Cytogenetic characterization of three cell lines derived from primary cervical tumors of different histologic grade

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

Hann, E.; Beauregard, L.; Mikumo, R.

Braum et al.(1993) established three cell lines from keratinizing and nonkeratinizing cervical carcinomas. These cell lines were subsequently analyzed for growth properties and the physical state of the human papillomavirus type 16 genome. TC140, derived from a keratinizing cervical tumor, contains human papillomavirus type 16 in the episomal state. TC-146A and TC-146B, derived from a nonkeratinizing large-cell cervical carcinoma, contain human papillomavirus type 16 in the integrated state. The goal of the present study was to cytogenetically characterize these cell lines, developed from cervical carcinoma with a defined histopathology, in order to shed additional light on the biological basis ofmore » the histological and clinical heterogeneity of cervical cancers. Information on solid tumors has been limited because they are often difficult to culture and the karyotypes on the available metaphases are often complex with unidentifiable markers. The chromosomes of these three cell lines were characterized in the present study using GTG-banding. For cell line 140, the most striking chromosomal abnormalities noted were the presence of an i(5p) or i(12p) marker, an isochromosome 8q marker and multiple copies of chromosome 9. For cell line 146A, the most notable chromosomal abnormalities noted were the presence of a marker chromosome 7 with additional materials present on the long arms, an isochomosome of the long arms of chromosome 8 and a question of chromosome 19 markers. For cell line 146B, the most notable chromosomal abnormalities were found to be a deleted X chromosome, a marker chromosome 7 with additional material on the long arm, an isochromosome 8q marker, and isochromosome 16q marker and one or more copies of an isochromosome 17q marker. Fluorescent in situ hybridization experiments performed using select probes further corroborate the results of the above-mentioned conventional cytogenetic studies.« less

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

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.

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

2010-12-01

The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

Increasing anthraquinone production by overexpression of 1-deoxy-D: -xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia.

PubMed

Quevedo, Carla; Perassolo, María; Alechine, Eugenia; Corach, Daniel; Giulietti, Ana María; Talou, Julián Rodriguez

2010-07-01

A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-D: -xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

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.

Defects in the Fanconi Anemia Pathway and Chromatid Cohesion in Head and Neck Cancer.

PubMed

Stoepker, Chantal; Ameziane, Najim; van der Lelij, Petra; Kooi, Irsan E; Oostra, Anneke B; Rooimans, Martin A; van Mil, Saskia E; Brink, Arjen; Dietrich, Ralf; Balk, Jesper A; Ylstra, Bauke; Joenje, Hans; Feller, Stephan M; Brakenhoff, Ruud H

2015-09-01

Failure to repair DNA damage or defective sister chromatid cohesion, a process essential for correct chromosome segregation, can be causative of chromosomal instability (CIN), which is a hallmark of many types of cancers. We investigated how frequent this occurs in head and neck squamous cell carcinoma (HNSCC) and whether specific mechanisms or genes could be linked to these phenotypes. The genomic instability syndrome Fanconi anemia is caused by mutations in any of at least 16 genes regulating DNA interstrand crosslink (ICL) repair. Since patients with Fanconi anemia have a high risk to develop HNSCC, we investigated whether and to which extent Fanconi anemia pathway inactivation underlies CIN in HNSCC of non-Fanconi anemia individuals. We observed ICL-induced chromosomal breakage in 9 of 17 (53%) HNSCC cell lines derived from patients without Fanconi anemia. In addition, defective sister chromatid cohesion was observed in five HNSCC cell lines. Inactivation of FANCM was responsible for chromosomal breakage in one cell line, whereas in two other cell lines, somatic mutations in PDS5A or STAG2 resulted in inadequate sister chromatid cohesion. In addition, FANCF methylation was found in one cell line by screening an additional panel of 39 HNSCC cell lines. Our data demonstrate that CIN in terms of ICL-induced chromosomal breakage and defective chromatid cohesion is frequently observed in HNSCC. Inactivation of known Fanconi anemia and chromatid cohesion genes does explain CIN in the minority of cases. These findings point to phenotypes that may be highly relevant in treatment response of HNSCC. ©2015 American Association for Cancer Research.

The vitamin C:vitamin K3 system - enhancers and inhibitors of the anticancer effect.

PubMed

Lamson, Davis W; Gu, Yu-Huan; Plaza, Steven M; Brignall, Matthew S; Brinton, Cathy A; Sadlon, Angela E

2010-12-01

The oxidizing anticancer system of vitamin C and vitamin K₃ (VC:VK₃, producing hydrogen peroxide via superoxide) was combined individually with melatonin, curcumin, quercetin, or cholecalciferol (VD₃) to determine interactions. Substrates were LNCaP and PC-3 prostate cancer cell lines. Three of the tested antioxidants displayed differences in cell line cytotoxicity. Melatonin combined with VC:VK₃ quenched the oxidizing effect, while VC:VK₃ applied 24 hours after melatonin showed no quenching. With increasing curcumin concentrations, an apparent combined effect of VC:VK₃ and curcumin occurred in LNCaP cells, but not PC-3 cells. Quercetin alone was cytotoxic on both cell lines, but demonstrated an additional 50-percent cytotoxicity on PC-3 cells when combined with VC:VK₃. VD₃ was effective against both cell lines, with more effect on PC-3. This effect was negated on LNCaP cells with the addition of VC:VK₃. In conclusion, a natural antioxidant can enhance or decrease the cytotoxicity of an oxidizing anticancer system in vitro, but generalizations about antioxidants cannot be made.

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

PubMed Central

Tosato, G; Tanner, J; Jones, K D; Revel, M; Pike, S E

1990-01-01

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions. Images PMID:2159561

Cytotoxic cardiac glycosides and other compounds from Asclepias syriaca.

PubMed

Araya, Juan J; Kindscher, Kelly; Timmermann, Barbara N

2012-03-23

Phytochemical investigation of the dried biomass of Asclepias syriaca afforded five new compounds (1-5), along with 19 known structures. Overall, the secondary metabolites isolated and identified from this plant showed a wide structural diversity including pentacyclic triterpenes, cardiac glycosides, flavonoid glycosides, lignans, a phenylethanoid, and a glycosylated megastigmane. In addition, the isolates were tested against the cancer breast cell line Hs578T, and those showing IC(50) values lower than 50 μM (1 and 6-9) were further investigated in three additional breast cancer cell lines (MCF-7, T47D, and Sk-Br-3) and the normal breast cell line Hs578Bst.

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells.

PubMed

Mayr, Christian; Wagner, Andrej; Neureiter, Daniel; Pichler, Martin; Jakab, Martin; Illig, Romana; Berr, Frieder; Kiesslich, Tobias

2015-06-23

The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG. The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1. EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 μM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2. EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines. Graphical abstract Summary illustration.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

PubMed

Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

2015-04-01

The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

PubMed

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

2002-03-01

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

Characteristics of cell lines established from human colorectal carcinoma.

PubMed

Park, J G; Oie, H K; Sugarbaker, P H; Henslee, J G; Chen, T R; Johnson, B E; Gazdar, A

1987-12-15

We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.

A human beta cell line with drug inducible excision of immortalizing transgenes

PubMed Central

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

Identification of Metastatic Tumor Stem Cell

DTIC Science & Technology

2010-09-01

addition to a tumor stem cell , an existence of a metastatic stem cell is predicted. Despite the critical importance of the concept, this idea has not been...isolating stem cell population from a unique set of breast tumor cell lines and by examining their metastatic behavior in an animal model. The overall...will (i) isolate stem - cell population from non-metastatic and metastatic cells of a pair of syngenic breast tumor cell lines, and test their metastatic

Mitoguazone induces apoptosis via a p53-independent mechanism.

PubMed

Davidson, K; Petit, T; Izbicka, E; Koester, S; Von Hoff, D D

1998-08-01

Mitoguazone (methylglyoxal bisguanylhydrazone, methyl-GAG or MGBG) is a synthetic polycarbonyl derivative with activity in patients with Hodgkin's and non-Hodgkin's lymphoma, head and neck cancer, prostate cancer, and esophageal cancer. Mitoguazone has also recently been documented to have activity in patients with AIDS-related lymphoma. Among anticancer drugs, mitoguazone has a unique mechanism of action via interference with the polyamine biosynthetic pathway. Polyamines stabilize DNA structure by non-covalent cross-bridging between phosphate groups on opposite strands. In addition, mitoguazone causes uncoupling of oxidative phosphorylation. In this study, the ability of mitoguazone to induce apoptosis by inhibiting the polyamine pathway was assessed in three Burkitt's lymphoma cell lines (Raji, Ramos and Daudi) and one prostate carcinoma cell line (MPC 3). Additional evaluations were performed in two human breast cancer cell lines (MCF7 with wild-type p53 and VM4K with mutated p53) to determine whether the p53 tumor suppressor gene was required for efficient apoptosis induction. The present study demonstrated that mitoguazone induces apoptosis in all the different human cancer cell lines tested in a concentration- and time-dependent way, and triggers a p53-independent programmed cell death in the human breast cancer MCF7 cell line.

Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo.

PubMed Central

Riedlinger, J; Grencis, R K; Wakelin, D

1986-01-01

T-cell lines specific for infective muscle larvae antigens of the intestinal nematode Trichinella spiralis have been generated in vitro. These antigen-specific T-cell lines express the L3T4+ Ly2- phenotype and secrete the lymphokines IL-2, IL-3 and gamma-IFN. They are stable in culture for up to 15 weeks and are protective when adoptively transferred into naive recipients. As few as 2 x 10(5) T. spiralis-specific tract. In addition, intestinal mastocytosis and peripheral blood eosinophilia were accelerated after adoptive transfer of T. spiralis-specific T-cell lines. PMID:2423438

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.

Estrogen effects of Daldinia concentrica and Psathyrella efflorescens extracts in vitro.

PubMed

Benie, Tanon; Kouakou, Koffi; Thieulant, Marie-Lise

2008-02-28

Daldinia concentrica and Psathyrella efflorescens are two fungi used in African traditional medicine. In the present study, their extracts were evaluated for their steroid activities in estrogen- or androgen-dependent cell lines using as endpoints steroid-dependent transcriptional activity and cell proliferation. Treatment of human breast cancer MCF-7 cells with 15 or 30 microg/ml of Daldinia concentrica or Psathyrellaefflorescens extracts in the absence of 17beta-estradiol (E2) significantly increased the transcriptional activity of an estrogen receptor (ER)-dependent reporter gene, in the same range as E2. Similar data were obtained in gonadotrope cell line alpha-T3-1. All the effects were prevented by the pure estrogen antagonist, ICI 182,780. In the absence of steroid addition, the two extracts induced cell proliferation of ER-dependent MCF-7 and Ishikawa Var-I cell lines by approximately 100% of the E2 response. Combination treatments with E2 showed no competitive or additive effects in the two latter cell lines. Interestingly, the extracts had no androgen-like response in androgen receptor (AR)-positive and ER-negative MDA-MB231 cells, suggesting that fungi effects are estrogen specific and extracts are not toxic at used concentrations. Results provided evidence that Daldinia concentrica or Psathyrellaefflorescens extracts induce estrogen-like effects in ER-positive cell lines, which could be responsible of the effects observed in vivo.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

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

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

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

PubMed

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

2015-06-12

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

Cell 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.

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.

Lidocaine and ropivacaine, but not bupivacaine, demethylate deoxyribonucleic acid in breast cancer cells in vitro.

PubMed

Lirk, P; Hollmann, M W; Fleischer, M; Weber, N C; Fiegl, H

2014-07-01

Lidocaine demethylates deoxyribonucleic acid (DNA) in breast cancer cells. This modification of epigenetic information may be of therapeutic relevance in the perioperative period, because a decrease in methylation can reactivate tumour suppressor genes and inhibit tumour growth. The objectives of this study were to determine the effect of two amide local anaesthetics, ropivacaine and bupivacaine, on methylation in two breast cancer cell lines and to detect whether the combination of lidocaine with the chemotherapy agent 5-aza-2'-deoxycytidine (DAC) would result in additive demethylating effects. Breast cancer cell lines BT-20 [oestrogen receptor (ER)-negative] and MCF-7 (ER-positive) were incubated with lidocaine, bupivacaine, and ropivacaine to assess demethylating properties. Then, we tested varying concentrations of lidocaine and DAC to assess whether their demethylating effects were additive. Cell numbers and global methylation status were analysed. Lidocaine decreased methylation in BT-20 and MCF-7 cells, ropivacaine decreased methylation in BT-20 cells, and bupivacaine had no demethylating effect. When combined, lidocaine and DAC had additive demethylating effects. At clinically relevant doses, lidocaine and ropivacaine exert demethylating effects on specific breast cancer cell lines, but bupivacaine does not. The demethylating effects of lidocaine and DAC are indeed additive. © The Author [2014]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of total hydroalcholic extract of Nigella sativa and its n-hexane and ethyl acetate fractions on ACHN and GP-293 cell lines.

