

PubMed Central

Lodrini, Marco; Sprüssel, Annika; Astrahantseff, Kathy; Tiburtius, Daniela; Konschak, Robert; Lode, Holger N.; Fischer, Matthias; Keilholz, Ulrich; Eggert, Angelika; Deubzer, Hedwig E.

2017-01-01

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients. PMID:29156716

Mass Spectrometry of Human Leukocyte Antigen Class I Peptidomes Reveals Strong Effects of Protein Abundance and Turnover on Antigen Presentation*

PubMed Central

Bassani-Sternberg, Michal; Pletscher-Frankild, Sune; Jensen, Lars Juhl; Mann, Matthias

2015-01-01

HLA class I molecules reflect the health state of cells to cytotoxic T cells by presenting a repertoire of endogenously derived peptides. However, the extent to which the proteome shapes the peptidome is still largely unknown. Here we present a high-throughput mass-spectrometry-based workflow that allows stringent and accurate identification of thousands of such peptides and direct determination of binding motifs. Applying the workflow to seven cancer cell lines and primary cells, yielded more than 22,000 unique HLA peptides across different allelic binding specificities. By computing a score representing the HLA-I sampling density, we show a strong link between protein abundance and HLA-presentation (p < 0.0001). When analyzing overpresented proteins – those with at least fivefold higher density score than expected for their abundance – we noticed that they are degraded almost 3 h faster than similar but nonpresented proteins (top 20% abundance class; median half-life 20.8h versus 23.6h, p < 0.0001). This validates protein degradation as an important factor for HLA presentation. Ribosomal, mitochondrial respiratory chain, and nucleosomal proteins are particularly well presented. Taking a set of proteins associated with cancer, we compared the predicted immunogenicity of previously validated T-cell epitopes with other peptides from these proteins in our data set. The validated epitopes indeed tend to have higher immunogenic scores than the other detected HLA peptides. Remarkably, we identified five mutated peptides from a human colon cancer cell line, which have very recently been predicted to be HLA-I binders. Altogether, we demonstrate the usefulness of combining MS-analysis with immunogenesis prediction for identifying, ranking, and selecting peptides for therapeutic use. PMID:25576301

Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

PubMed

Malek, Sri Nurestri Abdul; Shin, Sim Kae; Wahab, Norhanom Abdul; Yaacob, Hashim

2009-05-06

Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Comparative study of the photodynamic effect in tumor and nontumor animal cell lines

NASA Astrophysics Data System (ADS)

Stoykova, Elena V.; Alexandrova, R.; Shurulinkov, Stanislav; Sabotinov, O.; Minchev, Georgi

2004-09-01

In this study we evaluate the cytotoxicity of two photosensitisers with absorption peaks in the green and red part of the spectrum on animal cell lines. The cytotoxicity assessment was performed for a tumor cell line LSCC-SF-Mc29, obtained from a transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29, a tumor line LSR-SF-SR, obtained from a transplantable sarcoma in rat induced by Rous sarcoma virus strain Schmidt-Ruppin and for normal mouse and bovine cell lines. Up to now the effect of the photodynamic therapy on virus-induced cancers has not been clarified. The cells were treated with 5,10,15,20 - tetra (4-sulfophenyl) porphyrin with main absorption peak at 519 nm and a dye activated with a red light. The cells were seeded in 96-well plates at 2 x 104 cells/well. The cells were exposed to irradiation from a pulsed CuBr vapor laser at 510.6 nm and 578.2 nm and exposure rate 50 mW/cm2, from an Ar-ion laser at 514 nm and 1 mW/cm2 and to 655 nm-irradiation from a semiconductor laser at 10 mW/cm2. The biological activity of the tested compounds was measured by the neutral red uptake cytotoxicity test. The light dose-response curves and light exposures that ensure 50% drop in the treated cells viability in comparison with the cells grown in non-modified medium were obtained for each cell line. The cytotoxic effect of both photosensitisers is most distinguished for the tumor line LSCC-SF-Mc29. The 2-4 times higher viability of the normal cell lines in comparison with the tumor lines is established. The bovine cell lines are more vulnerable than the mouse lines.

Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia.