PubMed

Shahraki, Samira; Khajavirad, Abolfazl; Shafei, Mohammad Naser; Mahmoudi, Mahmoud; Tabasi, Nafisa Sadat

2016-01-01

Medicinal plants are noted for their many advantages including the ability to treat diseases such as cancer. In this study, we examined the antitumor effect of the medicinal plant Nigella sativa on the morphology, survival, and apoptosis of ACHN (human renal adenocarcinoma) and GP-293 (normal renal epithelial) cell lines. From a hydroalcoholic extract of N. sativa, n-hexane and ethyl acetate fractions were extracted. Cells were treated with various concentrations of total hydroalcholic extract and n-hexane and ethyl acetate fractions; cell viability, morphological changes, and apoptosis were then determined. Results were presented as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was applied for the statistical analysis of the data. The total extract and the fractions in a dose- and time-dependent manner reduced the cell viability in ACHN with no effect on the GP-293 cell line. In addition, the total extract resulted in more morphological changes in the ACHN cells compared to the GP-293 cells. The effect of the total extract in inducing apoptosis after 48 hours in the ACHN cell line was greater than in GP-293. In addition, the effect of the two fractions was lower than the total extract at all used concentrations. Therefore, the effect of total extract and n-hexane and ethyl acetate fractions of N. sativa on cell viability and apoptosis in the ACHN cell line is greater than in the GP-293 cell line. However, the effect of the total extract is higher than either of the two fractions on their own.

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).

[Biological behavior of hypopharyngeal carcinoma].

PubMed

Zhou, L X

1997-01-01

Hypopharyngeal squamous cell carcinomas (HPC) has an extremely poor prognosis. Characteristics of cell lines of head and neck squamous cell carcinomas including HPC were studied by various methods, e.g., chemosensitivity test and the immunohistochemistry staining method, to determine whether this poor prognosis is due to the biological behavior of this cancer. An HPC cell line was found to be resistant to anti tumor drugs, i.e., PEP, MTX and CPM and moderately sensitive to CDDP, 5-FU and ADM. Thermoresistance to hyperthermatic treatment and weak expression of ICAM-1 on the HPC cell line were observed. DNA synthesis by the HPC cell line was induced by stimulation with a low concentration of EGF and the amount of EGFR on these HPC cells was very high. In addition, cyclinD1 overexpression was found in the HPC cell line. Based on the above findings, further analysis of hypopharyngeal carcinoma cells and the development of a new treatment modality to control tumor growth and metastatic factors influencing the poor outcome are necessary to improve the prognosis of this cancer.

APELA promotes tumour growth and cell migration in ovarian cancer in a p53-dependent manner.

PubMed

Yi, Yuyin; Tsai, Shu-Huei; Cheng, Jung-Chien; Wang, Evan Y; Anglesio, Michael S; Cochrane, Dawn R; Fuller, Megan; Gibb, Ewan A; Wei, Wei; Huntsman, David G; Karsan, Aly; Hoodless, Pamela A

2017-12-01

APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

PubMed

Dussault, Nathalie; Ducas, Eric; Racine, Claudia; Jacques, Annie; Paré, Isabelle; Côté, Serge; Néron, Sonia

2008-11-01

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

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

PubMed

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

1996-01-01

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

A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro

PubMed Central

Rathore, Kusum; Cekanova, Maria

2015-01-01

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo. PMID:26451087

A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

PubMed

Rathore, Kusum; Cekanova, Maria

2015-01-01

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

Pediatric Glioblastoma Therapies Based on Patient-Derived Stem Cell Resources

DTIC Science & Technology

2013-10-01

stem cell lines have been successfully isolated from adults, in this proposal we aim to isolate and characterize GSC populations from pediatric patients. In the past two years we have successfully derived and cultured eight patient-derived pediatric glioma stem cell lines. In the past year we have continued molecular and phenotypic characterization of these lines. This characterization included analysis of gene expression and patient-specific gene mutations, and also proof-of-concept shRNA screens. In addition we have begun to identify candidate

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

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

Wairagu, Peninah M.; Institute of Lifestyle Medicine, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do 220-701; Nuclear Receptor Research Consortium, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do 220-701

2014-05-09

Highlights: • The 48 NR genes and 48 biological anti-cancer targets are profiled in paired-cells. • Growth inhibition by NR ligands or TKIs is target receptor level-dependent. • T0901317 with gefitinib/PHA665752 shows additive growth inhibition in lung cells. - Abstract: Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where eachmore » pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.« less

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes.

PubMed

Tomoyasu, Chihiro; Imamura, Toshihiko; Tomii, Toshihiro; Yano, Mio; Asai, Daisuke; Goto, Hiroaki; Shimada, Akira; Sanada, Masashi; Iwamoto, Shotaro; Takita, Junko; Minegishi, Masayoshi; Inukai, Takeshi; Sugita, Kanji; Hosoi, Hajime

2018-05-21

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph + B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph - B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.

Establishment of two new scirrhous gastric cancer cell lines: analysis of factors associated with disseminated metastasis.

PubMed Central

Yashiro, M.; Chung, Y. S.; Nishimura, S.; Inoue, T.; Sowa, M.

1995-01-01

Determination of the differences between cell lines which are derived from a primary tumour and a disseminated metastatic lesion from the same patient may aid in elucidating the factors associated with disseminated metastases. We report on the establishment and characterisation of two new scirrhous gastric cancer cell lines, designated OCUM-2M and OCUM-2D, derived from a 49-year-old female. OCUM-2M was derived from a primary gastric tumour, and OCUM-2D was derived from a sample of disseminated metastasis. The two cell lines were derived from the same patient. We investigated biological differences between the two cell lines to study mechanisms involved in disseminated metastasis. The growth activity of OCUM-2D cells as determined by doubling time and tumorigenicity was greater than that of OCUM-2M cells. The level of epidermal growth factor receptor (EGFR) expression in OCUM-2D cells was about twice that of OCUM-2M cells and the growth of OCUM-2D cells was stimulated more by epidermal growth factor (EGF) than that of OCUM-2M cells. The invasive activity of OCUM-2D cells was higher than that of OCUM-2M cells and was increased after addition of transforming growth factor-beta 1 (TGF-beta 1). An increase in the number of attached and spreading cells was found following the addition of 10 ng ml-1 TGF-beta 1. These findings suggest that high growth and invasive activity may play an important role in disseminated metastasis and that EGF and TGF-beta 1, which affect the growth and invasive activity of OCUM-2D cells, might be factors associated with metastasis in scirrhous gastric carcinoma. The two cell lines OCUM-2M and OCUM-2D should be beneficial for analysing mechanisms of tumour progression. Images Figure 1 Figure 5 Figure 6 Figure 7 Figure 10 PMID:7577468

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

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

Population differences in the rate of proliferation of international HapMap cell lines.

PubMed

Stark, Amy L; Zhang, Wei; Zhou, Tong; O'Donnell, Peter H; Beiswanger, Christine M; Huang, R Stephanie; Cox, Nancy J; Dolan, M Eileen

2010-12-10

The International HapMap Project is a resource for researchers containing genotype, sequencing, and expression information for EBV-transformed lymphoblastoid cell lines derived from populations across the world. The expansion of the HapMap beyond the four initial populations of Phase 2, referred to as Phase 3, has increased the sample number and ethnic diversity available for investigation. However, differences in the rate of cellular proliferation between the populations can serve as confounders in phenotype-genotype studies using these cell lines. Within the Phase 2 populations, the JPT and CHB cell lines grow faster (p < 0.0001) than the CEU or YRI cell lines. Phase 3 YRI cell lines grow significantly slower than Phase 2 YRI lines (p < 0.0001), with no widespread genetic differences based on common SNPs. In addition, we found significant growth differences between the cell lines in the Phase 2 ASN populations and the Han Chinese from the Denver metropolitan area panel in Phase 3 (p < 0.0001). Therefore, studies that separate HapMap panels into discovery and replication sets must take this into consideration. Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells

PubMed Central

Vié, Nadia; Copois, Virginie; Bascoul-Mollevi, Caroline; Denis, Vincent; Bec, Nicole; Robert, Bruno; Fraslon, Caroline; Conseiller, Emmanuel; Molina, Franck; Larroque, Christian; Martineau, Pierre; Del Rio, Maguy; Gongora, Céline

2008-01-01

Background Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. Results We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. Conclusion These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy. PMID:18221502

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

PubMed

Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

2006-02-17

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

PubMed

Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-07-01

Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

Pharmacogenomic Characterization and Isobologram Analysis of the Combination of Ascorbic Acid and Curcumin-Two Main Metabolites of Curcuma longa-in Cancer Cells.

PubMed

Ooko, Edna; Kadioglu, Onat; Greten, Henry J; Efferth, Thomas

2017-01-01

Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa . This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53 +/+ and HCT116p53 -/- colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription ( TFAM, TCERG1, RGS13, C11orf31 ), apoptosis-regulation ( CRADD, CDK7, CDK19, CD81, TOM1 ) signal transduction ( NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27 ) DNA repair ( TOPBP1, RPA2 ), mRNA metabolism ( RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2 ), and transporter genes ( ABCA1 ) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA.

Pharmacogenomic Characterization and Isobologram Analysis of the Combination of Ascorbic Acid and Curcumin—Two Main Metabolites of Curcuma longa—in Cancer Cells

PubMed Central

Ooko, Edna; Kadioglu, Onat; Greten, Henry J.; Efferth, Thomas

2017-01-01

Curcuma longa has long been used in China and India as anti-inflammatory agent to treat a wide variety of conditions and also as a spice for varied curry preparations. The chemoprofile of the Curcuma species exhibits the presence of varied phytochemicals with curcumin being present in all three species but AA only being shown in C. longa. This study explored the effect of a curcumin/AA combination on human cancer cell lines. The curcumin/AA combination was assessed by isobologram analysis using the Loewe additivity drug interaction model. The drug combination showed additive cytotoxicity toward CCRF-CEM and CEM/ADR5000 leukemia cell lines and HCT116p53+/+ and HCT116p53−/− colon cancer cell line, while the glioblastoma cell lines U87MG and U87MG.ΔEGFR showed additive to supra-additive cytotoxicity. Gene expression profiles predicting sensitivity and resistance of tumor cells to induction by curcumin and AA were determined by microarray-based mRNA expressions, COMPARE, and hierarchical cluster analyses. Numerous genes involved in transcription (TFAM, TCERG1, RGS13, C11orf31), apoptosis-regulation (CRADD, CDK7, CDK19, CD81, TOM1) signal transduction (NR1D2, HMGN1, ABCA1, DE4ND4B, TRIM27) DNA repair (TOPBP1, RPA2), mRNA metabolism (RBBP4, HNRNPR, SRSF4, NR2F2, PDK1, TGM2), and transporter genes (ABCA1) correlated with cellular responsiveness to curcumin and ascorbic acid. In conclusion, this study shows the effect of the curcumin/AA combination and identifies several candidate genes that may regulate the response of varied cancer cells to curcumin and AA. PMID:28210221

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.

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus.

PubMed

Ganassin, R C; Sanders, S M; Kennedy, C J; Joyce, E M; Bols, N C

1999-01-01

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.

Rhizome of Anemarrhena asphodeloides as mediators of the eco-friendly synthesis of silver and gold spherical, face-centred cubic nanocrystals and its anti-migratory and cytotoxic potential in normal and cancer cell lines.

PubMed

Lee, Hyun A; Castro-Aceituno, Veronica; Abbai, Ragavendran; Moon, Seong Soo; Kim, Yeon-Ju; Simu, Shakina Yesmin; Yang, Deok Chun

2018-03-29

The water extract of Anemarrhena asphodeloides, the traditional oriental medicinal plant, mediated the eco-friendly synthesis of silver nanoparticles (Aa-AgNPs) and gold nanoparticles (Aa-AuNPs). First, its therapeutic rhizome was powdered prior to water extraction and then silver, gold nanoparticles were synthesized. Aa-AgNPs and Aa-AuNPs were found to be spherical, face-centred cubic nanocrystals with a Z-average hydrodynamic diameter of 190 and 258 nm, respectively. In addition, proteins and aromatic biomolecules were the plausible players associated with the production and stabilization of Aa-AgNPs; instead, phenolic compounds were responsible for the synthesis and stability of Aa-AuNPs. In vitro cytotoxic analysis revealed that up to 50 μg.mL -1 concentration Aa-AuNPs did not exhibit any toxicity on 3T3-L1, HT29 and MCF7 cell lines, while being specifically cytotoxic to A549 cell line. On the contrary, Aa-AgNPs displayed a significantly higher toxicity in comparison to Aa-AuNPs in all cell lines specially MCF7 cell line. Since cancer cells were more sensitive to Aa-Au/AgNPs treatments, further evaluation was done in order to determine their anticancer potential. Reactive oxygen species (ROS) generation was not affected by Aa-AuNPs, on the other hand, Aa-AgNPs treatment exhibited a higher potential to induce oxidative stress in A549 cells than HT29 and MCF7 cells. In addition, Aa-Ag/AuNPs reduced cell migration in A549 cells at 10 and 50 μg.mL -1 , respectively. So far, this is the only report uncovering the ability of A. asphodeloides to synthesize silver and gold nanoparticles with anticancer potential and also indirectly enabling its large-scale utilization with value addition.

Establishment and characterization of the NCC-SS1-C1 synovial sarcoma cell line.