PubMed

Hamid, Sharifah; Lim, Kue Peng; Zain, Rosnah Binti; Ismail, Siti Mazlipah; Lau, Shin Hin; Mustafa, Wan Mahadzir Wan; Abraham, M Thomas; Nam, Noor Akmar; Teo, Soo-Hwang; Cheong, Sok Ching

2007-03-01

We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

Bio-Physicochemical Interactions of Engineered Nanomaterials in In Vitro Cell Culture Model

DTIC Science & Technology

2012-08-14

viz. human hepatocarcinoma cell line (Hep G2), human adinocarcinoma cell line (A549), human embryonic kidney cell line (HEK 293), human neuroblastoma...Glutamine, 1% Na-Pyruvate and 10 ml/L antibiotic solution at 37oC under a humidified atmosphere of 5% CO2/95% air.  Human hepatocarcinoma cell line

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

PubMed

Goswami, M; Sharma, B S; Tripathi, A K; Yadav, Kamalendra; Bahuguna, S N; Nagpure, N S; Lakra, W S; Jena, J K

2012-05-25

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation

PubMed Central

Quentmeier, Hilmar; Pommerenke, Claudia; Ammerpohl, Ole; Geffers, Robert; Hauer, Vivien; MacLeod, Roderick AF; Nagel, Stefan; Romani, Julia; Rosati, Emanuela; Rosén, Anders; Uphoff, Cord C; Zaborski, Margarete; Drexler, Hans G

2016-01-01

Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies. PMID:27566572

The Cellosaurus, a Cell-Line Knowledge Resource

PubMed Central

Bairoch, Amos

2018-01-01

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

Expression and rearrangement of the ROS1 gene in human glioblastoma cells

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

Birchmeier, C.; Sharma, S.; Wigler, M.

1987-12-01

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, the authors found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, they detected a potentially activating mutation at the ROS1 locus.

Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

PubMed

Hoshino, Keita; Isawa, Haruhiko; Kuwata, Ryusei; Tajima, Shigeru; Takasaki, Tomohiko; Iwabuchi, Kikuo; Sawabe, Kyoko; Kobayashi, Mutsuo; Sasaki, Toshinori

2015-08-01

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.

PubMed

Wasielewski, Marijke; Hanifi-Moghaddam, Pejman; Hollestelle, Antoinette; Merajver, Sofia D; van den Ouweland, Ans; Klijn, Jan G M; Ethier, Stephen P; Schutte, Mieke

2009-01-01

The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.

Reflector system for a lighting fixture

DOEpatents

Siminovitch, M.J.; Page, E.; Gould, C.T.

1998-09-08

Disclosed herein is a reflector system for a lighting fixture having a illumination source surrounded by an envelope. The reflector system includes a first reflector surrounding the illumination source. The reflector system also includes a second reflector which is non-contiguous with the first reflector and which surrounds the illumination source. The illumination source creates light rays which are reflected by the first and second reflectors. The first reflector directs light rays toward the center line of the fixture. However, the reflected rays despite being so reflected do not substantially intersect the envelope. The reflected light rays from the second reflector being directed so that they diverge from the center line of the fixture avoiding intersection with the semi-transparent envelope. 5 figs.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

PubMed

Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-11-22

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Satoh, Motonobu; Kohara, Arihiro

2018-05-17

Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.

PubMed Central

Reeve, J. G.; Xiong, J.; Morgan, J.; Bleehen, N. M.

1996-01-01

As a first step towards elucidating the potential role(s) of bcl-2 and bcl-2-related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-x1-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl-2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8630278

RON kinase isoforms demonstrate variable cell motility in normal cells.

PubMed

Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

2016-09-01

Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

Establishment and characterization of a new fish cell line from head kidney of half-smooth tongue sole (Cynoglossus semilaevis).

PubMed

Zheng, Yuan; Wang, Na; Xie, Ming-Shu; Sha, Zhen-Xia; Chen, Song-Lin

2012-12-01

A new cell line (TSHKC) derived from half-smooth tongue sole (Cynoglossus semilaevis) head kidney was developed. The cell line was subcultured for 40 passages over a period of 360 days. The cell line was optimally maintained in minimum essential medium supplemented with HEPES, antibiotics, fetal bovine serum, 2-Mercaptoethanol (2-Me), sodium pyruvate and basic fibroblast growth factor. The suitable growth temperature for TSHKC cells was 24 °C, and microscopically, TSHKC cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TSHKC cell line had a normal diploid karyotype with 2n = 42, contained the heterogametic W chromosome. The TSHKC cell line was found to be susceptible to lymphocystis disease virus. The fluorescent signals were observed in TSHKC when the cells were transfected with green fluorescent protein and red fluorescent protein reporter plasmids.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

NASA Astrophysics Data System (ADS)

Armitage, Mark

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line

PubMed Central

STOCZYNSKA-FIDELUS, EWELINA; PIASKOWSKI, SYLWESTER; PAWLOWSKA, ROZA; SZYBKA, MALGORZATA; PECIAK, JOANNA; HULAS-BIGOSZEWSKA, KRYSTYNA; WINIECKA-KLIMEK, MARTA; RIESKE, PIOTR

2016-01-01

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research. PMID:26870252

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

PubMed Central

Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

2013-01-01

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

History of leukemia-lymphoma cell lines.

PubMed

Drexler, Hans G; Macleod, Roderick A F

2010-08-01

We outline the near 50-year history of leukemia-lymphoma (LL) cell lines - a key model system in biomedicine. Due to the detailed documentation of their oncogenomic and transcriptional alterations via recent advances in molecular medicine, LL cell lines may be fitted to parent tumors with a degree of precision unattainable in other cancers. We have surveyed the corpus of published LL cell lines and found 637 examples that meet minimum standards of authentication and characterization. Alarmingly, the rate of establishment of new LL cell lines has plummeted over the last decade. Although the main hematopoietic developmental cell types are represented by cell lines, some LL categories stubbornly resist establishment in vitro. The advent of engineering techniques for immortalizing primary human cells that maintain differentiation means the time is ripe for renewed search for in vitro models from un(der)represented hematologic entities. Given their manifold applications in biomedicine, there is little doubt that LL-derived cell lines will continue to play a vital part well into the next half-century as well. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Molecular characterization of immortalized normal and dysplastic oral cell lines.

PubMed

Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

2015-05-01

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

PubMed Central

Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

2016-01-01

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

PubMed

Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

2015-02-03

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

PubMed

Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

2018-01-12

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

Cancer.gov

SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Establishment and characterization of five immortalized human scalp dermal papilla cell lines.

PubMed

Kwack, Mi Hee; Yang, Jung Min; Won, Gong Hee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2018-02-05

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research. Copyright © 2018 Elsevier Inc. All rights reserved.

76 FR 42678 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-19

... collection). Burden Hours: 250. Number of Respondents: 100 (15 cell line limit). Average Hours per Response: 2 hours and 30 minutes (10 minutes/cell line x 15 cell lines). Needs and Uses: The NIST Biochemical...: National Institute of Standards and Technology (NIST) Title: Identification of Human Cell Lines Project...

Laser-generated ultrasound for high-precision cutting of tissue-mimicking gels (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Taehwa; Luo, Wei; Li, Qiaochu; Guo, L. Jay

2017-03-01

Laser-generated focused ultrasound has shown great promise in precisely treating cells and tissues by producing controlled micro-cavitation within the acoustic focal volume (<100 um). However, the previous demonstration used cells and tissues cultured on glass substrates. The glass substrates were found to be critical to cavitation, because ultrasound amplitude doubles due to the reflection from the substrate, thus allowing for reaching pressure amplitude to cavitation threshold. In other words, without the sound reflecting substrate, pressure amplitude may not be strong enough to create cavitation, thus limiting its application to only cultured biomaterials on the rigid substrates. By using laser-generated focused ultrasound without relying on sound-reflecting substrates, we demonstrate free-field cavitation in water and its application to high-precision cutting of tissue-mimicking gels. In the absence of a rigid boundary, strong pressure for cavitation was enabled by recently optimized photoacoustic lens with increased focal gain (>30 MPa, negative pressure amplitude). By moving cavitation spots along pre-defined paths through a motorized stage, tissue-mimicking gels of different elastic moduli were cut into different shapes (rectangle, triangle, and circle), leaving behind the same shape of holes, whose sizes are less than 1 mm. The cut line width is estimated to be less than 50 um (corresponding to localized cavitation region), allowing for accurate cutting. This novel approach could open new possibility for in-vivo treatment of diseased tissues in a high-precision manner (i.e., high-precision invisible sonic scalpel).

Complex nature of SNP genotype effects on gene expression in primary human leucocytes.