PubMed

Kito, Fusako; Oyama, Rieko; Takai, Yoko; Sakumoto, Marimu; Shiozawa, Kumiko; Qiao, Zhiwei; Uehara, Takenori; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-04-01

Synovial sarcoma is an aggressive mesenchymal malignancy characterized by unique gene fusions. Tissue culture cells are essential tools for further understanding tumorigenesis and anti-cancer drug development; however, only a limited number of well-characterized synovial sarcoma cell lines exist. Thus, the objective of this study was to establish a patient-derived synovial sarcoma cell line. We established a synovial sarcoma cell line from tumor tissue isolated from a 72-year-old female patient. Prepared cells were analyzed for the presence of gene fusions by fluorescence in situ hybridization, RT-PCR, and karyotyping. In addition, the resulting cell line was characterized by viability, short tandem repeat, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell line were examined by mass spectrometric protein expression profiling and KEGG pathway analysis. Our analyses revealed that the primary tumor and NCC-SS1-C1 cell line harbored the SS18-SSX1 fusion gene typical of synovial sarcoma and similar proteomics profiles. In vitro analyses also confirmed that the established cell line harbored invasive, colony-forming, and spheroid-forming potentials. Moreover, drug screening with chemotherapeutic agents and tyrosine kinase inhibitors revealed that doxorubicin, a subset of tyrosine kinase inhibitors, and several molecular targeting drugs markedly decreased NCC-SS1-C1 cell viability. Results from the present study support that the NCC-SS1-C1 cell line will be an effective tool for sarcoma research.

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

NASA Astrophysics Data System (ADS)

Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

2006-06-01

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

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)

Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

2014-03-01

Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line's higher tolerance of nsPEF exposure.

GE132+Natural: Novel promising dietetic supplement with antiproliferative influence on prostate, colon, and breast cancer cells.

PubMed

Okic-Djordjevic, I; Trivanovic, D; Krstic, J; Jaukovic, A; Mojsilovic, S; Santibanez, J F; Terzic, M; Vesovic, D; Bugarski, D

2013-01-01

Natural products have been investigated for promising new leads in pharmaceutical development. The purpose of this study was to analyze the biological effect of GE132+Natural, a novel supplement consisting of 5 compounds: Resveratrol, Ganoderma lucidum, Sulforaphane, Lycopene and Royal jelly. The antiproliferative activity of GE132+Natural was tested on 3 different human cancer cell lines: MCF7 (breast cancer cells), PC3 (prostate cancer cells), and SW480 (colon cancer cells), as well as on EA.hy 926 (normal human endothelial cell line). In addition, the cytotoxicity of GE132+- Natural on the proliferation of primary human mesenchymal stem cells isolated from dental pulp (DP=MSC), along with its in vitro impact on different peripheral blood parameters, was determined. The results revealed high antiproliferative activity of GE132+Natural on all tested cancer cell lines (PC3, MCF7 and SW480), as well as on the EA.hy 926 endothelial cell line in a dose-dependent manner. However, applied in a wide range of concentrations GE132+Natural did not affect both the proliferation of primary mesenchymal stem cells and the peripheral blood cells counts. The data obtained demonstrated that GE132+Natural is effective in inhibiting cancer cell proliferation, indicating its potential beneficial health effects. In addition, the results pointed that adult mesenchymal stem cells might be valuable as a test system for evaluating the toxicity and efficacy of new medicines or chemicals.

Characterization of three new serous epithelial ovarian cancer cell lines

PubMed Central

Ouellet, Véronique; Zietarska, Magdalena; Portelance, Lise; Lafontaine, Julie; Madore, Jason; Puiffe, Marie-Line; Arcand, Suzanna L; Shen, Zhen; Hébert, Josée; Tonin, Patricia N; Provencher, Diane M; Mes-Masson, Anne-Marie

2008-01-01

Background Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946). Methods In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice. Results While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease. Conclusion This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. PMID:18507860

Antiproliferative activity of synthetic fatty acid amides from renewable resources.

PubMed

dos Santos, Daiane S; Piovesan, Luciana A; D'Oca, Caroline R Montes; Hack, Carolina R Lopes; Treptow, Tamara G M; Rodrigues, Marieli O; Vendramini-Costa, Débora B; Ruiz, Ana Lucia T G; de Carvalho, João Ernesto; D'Oca, Marcelo G Montes

2015-01-15

In the work, the in vitro antiproliferative activity of a series of synthetic fatty acid amides were investigated in seven cancer cell lines. The study revealed that most of the compounds showed antiproliferative activity against tested tumor cell lines, mainly on human glioma cells (U251) and human ovarian cancer cells with a multiple drug-resistant phenotype (NCI-ADR/RES). In addition, the fatty methyl benzylamide derived from ricinoleic acid (with the fatty acid obtained from castor oil, a renewable resource) showed a high selectivity with potent growth inhibition and cell death for the glioma cell line-the most aggressive CNS cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.

PubMed Central

Burns, J. E.; Baird, M. C.; Clark, L. J.; Burns, P. A.; Edington, K.; Chapman, C.; Mitchell, R.; Robertson, G.; Soutar, D.; Parkinson, E. K.

1993-01-01

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8390283

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

PubMed

Dehler, Carola E; Boudinot, Pierre; Martin, Samuel A M; Collet, Bertrand

2016-08-01

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

PubMed

Kojima, Reiji; Ohno, Tatsukuni; Iikura, Motoyasu; Niki, Toshiro; Hirashima, Mitsuomi; Iwaya, Keichi; Tsuda, Hitoshi; Nonoyama, Shigeaki; Matsuda, Akio; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

2014-01-01

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

Subcloning of three osteoblastic cell lines with distinct differentiation phenotypes from the mouse osteoblastic cell line KS-4.

PubMed

Yamashita, T; Ishii, H; Shimoda, K; Sampath, T K; Katagiri, T; Wada, M; Osawa, T; Suda, T

1996-11-01

Three distinct osteoblastic cell lines (KS418, KS460, and KS483) were subcloned from the mouse osteoblastic KS-4 cells, which possessed the abilities not only to differentiate into mature osteoblasts, but also to support osteoclast differentiation in coculture with spleen cells. The order of the magnitude of the basal alkaline phosphatase (ALP) activity was KS483 > KS418 > KS460. KS483 cells were also more differentiated than KS418 and KS460 in terms of ALP activity and osteocalcin production, when cultured in growth medium containing 10% fetal bovine serum. In long-term culture, KS418 and KS483 apparently differentiated into mature osteoblasts and formed calcified nodules without addition of beta-glycerophosphate. Electron microscopic analysis demonstrated that calcification occurring in the nodules was initiated in the matrix vesicles as observed in bone formation in vivo. Nodule formation and mineral deposition occurred simultaneously in the presence of beta-glycerophosphate, but the former always preceded the latter without addition of beta-glycerophosphate. In contrast, KS460 cells did not show time-dependent increases of ALP activity, type I collagen expression and osteocalcin production, which were induced by treatment with recombinant osteogenic protein-1 (OP-1). The three cell lines similarly supported osteoclast differentiation in coculture with spleen cells in response to 1,25-dihydroxyvitamin D3. These results indicate that the three cell lines subcloned from the original KS-4 cells represent phenotypically distinct osteoblasts during osteoblast differentiation, but are equipped similarly with the capacity to support osteoclast differentiation. The subcloned cells of the KS-4 series may provide useful systems in which to study osteoblast differentiation and function.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

CellLineNavigator: a workbench for cancer cell line analysis

PubMed Central

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

2013-01-01

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

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.

Human Monocytes in the Presence of Interferons Alpha2a and Gamma Are Potent Killers of Serous Ovarian Cancer Cell Lines in Combination with Paclitaxel and Carboplatin

PubMed Central

Johnson, Chase L.; Zoon, Kathryn C.

2015-01-01

Interferons (IFNs) play an important role in immune surveillance of tumors; however, their efficacy in the treatment of malignancies has been limited. Monocytes are mononuclear phagocytes that are critical to the generation of an innate immune response to tumors. The authors and others have shown that treatment of tumor cell lines in vitro and in vivo with human monocytes primed with type I and type II IFNs results in killing. We now expand on this work, in an extended panel of ovarian cancer cell lines. In this study, we hypothesized that there would be variable sensitivity amongst cell lines to the killing properties of monocytes and IFNs. To this end, we explored the interactions of IFN primed monocytes in conjunction with the standard of therapy for ovarian cancer, taxane, and platinum-based chemotherapeutics. Using 6 ovarian cancer cell lines, we demonstrated that there is variation from cell line to cell line in the ability of IFN-α2a and IFN-γ primed monocytes to synergistically kill target tumor cells, and further, there is an additive killing effect when target cells are treated with both IFN primed monocytes and chemotherapy. PMID:25068849

Pharmacological importance, characterization and applications of gold and silver nanoparticles synthesized by Panax ginseng fresh leaves.

PubMed

Singh, Priyanka; Singh, Hina; Ahn, Sungeun; Castro-Aceituno, Verónica; Jiménez, Zuly; Simu, Shakina Yesmin; Kim, Yeon Ju; Yang, Deok Chun

2017-11-01

Previously, we showed the rapid and eco-friendly synthesis of gold and silver nanoparticles within 3 and 45 min by fresh leaves extract of herbal medicinal plant Panax ginseng. In addition, we characterized the nanoparticles in terms of shape, size, morphology and stability by FE-TEM, EDX, elemental mapping, SEAD, XRD and particles size analysis. In addition of this, we showed their antimicrobial, anti-coagulant, and biofilm inhibition activity of nanoparticles. Continuing our previous study, here we highlight the further characterization and biomedical applications of P. ginseng leaf-mediated gold and silver nanoparticles. We characterized the nanoparticles further in terms of active functional group and capping layer, surface charge, and temperature stability. Based on these factors, we explored the nanoparticles for antioxidant efficacy, biocompatibility in HaCaT cells, 3T3-L1 pre-adipocytes cells, for anticancer efficacy in A549 lung cancer and B16BL6 skin melenoma cancer cell lines and for anti-inflammation efficacy in RAW 264.7 cell lines. Based on our findings, we suggest that the P. ginseng-mediated gold nanoparticles have high antioxidant activity and highly biocompatibility in HaCaT cells, 3T3-L1 pre-adipocytes cells, RAW 264.7 cells lines and could be considered for future drug delivery carriers. The silver nanoparticles also showed high potent antioxidant efficacy, additionally it showed high anticancer effect in A549 lung cancer and B16BL6 skin melenoma cancer cell lines as compared to precursor salts. Moreover, both gold and silver nanoparticles have anti-inflammatory efficacies in RAW 264.7 cells. Thus, the study may provide useful insights of P. ginseng leaves extract-mediated biocompatible gold and silver nanoparticles and improving their applicability in designing nanoparticles carrier systems for drug delivery applications.

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

PubMed

Grobarczyk, Benjamin; Franco, Bénédicte; Hanon, Kevin; Malgrange, Brigitte

2015-10-01

Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

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

PubMed Central

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

2017-01-01

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

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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

Toki, Yasumichi; Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp; Tanaka, Hiroki

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncatedmore » peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.« less

Characterization and differentiation of human embryonic stem cells.

PubMed

Carpenter, M K; Rosler, E; Rao, M S

2003-01-01

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line

PubMed Central

Foreman, Jennifer E.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2009-01-01

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis. PMID:19660859

A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

PubMed

Solarte, Víctor A; Rosas, Jaiver E; Rivera, Zuly J; Arango-Rodríguez, Martha L; García, Javier E; Vernot, Jean-Paul

2015-01-01

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

No preclinical rationale for IGF1R directed therapy in chondrosarcoma of bone.

PubMed

Peterse, Elisabeth F P; Cleven, Arjen H G; De Jong, Yvonne; Briaire-de Bruijn, Inge; Fletcher, Jonathan A; Danen, Erik H J; Cleton-Jansen, Anne-Marie; Bovée, Judith V M G

2016-07-14

Chondrosarcoma is a malignant cartilage forming bone tumour for which no effective systemic treatment is available. Previous studies illustrate the need for a better understanding of the role of the IGF pathway in chondrosarcoma to determine if it can be a target for therapy, which was therefore explored in this study. Expression of mediators of IGF1R signalling and phosphorylation status of IRS1 was determined in chondrosarcoma cell lines by qRT-PCR and western blot. The effect of activation and inhibition of IGF1R signalling on downstream targets was assessed by western blot. Ten chondrosarcoma cell lines were treated with OSI-906 (IGF1R and IR dual inhibitor) after which cell proliferation and migration were determined by a viability assay and the xCELLigence system, respectively. In addition, four chondrosarcoma cell lines were treated with a combination of doxorubicin and OSI-906. By immunohistochemistry, IGF1R expression levels were determined in tissue microarrays of 187 cartilage tumours and ten paraffin embedded cell lines. Mediators of IGF1R signalling are heterogeneously expressed and phosphorylated IRS1 was detected in 67 % of the tested chondrosarcoma cell lines, suggesting that IGF1R signalling is active in a subset of chondrosarcoma cell lines. In the cell lines with phosphorylated IRS1, inhibition of IGF1R signalling decreased phosphorylated Akt levels and increased IGF1R expression, but it did not influence MAPK or S6 activity. In line with these findings, treatment with IGF1R/IR inhibitors did not impact proliferation or migration in any of the chondrosarcoma cell lines, even upon stimulation with IGF1. Although synergistic effects of IGF1R/IR inhibition with doxorubicin are described for other cancers, our results demonstrate that this was not the case for chondrosarcoma. In addition, we found minimal IGF1R expression in primary tumours in contrast to the high expression detected in chondrosarcoma cell lines, even if both were derived from the same tumour, suggesting that in vitro culturing upregulates IGF1R expression. The results from this study indicate that the IGF pathway is not essential for chondrosarcoma growth, migration or chemoresistance. Furthermore, IGF1R is only minimally expressed in chondrosarcoma primary tumours. Therefore, the IGF pathway is not expected to be an effective therapeutic target for chondrosarcoma of bone.

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

PubMed Central

Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure

PubMed Central

Kuijk, Ewart W.; Rasmussen, Shauna; Blokzijl, Francis; Huch, Meritxell; Gehart, Helmuth; Toonen, Pim; Begthel, Harry; Clevers, Hans; Geurts, Aron M.; Cuppen, Edwin

2016-01-01

The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah−/− Il2rg−/− rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat. PMID:26915950

RERG suppresses cell proliferation, migration and angiogenesis through ERK/NF-κB signaling pathway in nasopharyngeal carcinoma.

PubMed

Zhao, Weilin; Ma, Ning; Wang, Shumin; Mo, Yingxi; Zhang, Zhe; Huang, Guangwu; Midorikawa, Kaoru; Hiraku, Yusuke; Oikawa, Shinji; Murata, Mariko; Takeuchi, Kazuhiko

2017-06-28

Nasopharyngeal carcinoma (NPC) is a malignancy of the head and neck that is prevalent in Southeast Asia and southern China. Recent studies in epigenetics suggest that DNA methylation plays a pivotal role in the onset and progression of cancer. Combining the methyl-DNA binding domain capture technique and cDNA microarray analysis, we identified a unique hypermethylated gene, RERG (Ras-like estrogen-regulated growth inhibitor), that was down-regulated in NPC tissues. RERG is a tumor suppressor gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types. RERG expression was assessed in human subjects (NPC primary tissues and non-cancer tissues) and cell lines (NPC cell lines and an immortalized epithelial cell line NP460). Further, we investigated the methylation rate of RERG in both human subject and cell lines. 5-Aza-2'-deoxycytidine (Aza) or combined with trichostatin A (TSA) were treated to three NPC cell lines (HK1, C666-1 and HK1_EBV). In addition, the role of RERG in NPC cells and its underlying mechanisms were explored by overexpression of RERG in NPC cell lines. RERG was significantly down-regulated in NPC cancer nests compared to normal nasopharyngeal epithelium cells. Furthermore, the RERG promoter was frequently methylated in NPC tissues and cell lines. The RERG methylation rate yielded an area under the curve (AUC) of receiver operating characteristic (ROC) curve was 0.897 (95%CI: 0.818-0.976). The down-regulation of RERG was restored in NPC cells treated with Aza and TSA. In addition, ectopic expression of RERG in NPC cell lines resulted in a significant suppression of cell proliferation, clonogenicity, migration and invasion. RERG-overexpressing cells showed significantly slower growth and less angiogenesis in tumor xenografts in nude mice. RERG suppressed the ERK/NF-κB signaling pathway and inhibited tumor growth and angiogenesis with down-regulation of MMPs and IL8 in tumors of nude mouse xenografts. Our results suggest that RERG is frequently silenced by promoter CpG methylation in NPC, and acts as a functional tumor suppressor by suppressing the ERK/NF-κB signaling pathway. These findings support the potential use of RERG as a novel molecular target in NPC therapy.

An immortalized steroidogenic goat granulosa cell line as a model system to study the effect of the endoplasmic reticulum (ER)-stress response on steroidogenesis.

PubMed

Yang, Diqi; Wang, Lei; Lin, Pengfei; Jiang, Tingting; Wang, Nan; Zhao, Fan; Chen, Huatao; Tang, Keqiong; Zhou, Dong; Wang, Aihua; Jin, Yaping

2017-02-16

With granulosa and theca cells, the ovaries are responsible for producing oocytes and secreting sex steroids such as estrogen and progesterone. Endoplasmic reticulum stress (ERS) plays an important role in follicle atresia and embryo implantation. In this study, goat granulosa cells were isolated from medium-sized (4-6 mm) healthy follicles. Primary granulosa cells were immortalized by transfection with human telomerase reverse transcriptase (hTERT) to establish a goat granulosa cell line (hTERT-GGCs). These hTERT-GGCs expressed hTERT and had relatively long telomeres at passage 50. Furthermore, hTERT-GGCs expressed the gonadotropin receptor genes CYP11A1, StAR, and CYP19A1, which are involved in steroidogenesis. Additionally, progesterone was detectable in hTERT-GGCs. Although the proliferation potential of hTERT-GGCs significantly improved, there was no evidence to suggest that the hTERT-GGCs are tumorigenic. In addition, thapsigargin (Tg) treatment led to a significant dose-dependent decrease in progesterone concentration and steroidogenic enzyme expression. In summary, we successfully generated a stable goat granulosa cell line. We found that Tg induced ERS in hTERT-GGCs, which reduced progesterone production and steroidogenic enzyme expression. Future studies may benefit from using this cell line as a model to explore the molecular mechanisms regulating steroidogenesis and apoptosis in goat granulosa cells.

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

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

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

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Data set of the protein expression profiles of Luminal A, Claudin-low and overexpressing HER2+ breast cancer cell lines by iTRAQ labelling and tandem mass spectrometry

PubMed Central

Calderón-González, Karla Grisel; Valero Rustarazo, Ma Luz; Labra-Barrios, Maria Luisa; Bazán-Méndez, César Isaac; Tavera-Tapia, Alejandra; Herrera-Aguirre, Marí;aEsther; Sánchez del Pino, Manuel M.; Gallegos-Pérez, José Luis; González-Márquez, Humberto; Hernández-Hernández, Jose Manuel; León-Ávila, Gloria; Rodríguez-Cuevas, Sergio; Guisa-Hohenstein, Fernando; Luna-Arias, Juan Pedro

2015-01-01

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article “Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry” (Calderón-González et al. [1] in press). PMID:26217805

Dual effects of N-acetyl-L-cysteine dependent on NQO1 activity: Suppressive or promotive of 9,10-phenanthrenequinone-induced toxicity

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

Toyooka, Tatsushi; Shinmen, Takuya; Aarts, Jac M.M.J.G.

2012-11-01

A typical antioxidant, N-acetyl-L-cysteine (NAC) generally protects cells from oxidative damage induced by reactive oxygen species (ROS). 9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, produces ROS in redox cycling following two-electron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1), which has been considered as a cause of its cyto- and genotoxicity. In this study, we show that NAC unexpectedly augments the toxicity of 9,10-PQ in cells with low NQO1 activity. In four human skin cell lines, the expression and the activity of NQO1 were lower than in human adenocarcinoma cell lines, A549 and MCF7. In the skin cells, the cytotoxicitymore » of 9,10-PQ was significantly enhanced by addition of NAC. The formation of DNA double strand breaks accompanying phosphorylation of histone H2AX, was also remarkably augmented. On the other hand, the cyto- and genotoxicity were suppressed by addition of NAC in the adenocarcinoma cells. Two contrasting experiments: overexpression of NQO1 in CHO-K1 cells which originally expressed low NQO1 levels, and knock‐down of NQO1 in the adenocarcinoma cell line A549 by transfection of RNAi, also showed that NAC suppressed 9,10-PQ-induced toxicity in cell lines expressing high NQO1 activity and enhanced it in cell lines with low NQO1 activity. The results suggested that dual effects of NAC on the cyto- and genotoxicity of 9,10-PQ were dependent on tissue-specific NQO1 activity. -- Highlights: ► NAC augmented the cytotoxicity of 9,10-PQ in skin cell lines. ► 9,10-PQ-induced DSBs accompanying γ-H2AX were also augmented by NAC. ► NAC suppressed the cyto- and genotoxicity of 9,10-PQ in adenocarcinoma cell lines. ► The dual effects of NAC on toxicity of 9,10-PQ were dependent on NQO1 activity.« less

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

Cytotoxic effects of ultra-diluted remedies on breast cancer cells.

PubMed

Frenkel, Moshe; Mishra, Bal Mukund; Sen, Subrata; Yang, Peiying; Pawlus, Alison; Vence, Luis; Leblanc, Aimee; Cohen, Lorenzo; Banerji, Pratip; Banerji, Prasanta

2010-02-01

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers.

PubMed Central

Kukowska-Latallo, J F; Bielinska, A U; Johnson, J; Spindler, R; Tomalia, D A; Baker, J R

1996-01-01

Starburst polyamidoamine dendrimers are a new class of synthetic polymers with unique structural and physical characteristics. These polymers were investigated for the ability to bind DNA and enhance DNA transfer and expression in a variety of mammalian cell lines. Twenty different types of polyamidoamine dendrimers were synthesized, and the polymer structure was confirmed using well-defined analytical techniques. The efficiency of plasmid DNA transfection using dendrimers was examined using two reporter gene systems: firefly luciferase and bacterial beta-galactosidase. The transfections were performed using various dendrimers, and levels of expression of the reporter protein were determined. Highly efficient transfection of a broad range of eukaryotic cells and cell lines was achieved with minimal cytotoxicity using the DNA/dendrimer complexes. However, the ability to transfect cells was restricted to certain types of dendrimers and in some situations required the presence of additional compounds, such as DEAE-dextran, that appeared to alter the nature of the complex. A few cell lines demonstrated enhanced transfection with the addition of chloroquine, indicating endosomal localization of the complexes. The capability of a dendrimer to transfect cells appeared to depend on the size, shape, and number of primary amino groups on the surface of the polymer. However, the specific dendrimer most efficient in achieving transfection varied between different types of cells. These studies demonstrate that Starburst dendrimers can transfect a wide variety of cell types in vitro and offer an efficient method for producing permanently transfected cell lines. Images Fig. 1 Fig. 2 Fig. 4 PMID:8643500

Growth Inhibitory Effect of Palatine Tonsil-derived Mesenchymal Stem Cells on Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Lim, Yun-Sung; Lee, Jin-Choon; Lee, Yoon Se; Wang, Soo-Geun

2012-01-01

Objectives Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. However, the effect of human MSCs on the growth of human tumors is not well understood. The purpose of this study is to confirm the growth effect of palatine tonsil-derived MSCs (TD-MSCs) on head and neck squamous cell carcinoma (HNSCC) cell lines and to elucidate the mechanism of their action. Methods TD-MSCs were isolated from patient with chronic tonsillitis and tonsillar hypertrophy. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied and cocultured with isolated palatine tonsil-derived MSC. The growth inhibitory effect of MSCs on HNSCC cell lines was tested through methylthiazolyldiphenyl-tetrazolium (MTT) assay. The apoptosis induction effect of MSCs on cell lines was assessed with flow cytometry and reverse transcriptase (RT)-PCR. Results Palatine tonsil-derived MSCs exhibited a growth inhibitory effect on both cell lines. Cell cycle analysis showed an accumulation of tumor cells predominantly in G0/G1 phase with an increase in concentration of TD-MSCs, which was confirmed by increased mRNA expression of cell cycle negative regulator p21. Apoptosis of tumor cells increased significantly as concentration of cocultured TD-MSCs increased. Additionally, mRNA expression of caspase 3 was upregulated with increased concentration of TD-MSCs. Conclusion TD-MSCs have a potential growth inhibitory effect on HNSCC cell lines in vitro by inducing apoptotic cell death and G1 phase arrest of cell lines. PMID:22737289

Developing global regression models for metabolite concentration prediction regardless of cell line.

PubMed

André, Silvère; Lagresle, Sylvain; Da Sliva, Anthony; Heimendinger, Pierre; Hannas, Zahia; Calvosa, Éric; Duponchel, Ludovic

2017-11-01

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Effects of Newcastle Disease Virus Strains AF2240 and V4-UPM on Cytolysis and Apoptosis of Leukemia Cell Lines

PubMed Central

Alabsi, Aied M.; Bakar, Siti Aishah Abu; Ali, Rola; Omar, Abdul Rahman; Bejo, Mohd Hair; Ideris, Aini; Ali, Abdul Manaf

2011-01-01

Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD50 values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line. PMID:22272097

Selective Targeting of CTNNB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs

PubMed Central

Uitdehaag, Joost C. M.; de Roos, Jeroen A. D. M.; van Doornmalen, Antoon M.; Prinsen, Martine B. W.; Spijkers-Hagelstein, Jill A. P.; de Vetter, Judith R. F.; de Man, Jos; Buijsman, Rogier C.; Zaman, Guido J. R.

2015-01-01

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes. PMID:26018524

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

PubMed Central

Swift, Brenna E.; Williams, Brent A.; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A.; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

2012-01-01

Background Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. Design and Methods The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89–99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted. PMID:22271890

Neural Stem Cells Injected into the Sound-Damaged Cochlea Migrate Throughout the Cochlea and Express Markers of Hair Cells, Supporting Cells, and Spiral Ganglion Cells

PubMed Central

Corliss, Deborah A.; Gray, Brianna; Anderson, Julia K.; Bobbin, Richard P.; Snyder, Evan Y.; Cotanche, Douglas A.