PubMed

Heap, Graham A; Trynka, Gosia; Jansen, Ritsert C; Bruinenberg, Marcel; Swertz, Morris A; Dinesen, Lotte C; Hunt, Karen A; Wijmenga, Cisca; Vanheel, David A; Franke, Lude

2009-01-07

Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown. We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects. In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected. In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination.

PubMed

Korch, Christopher; Spillman, Monique A; Jackson, Twila A; Jacobsen, Britta M; Murphy, Susan K; Lessey, Bruce A; Jordan, V Craig; Bradford, Andrew P

2012-10-01

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

NASA Astrophysics Data System (ADS)

Netchareonsirisuk, Ponsawan; Puthong, Songchan; Dubas, Stephan; Palaga, Tanapat; Komolpis, Kittinan

2016-11-01

Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5-15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO3 alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC50), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84-90 %) than necrosis (8-12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

The combination effect of sodium butyrate and 5-Aza-2'-deoxycytidine on radiosensitivity in RKO colorectal cancer and MCF-7 breast cancer cell lines.

PubMed

Cho, Hang Joo; Kim, Sin Young; Kim, Kee Hwan; Kang, Won Kyung; Kim, Ji Il; Oh, Seong Tack; Kim, Jeong Soo; An, Chang Hyeok

2009-05-21

The overall level of chromatin compaction is an important mechanism of radiosensitivity, and modification of DNA methylation and histone deacetylation may increase radiosensitivity by altering chromatin compaction. In this study, we investigated the effect of a demethylating agent, a histone deacetylase(HDAC) inhibitor, and the two agents combined on radiosensitivity in human colon and breast cancer cell lines. In this study, we used RKO colorectal cancer cell line and MCF-7 breast cancer cell lines and normal colon cell lines. On each of the cell lines, we used three different agents: the HDAC inhibitor sodium butyrate(SB), the demethylating agent 5-Aza-2'-deoxycytidine(5-aza-DC), and radiation. We then estimated the percentage of the cell survival using the XTT method and experimented to determine if there was an augmentation in the therapeutic effect by using different combinations of the two or three of the treatment methods. After treatment of each cell lines with 5-aza-DC, SB and 6 grays of radiation, we observed that the survival fraction was lower after the treatment with 5-aza-DC or SB than with radiation alone in RKO and MCF-7 cell lines(p < 0.001). The survival fraction was lowest when the two agents, 5-aza-DC and SB were combined with radiation in both RKO and MCF-cell lines. In conclusion, 5-aza-DC and SB can enhance radiosensitivity in both MCF-7 and RKO cell lines. The combination effect of a demethylating agent and an HDAC inhibitor is more effective than that of single agent treatment in both breast and colon cancer cell lines.

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives

PubMed Central

Dumont, Jennifer; Euwart, Don; Mei, Baisong; Estes, Scott; Kshirsagar, Rashmi

2016-01-01

Abstract Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins. PMID:26383226

Honoring the work and life of Leroy C. Stevens. A symposium as part of the International Stem Cell Initiative Workshop.

PubMed

Graham, Christopher F; Solter, Davor; Gearhart, John D; Nadeau, Joseph H; Knowles, Barbara B

2016-01-01

In 2016, a symposium was convened in Leroy C. Stevens' honor, in association with a meeting of the International Stem Cell Initiative (ISCI). ISCI, funded internationally, is composed of a group of ~100 scientists from many countries, under the leadership of Peter Andrews, who have worked together to characterize a significant number of human pluripotent stem cell lines, to monitor their genetic stability and their differentiation into mature cell types and tissues in vitro and in vivo. Those at the ISCI meeting puzzled through one of the thorniest problems in the therapeutic use of the differentiated derivatives of pluripotent stem cells for human therapy; namely, pluripotent stem cells can differentiate into any cell type in the adult organism, but they also have the capacity for unlimited self-renewal, hence if mutated they may have tumorigenic potential. The meeting considered how these cells might become genetically or epigenetically abnormal and how the safety of these cells for human therapeutic uses could be assessed and assured. The symposium was an opportunity to pay tribute to Leroy Stevens and to the basic science origins of this newest aspect of regenerative medicine. It was a time to reflect on the past and on how it can influence the future of our field.

Probing the Behaviors of Gold Nanorods in Metastatic Breast Cancer Cells Based on UV-vis-NIR Absorption Spectroscopy

PubMed Central

Zhang, Weiqi; Ji, Yinglu; Meng, Jie; Wu, Xiaochun; Xu, Haiyan

2012-01-01

In this work, behaviors of positively-charged AuNRs in a highly metastatic tumor cell line MDA-MB-231 are examined based on UV-vis-NIR absorption spectroscopy in combination with inductively coupled plasma mass spectrometry (ICP-MS), transmission electron microscopy (TEM) and dark-field microscopic observation. It is found that characteristic surface plasmon resonance (SPR) peaks of AuNRs can be detected using spectroscopic method within living cells that have taken up AuNRs. The peak area of transverse SPR band is shown to be proportionally related to the amount of AuNRs in the cells determined with ICP-MS, which suggests a facile and real time quantification method for AuNRs in living cells. The shape of longitudinal SPR band in UV-vis-NIR spectrum reflects the aggregation state of AuNRs in the cells during the incubation period, which is proved by TEM and microscopic observations. Experimental results reveal that AuNRs are internalized by the cells rapidly; the accumulation, distribution and aggregation of AuNRs in the cells compartments are time and dose dependent. The established spectroscopic analysis method can not only monitor the behaviors of AuNRs in living cells but may also be helpful in choosing the optimum laser stimulation wavelength for anti-tumor thermotherapy. PMID:22384113

Adaptation to statins restricts human tumour growth in Nude mice

PubMed Central

2011-01-01

Background Statins have long been used as anti-hypercholesterolemia drugs, but numerous lines of evidence suggest that they may also bear anti-tumour potential. We have recently demonstrated that it was possible to isolate cancer cells adapted to growth in the continuous presence of lovastatin. These cells grew more slowly than the statin-sensitive cells of origin. In the present study, we compared the ability of both statin-sensitive and statin-resistant cells to give rise to tumours in Nude mice. Methods HGT-1 human gastric cancer cells and L50 statin-resistant derivatives were injected subcutaneously into Nude mice and tumour growth was recorded. At the end of the experiment, tumours were recovered and marker proteins were analyzed by western blotting, RT-PCR and immunohistochemistry. Results L50 tumours grew more slowly, showed a strong decrease in cyclin B1, over-expressed collagen IV, and had reduced laminin 332, VEGF and CD34 levels, which, collectively, may have restricted cell division, cell adhesion and neoangiogenesis. Conclusions Taken together, these results showed that statin-resistant cells developed into smaller tumours than statin-sensitive cells. This may be reflective of the cancer restricting activity of statins in humans, as suggested from several retrospective studies with subjects undergoing statin therapy for several years. PMID:22107808

Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription

PubMed Central

Ng, Laura J.; Cropley, Jennifer E.; Pickett, Hilda A.; Reddel, Roger R.; Suter, Catherine M.

2009-01-01

Tumours and immortalized cells avoid telomere attrition by using either the ribonucleoprotein enzyme telomerase or a recombination-based alternative lengthening of telomeres (ALT) mechanism. Available evidence from mice suggests that the epigenetic state of the telomere may influence the mechanism of telomere maintenance, but this has not been directly tested in human cancer. Here we investigated cytosine methylation directly adjacent to the telomere as a marker of the telomere's epigenetic state in a panel of human cell lines. We find that while ALT cells show highly heterogeneous patterns of subtelomeric methylation, subtelomeric regions in telomerase-positive cells invariably show denser methylation than normal cells, being almost completely methylated. When compared to matched normal and ALT cells, telomerase-positive cells also exhibit reduced levels of the telomeric repeat-containing-RNA (TERRA), whose transcription originates in the subtelomere. Our results are consistent with the notion that TERRA may inhibit telomerase: the heavy cytosine methylation we observe in telomerase-positive cells may reflect selection for TERRA silencing in order to facilitate telomerase activity at the telomere. These data suggest that the epigenetic differences between telomerase-positive and ALT cells may underlie the mechanism of telomere maintenance in human tumorigenesis and highlight the broad reaching consequences of epigenetic dysregulation in cancer. PMID:19129228

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro.

PubMed

Griegel, S; Hong, C; Frötschl, R; Hülser, D F; Greger, V; Horsthemke, B; Rajewsky, M F

1990-07-15

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

X-Ray Reflected Spectra from Accretion Disk Models. II. Diagnostic Tools for X-Ray Observations

NASA Technical Reports Server (NTRS)

Garcia, J.; Kallman, T. R.; Mushotzky, R. F.