2007-01-01

Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, it suggests that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea. PMID:17659854

Metallated porphyrin-doped conjugated polymer nanoparticles for efficient photodynamic therapy of brain and colorectal tumor cells.

PubMed

Ibarra, Luis Exequiel; Porcal, Gabriela Valeria; Macor, Lorena Paola; Ponzio, Rodrigo Andrés; Spada, Ramiro Martin; Lorente, Carolina; Chesta, Carlos Alberto; Rivarola, Viviana Alicia; Palacios, Rodrigo Emiliano

2018-03-01

 Assess biocompatibility, uptake and photodynamic therapy (PDT) mechanism of metallated porphyrin doped conjugated polymer nanoparticles (CPNs) in human brain and colorectal tumor cells and macrophages. CPNs were developed employing 9,9-dioctylfluorene-alt-benzothiadiazole, an amphiphilic polymer (PS-PEG-COOH),  and platinum octaethylporphyrin. T98G, SW480 and RAW 264.7 cell lines were exposed to CPNs to assess uptake and intracellular localization. Additionally, a PDT protocol using CPNs was employed for the in vitro killing of cancer and macrophage cell lines. CPNs were well incorporated into glioblastoma and macrophage cells with localization in lysosomes. SW480 cells were less efficient incorporating CPNs with localization in the plasma membrane. In all cell lines PDT treatment was efficient inducing oxidative stress that triggered apoptosis.

Production of Multiple Growth Factors by a Newly Established Human Thyroid Carcinoma Cell Line

PubMed Central

Yoshida, Yataro; Ohashi, Kensaku; Sano, Emiko; Kobayashi, Hisataka; Endo, Keigo; Naruto, Masanobu; Nakamura, Toru

1992-01-01

A multiple growth factor‐producing tumor cell line (NIM‐1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM‐1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM‐1‐conditioned medium (NIM‐1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony‐stimulating factor (CSF)‐dependent cell lines, NFS60‐KX and TF‐1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte‐CSF (G‐CSF), granulocyte/macrophage‐CSF (GM‐CSF) and interleukin(IL)‐6 mRNAs in NIM‐1 cells. Enzyme‐linked immunosorbent assays (ELISA) using NIM‐1CM also confirmed the production of IL‐la and a small amount of IL‐1β besides G‐CSF, GM‐CSF and IL‐6 in NIM‐1 cells. In addition, unexpected production of IL‐11 in NIM‐1 cells was detected by northern blot hybridization analysis and by bioassay using an IL‐11‐dependent cell line. Therefore, NIM‐1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM‐CSF, IL‐6 and IL‐11. PMID:1372885

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines.

PubMed

Nagakawa, O; Murakami, K; Yamaura, T; Fujiuchi, Y; Murata, J; Fuse, H; Saiki, I

2000-07-31

Membrane-type metalloproteinase-1 (MT1-MMP) is a transmembrane metalloproteinase, which activates proMMP-2 and expressed on the cell surface in many invasive cancer cells. We investigated the expression of MT1-MMP in prostate cancer cell lines. MT1-MMP protein and mRNA were expressed in PC-3, DU-145 and TSU-pr1 cells (androgen-independent prostate cancer cell lines), but in LNCaP cells (androgen-dependent prostate cancer cell line). MT1-MMP protein was negative and mRNA was low to detect by RT-PCR. Cell lysate of PC-3 cleaved proMMP-2 to the active form. In addition, both hepatocyte growth factor (HGF) and gastrin-releasing peptide (GRP) increased Matrigel invasion and induced the expression of MT1-MMP protein in DU-145 prostate cancer cells. These results suggest that MT1-MMP is indeed the tumor-specific activator of proMMP-2 in androgen-independent prostate cancer cells and plays an important role in the invasive properties of prostate cancer cells.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed

Hunt, L A

1980-08-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein.

Altered synthesis and processing of oligosaccharides of vesicular stomatitis virus glycoprotein in different lectin-resistant Chinese hamster ovary cell lines.

PubMed Central

Hunt, L A

1980-01-01

To determine the particular intracellular steps in the glycosylation of the vesicular stomatitis virus (VSV) glycoprotein that were altered in several lectin-resistant CHO cell lines, VSV-infected parental and mutant cells were pulse-labeled for 30 and 120 min with [3H]mannose and [3H]glucosamine. Cell-associated viral glycopeptides were analyzed by gel filtration combined with specific glycosidase digestions and compared with the corresponding mature virion oligosaccharides. The intracellular glycosylation of the VSV glycoprotein in a mutant cell line resistant to phytohemagglutinin was identical to that in the normal cells except for a complete block in processing at a specific step in the final trimming of the oligomannosyl core from five to three mannoses. The results demonstrated that a double-mutant cell line selected from the phytohemagglutinin-resistant cells for resistance to concanavalin A had an additional defect in one of the earliest stages of glycosylation, resulting in smaller precursor oligosaccharides linked to protein. Images PMID:6255177

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2).

PubMed

Kim, Joo-Shin; Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 microg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 microg/mL of each polysaccharide isolate to the cell line containing 80 microg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity.

Cytoprotective effect of polysaccharide isolated from different mushrooms against 7-ketocholesterol induced damage in mouse liver cell line (BNL CL. 2)

PubMed Central

Chung, Hau Yin; Na, Keun

2007-01-01

Cytoprotective ability of polysaccharides isolated from different edible mushrooms was investigated on the 7-ketocholesterol-induced damaged cell line. Polysaccharide extracts from six different edible mushrooms-Flammulina velutipes, Peurotus ostreatus, Lentinus edodes, Agrocybe aegerita, Agaricus blazei, and Cordyceps militaris- were prepared by hot water extraction and alcohol precipitation. Cytoprotective ability was evaluated by measuring the viable cells of the normal embryonic liver cell line (BNL CL. 2) in the presence of 7-ketocholesterol. At 80 µg/mL of 7-ketocholesterol, cytotoxicity was very high with a loss of 98% of viable cells after 20 h of incubation. With the addition of 200 µg/mL of each polysaccharide isolate to the cell line containing 80 µg/mL of 7-ketocholesterol, polysaccharide isolates from both Flammulina velutipes and Peurotus ostreatus could significantly inhibit the 7-ketochoelsterol-induced cytotoxicity in the cells. But other polysaccharide isolates were not effective in inhibiting cell damage caused by the oxLDL-induced cytotoxicity. PMID:20368935

Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.

PubMed

Mantovani, Fernanda B; Morrison, Jodi A; Mutsaers, Anthony J

2016-05-31

Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

Comparison of the effects of photon versus carbon ion irradiation when combined with chemotherapy in vitro.

PubMed

Schlaich, Fabian; Brons, Stephan; Haberer, Thomas; Debus, Jürgen; Combs, Stephanie E; Weber, Klaus-Josef

2013-11-06

Characterization of combination effects of chemotherapy drugs with carbon ions in comparison to photons in vitro. The human colon adenocarcinoma cell line WiDr was tested for combinations with camptothecin, cisplatin, gemcitabine and paclitaxel. In addition three other human tumour cell lines (A549: lung, LN-229: glioblastoma, PANC-1: pancreas) were tested for the combination with camptothecin. Cells were irradiated with photon doses of 2, 4, 6 and 8 Gy or carbon ion doses of 0.5, 1, 2 and 3 Gy. Cell survival was assessed using the clonogenic growth assay. Treatment dependent changes in cell cycle distribution (up to 12 hours post-treatment) were measured by FACS analysis after propidium-iodide staining. Apoptosis was monitored for up to 36 hours post-treatment by Nicoletti-assay (with qualitative verification using DAPI staining). All cell lines exhibited the well-known increase of killing efficacy per unit dose of carbon ion exposure, with relative biological efficiencies at 10% survival (RBE10) ranging from 2.3 to 3.7 for the different cell lines. In combination with chemotherapy additive toxicity was the prevailing effect. Only in combination with gemcitabine or cisplatin (WiDr) or camptothecin (all cell lines) the photon sensitivity was slightly enhanced, whereas purely independent toxicities were found with the carbon ion irradiation, in all cases. Radiation-induced cell cycle changes displayed the generally observed dose-dependent G2-arrest with little effect on S-phase fraction for all cell lines for photons and for carbon ions. Only paclitaxel showed a significant induction of apoptosis in WiDr cell line but independent of the used radiation quality. Combined effects of different chemotherapeutics with photons or with carbon ions do neither display qualitative nor substantial quantitative differences. Small radiosensitizing effects, when observed with photons are decreased with carbon ions. The data support the idea that a radiochemotherapy with common drugs and carbon ion irradiation might be as feasible as respective photon-based protocols. The present data serve as an important radiobiological basis for further combination experiments, as well as clinical studies on combination treatments.

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

NASA Technical Reports Server (NTRS)

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

1997-01-01

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

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.

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines.

PubMed

Watanabe, Masatada; Ohno, Shuji; Nakajin, Shizuo

2012-04-05

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA. Copyright © 2012 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

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

PubMed Central

Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

2010-01-01

Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

PubMed Central

Kasper, Grit; Reule, Matthias; Tschirschmann, Miriam; Dankert, Niels; Stout-Weider, Karen; Lauster, Roland; Schrock, Evelin; Mennerich, Detlev; Duda, Georg N; Lehmann, Kerstin E

2007-01-01

Background Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. Methods The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. Results Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. Conclusion These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis. PMID:17233884

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

Generation of stable cell line by using chitosan as gene delivery system.

PubMed

Şalva, Emine; Turan, Suna Özbaş; Ekentok, Ceyda; Akbuğa, Jülide

2016-08-01

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Somji, Seema; Zhou, Xu Dong; Mehus, Aaron; Sens, Mary Ann; Garrett, Scott H.; Lutz, Krista L.; Dunlevy, Jane R.; Zheng, Yun; Sens, Donald. A.

2009-01-01

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd+2) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd+2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd+2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd+2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd+2 transformed cells have both similarities and differences in their phenotype. PMID:19921857

Active microwave negative-index metamaterial transmission line with gain.

PubMed

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

2011-11-11

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

Comparative label-free LC-MS/MS analysis of colorectal adenocarcinoma and metastatic cells treated with 5-fluorouracil.

PubMed

Bauer, Kerry M; Lambert, Paul A; Hummon, Amanda B

2012-06-01

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC(50) 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increased with treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion-associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

1997-01-01

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

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines.

PubMed

Cirigliano, Stéfano M; Díaz Bessone, María I; Berardi, Damián E; Flumian, Carolina; Bal de Kier Joffé, Elisa D; Perea, Silvio E; Farina, Hernán G; Todaro, Laura B; Urtreger, Alejandro J

2017-01-01

Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment. The human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity. We demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment. Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

PubMed Central

Solarte, Víctor A.; Rosas, Jaiver E.; Rivera, Zuly J.; Arango-Rodríguez, Martha L.; García, Javier E.; Vernot, Jean-Paul

2015-01-01

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC. PMID:26609531

Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

NASA Astrophysics Data System (ADS)

Gordon, Geoffrey; Lo, Chun-Min

2007-03-01

Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

PubMed

Garcia, J A; Ferreira, H L; Vieira, F V; Gameiro, R; Andrade, A L; Eugênio, F R; Flores, E F; Cardoso, T C

2017-06-01

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy. © 2015 John Wiley & Sons Ltd.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

PubMed Central

Hematulin, Arunee; Meethang, Sutiwan; Utapom, Kitsana; Wongkham, Sopit; Sagan, Daniel

2018-01-01

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint. PMID:29541168

MELK as a potential target to control cell proliferation in triple-negative breast cancer MDA-MB-231 cells

PubMed Central

Li, Gang; Yang, Mei; Zuo, Li; Wang, Mei-Xing

2018-01-01

Maternal embryonic leucine zipper kinase (MELK) is an important regulator in tumorigenesis of human breast cancer, and if silenced leads to programmed cell death in specific breast cancer cell lines, including MDA-MB-231 cells. In the present study, RNA interference, proliferation assay and semi-quantification of cell cycle relative proteins were performed to determine the effects of MELK in human breast cancer cells. Data demonstrated that the highest level of MELK protein in the MDA-MB-231 cell line among eight breast cancer cell lines. The sensitivity of MELK small interfering-RNA varied in different breast cancer cell lines, but MELK silencing resulted in marked suppression of proliferation of triple-negative breast cancer (TNBC) and non-TNBC cells. Specific silencing of MELK caused G2 arrest in TNBC MDA-MB-231 and HCC1143 cells, and G1 arrest in non-TNBC T47D and MCF7 cells. Notably, the knockdown of MELK did not induce apoptosis in HCC1143 cells, indicated by the lack of caspase-3 expression. In addition, in response to MELK silencing, cyclin B and cyclin D1 were downregulated in four breast cancer cell lines. Furthermore, the silencing of MELK resulted in the upregulation of p21, p27 and phosphorylated (p)-c-Jun N-terminal kinase (JNK) in HCC1143 TNBC cells, and downregulation of p21 and p-JNK in T47D non-TNBC cells. Additionally, MELK protein was markedly suppressed in non-TNBC cells in response to estrogen deprivation. The findings from the present study suggested that MELK may be a potential target in MDA-MB-231 cells, although genetic knockdown of MELK resulted in inhibitory effects on proliferation of TNBC and non-TNBC cells. MELK exert its effect on different breast cancer cells via arrest of different cell cycle phases and therefore mediated by different mediators, which may be involved in the crosstalk with MELK signaling and with the estrogen receptor signaling pathway. PMID:29805690

A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.

PubMed

Wang, Fei; Guo, Tao; Jiang, Hongmei; Li, Ruobi; Wang, Ting; Zeng, Ni; Dong, Guanghui; Zeng, Xiaowen; Li, Daochuan; Xiao, Yongmei; Hu, Qiansheng; Chen, Wen; Xing, Xiumei; Wang, Qing

2018-06-01

Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

PubMed

Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

2015-05-01

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. © 2014 Wiley Periodicals, Inc.

Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

PubMed Central

Suchorolski, Martin T.; Paulson, Thomas G.; Sanchez, Carissa A.; Hockenbery, David; Reid, Brian J.

2013-01-01

Background Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. Methodology/Principal Findings We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. Conclusions/Significance Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells. PMID:23460817

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

PubMed

Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

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

Campos, D; Peeters, W; Bussink, J

2016-06-15

Purpose: To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. Methods: Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of amore » HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. Results: An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. Conclusion: The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.« less

Common genetic variation drives molecular heterogeneity in human iPSCs.

PubMed

Kilpinen, Helena; Goncalves, Angela; Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard; Stegle, Oliver; Gaffney, Daniel J

2017-06-15

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

Establishment and characterization of a novel dedifferentiated liposarcoma cell line, NDDLS-1.

PubMed

Ariizumi, Takashi; Ogose, Akira; Kawashima, Hiroyuki; Hotta, Tetsuo; Li, Guidong; Xu, Yongjun; Hirose, Takanori; Endo, Naoto

2011-08-01

We established a dedifferentiated liposarcoma cell line (NDDLS-1) that produces interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF). The parental tumor showed high leukemoid reactions. The NDDLS-1 cell line was established from a pleural effusion associated with a lung metastasis. Pleomorphic tumor cells arranged in a haphazard growth pattern were seen in xenograft tumors. Numerous inflammatory cells including neutrophils or eosinophils were present throughout the tumor cells. This finding resembled the dedifferentiated area of the parental tumor. The mice bearing NDDLS-1 showed marked leukocytosis. In addition, the NDDLS-1 cells expressed IL-6 and G-CSF at both the mRNA and protein levels, while the NDDLS-1 cells produced near normal levels of tumor necrosis factor alpha (TNF-α). In the cytogenetic analysis, both the parental tumor and the NDDLS-1 cells showed a ring or giant marker chromosomes. The NDDLS-1 cell line demonstrated the amplification and expression of both MDM2 and CDK4 by fluorescence in situ hybridization and immunohistochemical analysis. The NDDLS-1 cell line is consistent with the parental dedifferentiated liposarcoma, and it should therefore be useful for further investigations of human dedifferentiated liposarcomas. © 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

Henrietta Lacks, HeLa cells, and cell culture contamination.

PubMed

Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

2009-09-01

Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.

Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics of pig lines generated by crossing an MPS pulmonary lesion selected Landrace line and a highly immune capacity selected Large White line.

PubMed

Borjigin, Liushiqi; Shimazu, Tomoyuki; Katayama, Yuki; Watanabe, Kouichi; Kitazawa, Haruki; Roh, Sang-Gun; Aso, Hisashi; Katoh, Kazuo; Uchida, Takafumi; Suda, Yoshihito; Sakuma, Akiko; Nakajo, Mituru; Suzuki, Keiichi

2016-08-01

To understand the influence of crossbreeding on Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics, two crossbred lines were characterized. One crossbred line, LaWa, was generated by crossing the MPS pulmonary lesion selected Landrace line (La) and the highly immune-selected Large White line (Wa). The second crossbred line, LaWb, was generated by crossing the La line and the nonselected Large White line (Wb). The crossbred LbWb line (nonselected Landrace line × nonselected Large White line) and the La line were used as controls. The LaWa and LaWb lines had an intermediate level of MPS lung lesions between La and LbWb lines, although the difference was not statistically significant. After stimulation with sheep red blood cells (SRBCs), the LaWb and LaWa lines showed immune characteristics similar to that of the La line; the number of monocytes in peripheral blood increased, while B cells, T cells, secretion of SRBC-specific immunoglobulin G, and interleukin (IL)-13 decreased. Additionally, the number of natural killer (NK) cells and the expression of IL-4 and IL-17 were significantly higher in the LaWb and LaWa lines, respectively. These data suggested that crossbreeding of La and Wa lines resulted in the inheritance of some of the selected immune responses. © 2015 Japanese Society of Animal Science.

Effects of phytoestrogens on the trophoblast tumour cell lines BeWo and Jeg3.

PubMed

Plessow, D; Waldschläger, J; Richter, D U; Jeschke, U; Bruer, G; Briese, V; Friese, K

2003-01-01

Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind to estrogen receptors in humans. With this study we tested the effects of the phytoestrogens genistein and daidzein in cell proliferation and the production of progesterone and hCG in trophoblast tumour cells of the cell lines BeWo and Jeg3. The phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast tumour cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for progesterone and hCG. In addition we tested the effects of phytoestrogens on cell proliferation. Different concentrations of genistein and daidzein were cultivated with trophoblast tumour cells. After designated times, 1 microCi thymidin-(methyl-3H) was added. Methyl-3H thymidin incorporation was tested and compared to incorporation results of untreated cells. With this study we could show that the production of the steroid hormone progesterone and the protein hormone hCG is influenced by the phytoestrogens genistein and daidzein in trophoblast tumour cells of the cell lines BeWo and Jeg3. We found a correlation between the effects on the proliferation and the production of progesterone and hCG at high concentrations of genistein and daidzein in the cell lines tested. With low concentrations of genistein and daidzein we observed a stimulation of the production of hCG and a weak inhibition of proliferation in both cell lines BeWo and Jeg3. The results obtained with this study suggest that only high doses of phytoestrogens (> 1 mumol/ml) can reduce the proliferation of trophoblast tumour cells significantly. Low doses of phytoestrogens induced a higher hCG production in both cell lines tested. Although high hCG production did not lead to a higher proliferation rate of the tumour cells tested, hCG is able to induce neovascularisation in tumour cells. In summary, with this in vitro study we showed that high doses of phytoestrogens inhibit proliferation and progesterone production in trophoblast tumour cells. High doses of phytoestrogens could be useful candidates for special diet programs for prevention and surgery for patients with this type of disease. In addition we found a useful cell culture model for the testing of new types of phytoestrogens.

[New possibilities will open up in human gene therapy].

PubMed

Portin, Petter

2016-01-01

Gene therapy is divided into somatic and germ line therapy. The latter involves reproductive cells or their stem cells, and its results are heritable. The effects of somatic gene therapy are generally restricted to a single tissue of the patient in question. Until now, all gene therapies in the world have belonged to the regime of somatic therapy, germ line therapy having been a theoretical possibility only. Very recently, however, a method has been developed which is applicable to germ line therapy as well. In addition to technical challenges, severe ethical problems are associated with germ line therapy, demanding opinion statement.

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.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

PubMed

Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A

2016-01-01

The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.

Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

PubMed

Bao, Kai; Akguel, Baki; Bostanci, Nagihan

2014-01-01

In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. © 2014 S. Karger AG, Basel.

Synergistic Effect of Curcumin in Combination with Anticancer Agents in Human Retinoblastoma Cancer Cell Lines.

PubMed

Sreenivasan, Seethalakshmi; Krishnakumar, Subramanian

2015-01-01

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of the herb Curcuma longa, is known to have anti-proliferative and anti-tumor properties. In this study, we evaluated the cytotoxic effect of curcumin alone and in combination with individual drugs like carboplatin, etoposide, or vincristine in a human retinoblastoma (RB) cancer cell line. A drug-drug interaction was analyzed using the median effect/isobologram method and combination index values were used to characterize the interaction as synergistic or additive. We also performed the apoptosis and cell-cycle kinetics study with single drugs in combination with curcumin in a human RB cell lines (Y79 and Weri-Rb1). Curcumin caused concentration-dependent decrease in cell proliferation, cell kinetics, and also induced apoptosis in both the RB cell lines. When combination of curcumin with individual drugs like carboplatin or etoposide or vincristine was treated on to RB cells, both cell viability and cell cycling were reduced and increased apoptosis was noted, in comparison with single drug treatment. These effects were significant in both the cell lines, indicating the ability of curcumin to increase the sensitivity of RB cells to chemotherapy drugs. Our in vitro findings showed that the combination of curcumin with single drug treatment showed marked synergistic inhibitory effect against RB cell lines. These results suggest that curcumin can be used as a modulator which may have a potential therapeutic value for the treatment of RB cancer patients.

Arginase treatment prevents the recovery of canine lymphoma and osteosarcoma cells resistant to the toxic effects of prolonged arginine deprivation.

PubMed

Wells, James W; Evans, Christopher H; Scott, Milcah C; Rütgen, Barbara C; O'Brien, Timothy D; Modiano, Jaime F; Cvetkovic, Goran; Tepic, Slobodan

2013-01-01

Rapidly growing tumor cells require a nutrient-rich environment in order to thrive, therefore, restricting access to certain key amino acids, such as arginine, often results in the death of malignant cells, which frequently display defective cell cycle check-point control. Healthy cells, by contrast, become quiescent and remain viable under arginine restriction, displaying full recovery upon return to arginine-rich conditions. The use of arginase therapy to restrict available arginine for selectively targeting malignant cells is currently under investigation in human clinical trials. However, the suitability of this approach for veterinary uses is unexplored. As a prelude to in vivo studies in canine malignancies, we examined the in vitro effects of arginine-deprivation on canine lymphoid and osteosarcoma cell lines. Two lymphoid and 2 osteosarcoma cell lines were unable to recover following 6 days of arginine deprivation, but all remaining cell lines displayed full recovery upon return to arginine-rich culture conditions. These remaining cell lines all proved susceptible to cell death following the addition of arginase to the cultures. The lymphoid lines were particularly sensitive to arginase, becoming unrecoverable after just 3 days of treatment. Two of the osteosarcoma lines were also susceptible over this time-frame; however the other 3 lines required 6-8 days of arginase treatment to prevent recovery. In contrast, adult progenitor cells from the bone marrow of a healthy dog were able to recover fully following 9 days of culture in arginase. Over 3 days in culture, arginase was more effective than asparaginase in inducing the death of lymphoid lines. These results strongly suggest that short-term arginase treatment warrants further investigation as a therapy for lymphoid malignancies and osteosarcomas in dogs.

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

Gopalan, Vinod; Smith, Robert A.; Lam, Alfred K.-Y., E-mail: a.lam@griffith.edu.au

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expressionmore » by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA's effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas. - Highlights: • miR-498 is a non-coding RNA located in 19q13.41. • Colon cancer cell lines showed reduced expression of miR-498. • Mimic driven over expression of miR-498 in colon cancer cells resulted in lower cell proliferation. • miR-498 expression was down regulated in all colorectal adenocarcinoma tissues.« less

Production of medakafish chimeras from a stable embryonic stem cell line.

PubMed

Hong, Y; Winkler, C; Schartl, M

1998-03-31

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Production of medakafish chimeras from a stable embryonic stem cell line

PubMed Central

Hong, Yunhan; Winkler, Christoph; Schartl, Manfred

1998-01-01

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology. PMID:9520425

Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines.

PubMed

Yamamizu, Kohei; Sharov, Alexei A; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B; Schlessinger, David; Ko, Minoru S H

2016-05-06

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs.

Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

NASA Astrophysics Data System (ADS)

Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

An Inducible Endothelial Cell Surface Glycoprotein Mediates Melanoma Adhesion

NASA Astrophysics Data System (ADS)

Rice, G. Edgar; Bevilacqua, Michael P.

1989-12-01

Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7.

PubMed

Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2016-08-01

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC50=141.62μg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC50 induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Combination of PI3K/Akt/mTOR inhibitors and PDT in endothelial and tumor cells

NASA Astrophysics Data System (ADS)

Fateye, Babasola; Chen, Bin

2011-02-01

The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

Curcumin (Diferuloylmethane) Inhibits Cell Proliferation, Induces Apoptosis, and Decreases Hormone Levels and Secretion in Pituitary Tumor Cells

PubMed Central

Miller, Matthew; Chen, Shenglin; Woodliff, Jeffrey; Kansra, Sanjay

2008-01-01

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas. PMID:18450960

Curcumin (diferuloylmethane) inhibits cell proliferation, induces apoptosis, and decreases hormone levels and secretion in pituitary tumor cells.