2011-01-01

We present a comprehensive study of the emission spectra from accreting sources. We use our new reflection code to compute the reflected spectra from an accretion disk illuminated by X-rays. This set of models covers different values of ionization parameter, solar iron abundance and photon index for the illuminating spectrum. These models also include the most complete and recent atomic data for the inner-shell of the iron and oxygen isonuclear sequences. We concentrate our analysis to the 2 - 10 keV energy region, and in particular to the iron K-shell emission lines. We show the dependency of the equivalent width (EW) of the Fe Ka with the ionization parameter. The maximum value of the EW is approx. 800 eV for models with log Epsilon approx. 1.5, and decreases monotonically as Epsilon increases. For lower values of Epsilon the Fe K(alpha) EW decreases to a minimum near log Epsilon approx. 0.8. We produce simulated CCD observations based on our reflection models. For low ionized, reflection dominated cases, the 2 -10 keV energy region shows a very broad, curving continuum that cannot be represented by a simple power-law. We show that in addition to the Fe K-shell emission, there are other prominent features such as the Si and S L(alpha) lines, a blend of Ar VIII-XI lines, and the Ca x K(alpha) line. In some cases the S xv blends with the He-like Si RRC producing a broad feature that cannot be reproduced by a simple Gaussian profile. This could be used as a signature of reflection.

DNA Methylation as an Epigenetic Factor in the Development and Progression of Polycythemia Vera

DTIC Science & Technology

2008-10-01

vera and idiopathic myelofibrosis. Pathol Biol ( Paris ). 2001;49:164-166. 2. Spivak JL. Diagnosis of the myeloproliferative disorders: resolving...leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid...in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF- Services Email this article to a friend Download to

Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

PubMed Central

Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

2012-01-01

Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

Establishment and proteomic characterization of a novel synovial sarcoma cell line, NCC-SS2-C1.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Endo, Makoto; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-05-01

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18-SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18-SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18-SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.

C-Lines of Stocking for Southern Bottomland Hardwoods: A Guide to Identifying Insuffiecient Stocking

Treesearch

J.C.G. Goelz

1997-01-01

A B-line on a stocking chart represents suggested residual stocking after thinning, or minimum full stocking. A stand at the C-line on a stocking chart will achieve the B-line after a period of growth usually specified as 10 years. Four C-lines that reflect insufficient stocking of southern bottomland hardwoods are presented. These C-lines represent 10,15,20, and 25...

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells.

PubMed

Yuan, FangJun; Zhou, Wenbo; Zhang, Jifa; Zhang, Zhiyun; Zou, Can; Huang, Ling; Zhang, YouShun; Dai, Zongqing

2008-08-01

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from -15 to -80 degrees C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.

Linear morphoea follows Blaschko's lines.

PubMed

Weibel, L; Harper, J I

2008-07-01

The aetiology of morphoea (or localized scleroderma) remains unknown. It has previously been suggested that lesions of linear morphoea may follow Blaschko's lines and thus reflect an embryological development. However, the distribution of linear morphoea has never been accurately evaluated. We aimed to identify common patterns of clinical presentation in children with linear morphoea and to establish whether linear morphoea follows the lines of Blaschko. A retrospective chart review of 65 children with linear morphoea was performed. According to clinical photographs the skin lesions of these patients were plotted on to standardized head and body charts. With the aid of Adobe Illustrator a final figure was produced including an overlay of all individual lesions which was used for comparison with the published lines of Blaschko. Thirty-four (53%) patients had the en coup de sabre subtype, 27 (41%) presented with linear morphoea on the trunk and/or limbs and four (6%) children had a combination of the two. In 55 (85%) children the skin lesions were confined to one side of the body, showing no preference for either left or right side. On comparing the overlays of all body and head lesions with the original lines of Blaschko there was an excellent correlation. Our data indicate that linear morphoea follows the lines of Blaschko. We hypothesize that in patients with linear morphoea susceptible cells are present in a mosaic state and that exposure to some trigger factor may result in the development of this condition.

Participation of an abnormality in the transforming growth factor-beta signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor.