PubMed

Miller, Matthew; Chen, Shenglin; Woodliff, Jeffrey; Kansra, Sanjay

2008-08-01

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

PubMed

Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

2017-01-01

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells

PubMed Central

Luo, Lan; Kawakatsu, Miho; Guo, Chao-Wan; Urata, Yoshishige; Huang, Wen-Jing; Ali, Haytham; Doi, Hanako; Kitajima, Yuriko; Tanaka, Takayuki; Goto, Shinji; Ono, Yusuke; Xin, Hong-Bo; Hamano, Kimikazu; Li, Tao-Sheng

2014-01-01

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem (iPS) cells were investigated with two human iPS cell lines (201B7 and 253G1). Cells used in this study were expanded from a single colony of each cell line with the addition of proprietary antioxidant supplement or homemade antioxidant cocktail in medium, and maintained in parallel for 2 months. The cells grew well in all culture conditions and kept “stemness”. Although antioxidants modestly decreased the levels of intracellular reactive oxygen species, there were no differences in the expression of 53BP1 and pATM, two critical molecules related with DNA damage and repair, under various culture conditions. CGH analysis showed that the events of genetic aberrations were decreased only in the 253G1 iPS cells with the addition of homemade antioxidant cocktail. Long-term culture will be necessary to confirm whether low dose antioxidants improve the quality and genomic stability of iPS cells. PMID:24445363

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

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.

PubMed

Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S; Royer-Pokora, Brigitte

2016-01-01

We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15

PubMed Central

Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S.; Royer-Pokora, Brigitte

2016-01-01

We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells. PMID:27213811

Effects of nicotine on zebrafish: A comparative response between a newly established gill cell line and whole gills.

PubMed

Nathiga Nambi, K S; Abdul Majeed, S; Taju, G; Sivasubbu, Sridhar; Sarath Babu, V; Sahul Hameed, A S

2017-05-01

A novel cell line, Danio rerio gill (DrG), derived from the gill tissue of zebrafish, was established and characterized. The cells were able to grow at a wide range of temperatures from 25°C to 32°C in Leibovitz's L-15 medium. The DrG cell line consists of epithelial-like cells with a diameter of 18-22μm. The cell line was characterized by mitochondrial 12S rRNA gene. Acute toxicity tests were conducted on D. rerio by exposing them to nicotine for 96h under static conditions. In vitro cytotoxicity of nicotine was assessed in DrG cell line using multiple endpoints such as 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Linear correlations between each in vitro cytotoxicity assay and the in vivo mortality data were highly significant. Nicotine induced intracellular reactive oxygen species generation in DrG cell line in a concentration dependent manner. DrG cell line and zebrafish exposed to nicotine significantly increased the elevation of lipid peroxidation (LPO) while depletion of reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidise(GPx1a) was observed. In nicotine treated fish and cells a negative correlation between reduced glutathione and LPO was observed. In addition, the production of ROS and the resulting oxidative stress resulted in increased expression of apoptosis related genes p53 and cas3.Collectively, our result suggests that nicotine has the potential to induce reactive oxygen species (ROS) production, oxidative stress and apoptosis in DrG cell line and zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

The relationship of metabolic burden to productivity levels in CHO cell lines.

PubMed

Zou, Wu; Edros, Raihana; Al-Rubeai, Mohamed

2018-03-01

The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

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.

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

USGS Publications Warehouse

Batts, William N.; Polinski, Mark P.; Drennan, John D.; Ireland, Susan C.; Cain, Kenneth D.

2010-01-01

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle’s minimum essential medium with Earle’s salts (MEM) supplemented with GlutaMAX™, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg ml–1, respectively) did not negatively influence cell replication; however, the antimycotic Fungizone™ (2.5 µg ml–1, amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23°C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Establishment and partial characterization of a cell line from burbot Lota lota maculosa: susceptibility to IHNV, IPNV and VHSV.

PubMed

Polinski, Mark P; Drennan, John D; Batts, William N; Ireland, Susan C; Cain, Kenneth D

2010-05-18

This study describes the development and partial characterization of a continuous fibroblastic-like cell line (BEF-1) developed from late stage embryos of North American burbot Lota lota maculosa. This cell line has been maintained for over 5 yr and 100 passages in vitro. Cells were cultured using Eagle's minimum essential medium with Earle's salts (MEM) supplemented with GlutaMAX, and 10% fetal bovine serum (FBS), pH 7.4. The addition of penicillin-streptomycin-neomycin (PSN) antibiotic mixture (0.05, 0.05, 0.1 mg m(-1), respectively) did not negatively influence cell replication; however, the antimycotic FungizoneTM (2.5 microg m(-1), amphotericin B) caused cell rounding and resulted in a severe decrease in cell proliferation. Optimal incubation temperature has been observed between 15 and 23 degrees C, and at these temperatures cultures are routinely passed using standard trypsinization methods every 5 to 7 d at a split ratio of 1:3 or 1:4. The cell line was susceptible to isolates of the M and U North American genotypes of infectious hematopoietic necrosis virus (IHNV), and to isolates of genotypes I, IVa, and IVb of viral hemorrhagic septicemia virus (VHSV). In contrast, the cell line was refractory to infection by 2 North American isolates of infectious pancreatic necrosis virus (IPNV) from serotypes A1 and A9. This cell line provides a new laboratory tool, will allow further investigation into viral diseases of burbot and possibly other species, and is the first immortalized cell line reported from a species in the Gadidae (cod) family.

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures

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

Yim, L.K.; Stuart, W.D.

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake ofmore » L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system.« less

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

PubMed

Fazekas, Judit; Grunt, Thomas W; Jensen-Jarolim, Erika; Singer, Josef

2017-05-23

Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

Rapid Characterization of Candidate Biomarkers for Pancreatic Cancer Using Cell Microarrays (CMAs)

PubMed Central

Kim, Min-Sik; Kuppireddy, Sarada V.; Sakamuri, Sruthi; Singal, Mukul; Getnet, Derese; Harsha, H. C.; Goel, Renu; Balakrishnan, Lavanya; Jacob, Harrys K. C.; Kashyap, Manoj K.; Tankala, Shantal G.; Maitra, Anirban; Iacobuzio-Donahue, Christine A.; Jaffee, Elizabeth; Goggins, Michael G.; Velculescu, Victor E.; Hruban, Ralph H.; Pandey, Akhilesh

2013-01-01

Tissue microarrays have become a valuable tool for high-throughput analysis using immunohistochemical labeling. However, the large majority of biochemical studies are carried out in cell lines to further characterize candidate biomarkers or therapeutic targets with subsequent studies in animals or using primary tissues. Thus, cell line-based microarrays could be a useful screening tool in some situations. Here, we constructed a cell microarray (CMA) containing a panel of 40 pancreatic cancer cell lines available from American Type Culture Collection in addition to those locally available at Johns Hopkins. As proof of principle, we performed immunocytochemical labeling of an epithelial cell adhesion molecule (Ep-CAM), a molecule generally expressed in the epithelium, on this pancreatic cancer CMA. In addition, selected molecules that have been previously shown to be differentially expressed in pancreatic cancer in the literature were validated. For example, we observed strong labeling of CA19-9 antigen, a prognostic and predictive marker for pancreatic cancer. We also carried out a bioinformatics analysis of a literature curated catalog of pancreatic cancer biomarkers developed previously by our group and identified two candidate biomarkers, HLA class I and transmembrane protease, serine 4 (TMPRSS4), and examined their expression in the cell lines represented on the pancreatic cancer CMAs. Our results demonstrate the utility of CMAs as a useful resource for rapid screening of molecules of interest and suggest that CMAs can become a universal standard platform in cancer research. PMID:22985314

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

PubMed Central

Kustiawan, Paula M.; Puthong, Songchan; Arung, Enos T.; Chanchao, Chanpen

2014-01-01

Objective To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). Methods All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Results Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Conclusions Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s). PMID:25183275

Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid.

PubMed

Whelan, Stephen A; He, Jianbo; Lu, Ming; Souda, Puneet; Saxton, Romaine E; Faull, Kym F; Whitelegge, Julian P; Chang, Helena R

2012-10-05

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines.

PubMed

Kustiawan, Paula M; Puthong, Songchan; Arung, Enos T; Chanchao, Chanpen

2014-07-01

To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s).

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

PubMed

Lee, Jonathan K; Garbe, James C; Vrba, Lukas; Miyano, Masaru; Futscher, Bernard W; Stampfer, Martha R; LaBarge, Mark A

2015-01-01

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

PubMed Central

Panetta, J C; Evans, W E; Cheok, M H

2006-01-01

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance. PMID:16333308

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

PubMed

Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

2010-10-01

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Antimutagenicity of WR-1065 in L5178Y cells exposed to accelerated (56)Fe ions

NASA Technical Reports Server (NTRS)

Evans, H. H.; Evans, T. E.; Horng, M. F.

2002-01-01

The ability of the aminothiol WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] to protect L5178Y (LY) cells against the cytotoxic and mutagenic effects of exposure to accelerated (56)Fe ions (1.08 GeV/nucleon) was determined. It was found that while WR-1065 reduced the mutagenicity in both cell lines when it was present during the irradiation, the addition of WR-1065 after the exposure had no effect on the mutagenicity of the radiation in either cell line. No marked protection against the cytotoxic effects of exposure to (56)Fe ions was provided by WR-1065 when added either during or after irradiation in either cell line. We reported previously that WR-1065 protected the LY-S1 and LY-SR1 cell lines against both the cytotoxicity and mutagenicity of X radiation when present during exposure, but that its protection when administered after exposure was limited to the mutagenic effects in the radiation-hypersensitive cell line, LY-S1. The results indicate that the mechanisms involved differ in the protection against cytotoxic compared to mutagenic effects and in the protection against damage caused by accelerated (56)Fe ions compared to X radiation.

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.

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

PubMed Central

Donis, Ruben O.; Chen, i-Mei; Davis, C Todd; Foust, Angie; Hossain, M. Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2018-01-01

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. PMID:24975811

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

PubMed

Donis, Ruben O; Davis, C Todd; Foust, Angie; Hossain, M Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, Odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2014-11-12

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. Published by Elsevier Ltd.

Towards the elaboration of new gold-based optical theranostics.

PubMed

Doulain, Pierre-Emmanuel; Decréau, Richard; Racoeur, Cindy; Goncalves, Victor; Dubrez, Laurence; Bettaieb, Ali; Le Gendre, Pierre; Denat, Franck; Paul, Catherine; Goze, Christine; Bodio, Ewen

2015-03-21

Four new red BODIPY-gold(I) theranostic compounds were synthesized. Some of them were vectorized by tethering a biovector (glucose or bombesin derivatives) to the metallic center. Their photophysical properties were studied. Additionally, their cytotoxicity was examined on different cancer cell lines and on a normal cell line, they were tracked in vitro by fluorescence detection, and their uptake was evaluated by ICP-MS measurements.

CD10/NEP in non-small cell lung carcinomas. Relationship to cellular proliferation.

PubMed Central

Ganju, R K; Sunday, M; Tsarwhas, D G; Card, A; Shipp, M A

1994-01-01

The cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression. Images PMID:7962523

Vasculogenic mimicry in bladder cancer and its association with the aberrant expression of ZEB1

PubMed Central

Li, Baimou; Mao, Xiaopeng; Wang, Hua; Su, Guanyu; Mo, Chengqiang; Cao, Kaiyuan; Qiu, Shaopeng

2018-01-01

The aim of the present study was to investigate the associations between vasculogenic mimicry (VM) and zinc finger E-box binding homeobox 1 (ZEB1) in bladder cancer. VM structure and ZEB1 expression were analyzed by cluster of differentiation 34/periodic acid Schiff (PAS) double staining and immunohistochemical staining in 135 specimens from patients with bladder cancer, and a further 12 specimens from normal bladder tissues. Three-dimensional (3-D) culture was used to detect VM formation in the bladder transitional cancer cell lines UM-UC-3 and J82, and the immortalized human bladder epithelium cell line SV-HUC-1 in vitro. ZEB1 expression in these cell lines was compared by reverse transcription-quantitative polymerase chain reaction and western blot assays. In addition, small interfering RNA was used to inhibit ZEB1 in UM-UC-3 and J82 cells, followed by 3-D culturing of treated cell lines. As a result, VM was observed in 31.1% of specimens from bladder cancer tissues, and cases with high ZEB1 expression accounted for 60.0% of patients with bladder cancer. In addition, ZEB1 expression was closely associated with VM (r=0.189; P<0.05), and also increased as the grade and stage of the tumor developed. In an in vitro assay, UM-UC-3 and J82 cells exhibited VM formation, however, SV-HUC-1 did not. Furthermore, VM-forming cancer cell lines UM-UC-3 and J82 exhibited higher ZEB1 expression. Notably, VM formation was inhibited following knockdown of ZEB1. In conclusion, ZEB1 may be associated with VM in bladder cancer and serve an important role in the process of VM formation. However, its detailed mechanism requires further study. PMID:29552157

Effect of verteporfin-PDT on epithelial growth factor receptor (EGFR) signaling pathway in cholangiocarcinoma cell lines

NASA Astrophysics Data System (ADS)

Andreola, Fausto; Cerec, Virginie; Pereira, Stephen P.

2009-06-01

EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional chemo/radiotherapy have been reported in preclinical studies for different cancers but not cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii) response to combination of PDT and EGFR-targeting agents. We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic, respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor (Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA damage.

Type I Interferon Controls Propagation of Long Interspersed Element-1*

PubMed Central

Yu, Qiujing; Carbone, Christopher J.; Katlinskaya, Yuliya V.; Zheng, Hui; Zheng, Ke; Luo, Mengcheng; Wang, P. Jeremy; Greenberg, Roger A.; Fuchs, Serge Y.