PubMed

Zhang, Lei; Sato, Eiji; Amagasaki, Kenichi; Nakao, Atsuhito; Naganuma, Hirofumi

2006-07-01

Malignant glioma cells secrete and activate transforming growth factor-beta (TGFbeta) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFbeta was investigated. The authors examined the expression of downstream components of the TGFbeta receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFbeta1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFbeta-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFbeta1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase-4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFbeta1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFbeta1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21(cip1), p15(INK4B), CDK4, and cyclin D1 proteins was not altered by TGFbeta1, treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFbeta receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. These results suggest that the ability to resist TGFbeta-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFbeta signaling pathway.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

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.

Lung cancer cell lines: Useless artifacts or invaluable tools for medical science?

PubMed Central

Gazdar, Adi F.; Gao, Boning; Minna, John D.

2011-01-01

Multiple cell lines (estimated at 300–400) have been established from human small cell (SCLC) and non-small cell lung cancers (NSCLC). These cell lines have been widely dispersed to and used by the scientific community worldwide, with over 8000 citations resulting from their study. However, there remains considerable skepticism on the part of the scientific community as to the validity of research resulting from their use. These questions center around the genomic instability of cultured cells, lack of differentiation of cultured cells and absence of stromal–vascular–inflammatory cell compartments. In this report we discuss the advantages and disadvantages of the use of cell lines, address the issues of instability and lack of differentiation. Perhaps the most important finding is that every important, recurrent genetic and epigenetic change including gene mutations, deletions, amplifications, translocations and methylation-induced gene silencing found in tumors has been identified in cell lines and vice versa. These “driver mutations” represented in cell lines offer opportunities for biological characterization and application to translational research. Another potential shortcoming of cell lines is the difficulty of studying multistage pathogenesis in vitro.To overcome this problem, we have developed cultures from central and peripheral airways that serve as models for the multistage pathogenesis of tumors arising in these two very different compartments. Finally the issue of cell line contamination must be addressed and safeguarded against. A full understanding of the advantages and shortcomings of cell lines is required for the investigator to derive the maximum benefit from their use. PMID:20079948

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines.

PubMed

Hanu, Christine; Wong, Raimond; Sur, Ranjan K; Hayward, Joseph E; Seymour, Colin; Mothersill, Carmel

2017-02-01

To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure. Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells. We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1 Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5 Gy. These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.

Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.

PubMed

Hirabayashi, Masumi; Goto, Teppei; Tamura, Chihiro; Sanbo, Makoto; Hara, Hiromasa; Hochi, Shinichi

2014-03-07

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

PubMed

You, Seungkwon; Moon, Jai-Hee; Kim, Tae-Kyung; Kim, Sung-Chan; Kim, Jai-Woo; Yoon, Du-Hak; Kwak, Sungwook; Hong, Ki-Chang; Choi, Yun-Jaie; Kim, Hyunggee

2004-08-31

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

B7-H4 as a Target for Breast Cancer Immunotherapy

DTIC Science & Technology

2012-06-01

lymphoma and leukemia cell lines. CEM, Karpas 299, and TLBR -1, cell lines derived from acute T-cell lymphoblastic leukemia, large cell anaplastic...Accomplishments  Generation of human B7-H4-Fc fusion protein (antigen).  Discovery of a B7-H4 receptor on CEM, Karpas 299, and TLBR -1 cell lines...CEM Karpas 299 TLBR -1 Jurkat B7-H4R Figure 3. B7-H4 binding to human T-cell lymphoma cell lines. Red

Novel Tissue Protective Agents for the Treatment of Acute Radiation-induced BMF

DTIC Science & Technology

2013-03-01

induced apoptosis in the following hematopoietic cell lines: HL-60, NB-4 cells, 32Dc13 and EML cell line. Experimental design and methods: HL-60, a...et al., 1999). EML Cell line (ATCC CRL-11691), a bone marrow cell line obtained by immortalizing bone marrow cells from male BDF1 mice with a...Membrane preparations were made from HL-60, NB-4, 32Dc13 and EML cells attempts were made to co-immunoprecipitate the CD131 molecule with EPOR in

Human colon cancer HT-29 cell death responses to doxorubicin and Morus Alba leaves flavonoid extract.

PubMed

Fallah, S; Karimi, A; Panahi, G; Gerayesh Nejad, S; Fadaei, R; Seifi, M

2016-03-31

The mechanistic basis for the biological properties of Morus alba flavonoid extract (MFE) and chemotherapy drug of doxorubicin on human colon cancer HT-29 cell line death are unknown. The effect of doxorubicin and flavonoid extract on colon cancer HT-29 cell line death and identification of APC gene expression and PARP concentration of HT-29 cell line were investigated. The results showed that flavonoid extract and doxorubicin induce a dose dependent cell death in HT-29 cell line. MFE and doxorubicin exert a cytotoxic effect on human colon cancer HT-29 cell line by probably promoting or induction of apoptosis.

Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

PubMed

Norris, J S; Kohler, P O

1978-01-01

Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?

PubMed

Hughes, Peyton; Marshall, Damian; Reid, Yvonne; Parkes, Helen; Gelber, Cohava

2007-11-01

Increasing data demonstrate that cellular cross-contamination, misidentified cell lines, and the use of cultures at high-passage levels contribute to the generation of erroneous and misleading results as well as wasted research funds. Contamination of cell lines by other lines has been recognized and documented back to the 1950s. Based on submissions to major cell repositories in the last decade, it is estimated that between 18% and 36% of cell lines may be contaminated or misidentified. More recently, problems surrounding practices of over-subculturing cells are being identified. As a result of selective pressures and genetic drift, cell lines, when kept in culture too long, exhibit reduced or altered key functions and often no longer represent reliable models of their original source material. A review of papers showing significant experimental variances between low- and high-passage cell culture numbers, as well as contaminated lines, makes a strong case for using verified, tested cell lines at low- or defined passage numbers. In the absence of cell culture guidelines, mandates from the National Institutes of Health (NIH) and other funding agencies or journal requirements, it becomes the responsibility of the scientific community to perform due diligence to ensure the integrity of cell cultures used in research.

MS-HRM assay identifies high levels of epigenetic heterogeneity in human immortalized cell lines.

PubMed

Putnik, Milica; Wojdacz, Tomasz K; Pournara, Angeliki; Vahter, Marie; Wallberg, Annika E

2015-04-15

Immortalized cell lines are widely used in genetic and epigenetic studies, from exploration of basic molecular pathways to evaluation of disease-specific cellular properties. They are also used in biotechnology, e.g., in drug toxicity tests and vaccine production. Cellular and genetic uniformity is the main feature of immortalized cell lines and it has been particularly advantageous in functional genomic research, which has in recent years been expanded to include epigenetic mechanisms of gene expression regulation. Using the MS-HRM technique, we demonstrated heterogeneity in locus-specific methylation patterns in different cell cultures of four human cell lines: HEK293, HEK293T, LCL and DU145. Our results show that some human immortalized cell lines consist of cells that differ in the methylation status of specific loci, i.e., that they are epigenetically heterogeneous. We show that even two cultures of the same cell line obtained from different laboratories can differ in the methylation status of the specific loci. The results indicated that epigenetic uniformity of the cell lines cannot be assumed in experiments which utilize cell cultures and that the methylation status of the specific loci in the immortalized cell lines should be re-characterized and carefully profiled before epigenetic studies are performed. Copyright © 2015 Elsevier B.V. All rights reserved.

Potentiation by Tumor Necrosis Factor of Mitoxantrone Cytotoxicity to Human Ovarian Cancer Cell Lines

PubMed Central

Parodi, Silvio; Billi, Giovanna; Oliva, Cristina; Venturing, Marco; Noviello, Elvira; Conte, PierFranco

1992-01-01

The cytotoxic activity of human recombinant tumor necrosis factor (rHuTNF) (from 0.01 to 10000 U/ml) was assayed on six human ovarian cancer cell lines and one human cervical carcinoma cell line using a crystal violet assay. rHuTNF was cytotoxic to four cell lines (A2780, A2774, SW626, PAD, while 3 cell lines (IGROV1, SKOV3, Mel80) were marginally sensitive to its activity. However, under the same experimental conditions rHuTNF markedly enhanced the cytotoxicity of mitoxantrone, a chemotherapeutic drug targeted at DNA topoisomerase II, in six cell lines. The potentiation of mitoxantrone cytotoxicity was not caused by increased drug accumulation after rHuTNF treatment. No significant increase in cytotoxicity to Me180 cell line was seen when rHuTNF was added to mitoxantrone. PMID:1517145

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera).

PubMed

Goodman, Cynthia L; Ringbauer, Joseph A; Li, Yao-Fa; Lincoln, Tamra Reall; Stanley, David

2017-05-01

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.