2015-01-01

Type I interferons (IFN) including IFNα and IFNβ are critical for the cellular defense against viruses. Here we report that increased levels of IFNβ were found in testes from mice deficient in MOV10L1, a germ cell-specific RNA helicase that plays a key role in limiting the propagation of retrotransposons including Long Interspersed Element-1 (LINE-1). Additional experiments revealed that activation of LINE-1 retrotransposons increases the expression of IFNβ and of IFN-stimulated genes. Conversely, pretreatment of cells with IFN suppressed the replication of LINE-1. Furthermore, the efficacy of LINE-1 replication was increased in isogenic cell lines harboring inactivating mutations in diverse elements of the IFN signaling pathway. Knockdown of the IFN receptor chain IFNAR1 also stimulated LINE-1 propagation in vitro. Finally, a greater accumulation of LINE-1 was found in mice that lack IFNAR1 compared with wild type mice. We propose that LINE-1-induced IFN plays an important role in restricting LINE-1 propagation and discuss the putative role of IFN in preserving the genome stability. PMID:25716322

Conditioned media from a renal cell carcinoma cell line demonstrates the presence of basic fibroblast growth factor.

PubMed

Mydlo, J H; Zajac, J; Macchia, R J

1993-09-01

In a previous report, we demonstrated the isolation and purification of a heparin binding growth factor from human renal carcinoma, and suggested that this growth factor may play a role in the neovascularity and growth of the tumor. In this report, we demonstrate that the growth of the renal cell carcinoma cell line RC29 is stimulated by the addition of exogenous fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha). Also, media conditioned by this cell line was able to stimulate growth of the A431 vulvar tumor cell line, known for its high concentration of EGF receptors, 3T3 fibroblasts, human umbilical vein (HUV) cells and RC29 cells. Using heparin-sepharose chromatography and then SDS polyacrylamide gel electrophoresis (PAGE), we were able to demonstrate several proteins in the conditioned media of the RC29 cell line. Using Western blot analysis, we detected that at least one of the proteins expressed in this conditioned media was FGF and that it belongs to the basic, not acidic, family of fibroblast growth factors. These findings suggest that renal tumors may express growth factors that may play a direct role in maintaining their unrestricted proliferation.

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines.

PubMed

Witter, Lauren E; Gruber, Erika J; Lean, Fabian Z X; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor-replete or specific coagulation factor-deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X-deficient plasma; residual thrombin generation in factor VII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines

PubMed Central

Witter, Lauren E.; Gruber, Erika J.; Lean, Fabian Z. X.; Stokol, Tracy

2017-01-01

OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor–replete or specific coagulation factor–deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X–deficient plasma; residual thrombin generation in FVII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma. PMID:28029283

KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

PubMed

Suck, Garnet; Branch, Donald R; Smyth, Mark J; Miller, Richard G; Vergidis, Joanna; Fahim, Soad; Keating, Armand

2005-10-01

To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.

Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone).

PubMed

Isaka, Keiichi; Fujito, Atsuya; Sagawa, Yasukazu; Yudate, Tamaki; Nishi, Hirotaka; Ito, Hiroe; Takayama, Masaomi

2002-01-01

Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c-Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma.

Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

PubMed

Clerc, Jérôme; Florea, Bogdan I; Kraus, Marianne; Groll, Michael; Huber, Robert; Bachmann, André S; Dudler, Robert; Driessen, Christoph; Overkleeft, Herman S; Kaiser, Markus

2009-11-02

The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

Amyotrophic lateral sclerosis immunoglobulins increase intracellular calcium in a motoneuron cell line.

PubMed

Colom, L V; Alexianu, M E; Mosier, D R; Smith, R G; Appel, S H

1997-08-01

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca2+]i) in differentiated VSC4.1 cells. These transient [Ca2+]i increases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 microM) of omega-agatoxin IVa. The incidence of transient [Ca2+]i increases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca2+]i increases (addition of synthetic FTX or omega-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca2+]i increases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca2+]i changes induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

PubMed

Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

2011-06-01

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

PubMed

Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

2018-01-02

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Contamination of infectious RD-114 virus in vaccines produced using non-feline cell lines.

PubMed

Yoshikawa, Rokusuke; Sato, Eiji; Miyazawa, Takayuki

2011-01-01

All domestic cats have a replication-competent endogenous retrovirus, termed RD-114 virus, in their genome and several feline cell lines produce RD-114 viruses. Recently, we found that a portion of live attenuated feline and canine vaccines produced using feline cell lines was contaminated with infectious RD-114 viruses. In this study, we expanded our survey and examined canine vaccines produced using 'non-feline' cell lines. Consequently, we found two vaccines containing RD-114 viral RNA by reverse transcriptase (RT)-polymerase chain reaction (PCR) and real-time RT-PCR. We also confirmed the presence of infectious RD-114 virus in the vaccines by the LacZ marker rescue assay and PCR to detect proviral DNA in TE671 cells (human rhabdomyosarcoma cells) inoculated with the vaccines. It is impossible to investigate the definitive cause of contamination with RD-114 virus; however, we suspect that a seed canine parvovirus type 2 was contaminated with RD-114 virus, because many canine parvoviruses have been isolated and attenuated using feline cell lines. To exclude RD-114 virus from live attenuated vaccines, we must pay attention to the contamination of seed viruses with RD-114 virus in addition to avoiding feline cell lines producing RD-114 virus when manufacturing vaccines. Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

PubMed

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Li, Chuanfeng; Chen, Zongyan; Liu, Guangqing

2016-11-01

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

PubMed

Ekblad, Lars; Lindgren, Gustaf; Persson, Emma; Kjellén, Elisabeth; Wennerberg, Johan

2013-01-25

Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro. Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors. One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3. In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

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

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

Generation of stable PDX derived cell lines using conditional reprogramming.

PubMed

Borodovsky, Alexandra; McQuiston, Travis J; Stetson, Daniel; Ahmed, Ambar; Whitston, David; Zhang, Jingwen; Grondine, Michael; Lawson, Deborah; Challberg, Sharon S; Zinda, Michael; Pollok, Brian A; Dougherty, Brian A; D'Cruz, Celina M

2017-12-06

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.

Ell3 stimulates proliferation, drug resistance, and cancer stem cell properties of breast cancer cells via a MEK/ERK-dependent signaling pathway

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

Ahn, Hee-Jin; Kim, Gwangil; Park, Kyung-Soon, E-mail: kspark@cha.ac.kr

2013-08-09

Highlights: •Ell3 enhances proliferation and drug resistance of breast cancer cell lines. •Ell3 is related to the cancer stem cell characteristics of breast cancer cell lines. •Ell3 enhances oncogenicity of breast cancer through the ERK1/2 signaling pathway. -- Abstract: Ell3 is a RNA polymerase II transcription elongation factor that is enriched in testis. The C-terminal domain of Ell3 shows strong similarities to that of Ell (eleven−nineteen lysine-rich leukemia gene), which acts as a negative regulator of p53 and regulates cell proliferation and survival. Recent studies in our laboratory showed that Ell3 induces the differentiation of mouse embryonic stem cells bymore » protecting differentiating cells from apoptosis via the promotion of p53 degradation. In this study, we evaluated the function of Ell3 in breast cancer cell lines. MCF-7 cell lines overexpressing Ell3 were used to examine cell proliferation and cancer stem cell properties. Ectopic expression of Ell3 in breast cancer cell lines induces proliferation and 5-FU resistance. In addition, Ell3 expression increases the cancer stem cell population, which is characterized by CD44 (+) or ALDH1 (+) cells. Mammosphere-forming potential and migration ability were also increased upon Ell3 expression in breast cancer cell lines. Through biochemical and molecular biological analyses, we showed that Ell3 regulates proliferation, cancer stem cell properties and drug resistance in breast cancer cell lines partly through the MEK−extracellular signal-regulated kinase signaling pathway. Murine xenograft experiments showed that Ell3 expression promotes tumorigenesis in vivo. These results suggest that Ell3 may play a critical role in promoting oncogenesis in breast cancer by regulating cell proliferation and cancer stem cell properties via the ERK1/2 signaling pathway.« less

Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes.

PubMed

Kim, Ginah L; Wang, Xiaomei; Chalmers, Jennifer A; Thompson, David R; Dhillon, Sandeep S; Koletar, Margaret M; Belsham, Denise D

2011-01-01

The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

PubMed Central

2010-01-01

Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues. PMID:20964822

Oxytocin stimulates cell proliferation in vaginal cell line Vk2E6E7.

PubMed

Kallak, Theodora K; Uvnäs-Moberg, Kerstin

2017-03-01

Objective During and after menopause, the symptoms of vaginal atrophy cause great discomfort and necessitate effective treatment options. Currently, vaginally applied oxytocin is being investigated as a treatment for the symptoms of vaginal atrophy in postmenopausal women. To clarify the mechanisms behind oxytocins effects on vaginal atrophy, the present study investigated the effects of oxytocin on cell proliferation in the cells of the Vk2E6E7 line, a non-tumour vaginal cell line. The study also compared the effects of oxytocin with those of estradiol (E2). Study design The effects of both oxytocin and E2 on the proliferation of Vk2E6E7 cells were investigated using Cell Proliferation ELISA BrdU Colorimetric Assay. The expression of both oxytocin and oxytocin receptor was studied in Vk2E6E7 cells using quantitative real-time polymerase chain reaction and immunofluorescent staining. Main outcome measures Cell proliferation and gene expression. Results Oxytocin increased cell proliferation both time dependently and dose dependently. This differed from the effect pattern observed in cells treated with E2. In addition, in oxytocin-treated cells, the oxytocin receptor was found to be co-localized with caveolin-1, indicating pro-proliferative signalling within the cell. Conclusions Oxytocin stimulates cell proliferation and the co-localization of oxytocin receptor with caveolin-1 in oxytocin-treated cells, supporting the role of oxytocin signalling in cell proliferation. In addition, these findings suggest that increased cell proliferation is one mechanism by which local vaginal oxytocin treatment increases vaginal thickness and relieves vaginal symptoms in postmenopausal women with vaginal atrophy.

Proteomics of a new esophageal cancer cell line established from Persian patient.

PubMed

Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of IL11RA and MELK amplification in gastric cancer by comprehensive genomic profiling of gastric cancer cell lines

PubMed Central

Calcagno, Danielle Queiroz; Takeno, Sylvia Santomi; Gigek, Carolina Oliveira; Leal, Mariana Ferreira; Wisnieski, Fernanda; Chen, Elizabeth Suchi; Araújo, Taíssa Maíra Thomaz; Lima, Eleonidas Moura; Melaragno, Maria Isabel; Demachki, Samia; Assumpção, Paulo Pimentel; Burbano, Rommel Rodriguez; Smith, Marília Cardoso

2016-01-01

AIM To identify common copy number alterations on gastric cancer cell lines. METHODS Four gastric cancer cell lines (ACP02, ACP03, AGP01 and PG100) underwent chromosomal comparative genome hybridization and array comparative genome hybridization. We also confirmed the results by fluorescence in situ hybridization analysis using the bacterial artificial chromosome clone and quantitative real time PCR analysis. RESULTS The amplification of 9p13.3 was detected in all cell lines by both methodologies. An increase in the copy number of 9p13.3 was also confirmed by fluorescence in situ hybridization analysis. Moreover, the interleukin 11 receptor alpha (IL11RA) and maternal embryonic leucine zipper kinase (MELK) genes, which are present in the 9p13.3 amplicon, revealed gains of the MELK gene in all the cell lines studied. Additionally, a gain in the copy number of IL11RA and MELK was observed in 19.1% (13/68) and 55.9% (38/68) of primary gastric adenocarcinoma samples, respectively. CONCLUSION The characterization of a small gain region at 9p13.3 in gastric cancer cell lines and primary gastric adenocarcinoma samples has revealed MELK as a candidate target gene that is possibly related to the development of gastric cancer. PMID:27920471

Generation of monoclonal antibodies reacting with human epithelial ovarian cancer.

PubMed

Tagliabue, E; Mènard, S; Della Torre, G; Barbanti, P; Mariani-Costantini, R; Porro, G; Colnaghi, M I

1985-01-01

Fusion of the murine myeloma line P3-X63-Ag8-U1 with spleen cells from a mouse immunized with a membrane preparations (CM) of a mucinous ovarian cystoadenocarcinoma yielded two monoclonal antibodies, MOv1 and MOv2, which reacted by solid-phase radioimmunoassay with immunizing tumor CM but were unreactive with normal kidney CM as well as with plasma proteins and peripheral blood cells from the immunizing carcinoma patient. MOv1 and MOv2 were further tested by solid-phase radioimunoassay on a panel of different CM from fresh surgical specimens of ovarian and nonovarian carcinomas, benign ovarian tumors, normal ovary and kidney tissues, and on various tumor cell lines. In addition, the antibodies were characterized by immunofluorescence on live cells from cell lines and surgical specimens, and on frozen sections of benign and malignant ovarian tumors, of nonovarian tumors, and of normal tissues. The results obtained indicate that MOv1 and MOv2 recognize two different epitopes on molecules present on malignant and benign ovarian mucinous tumors and colonic glands. In addition, the antigen recognized by MOv2 was also detected in carcinmas of lung, colon, stomach, and breast; in gastrointestinal glands; and in the glandular lumina of normal lactating breast.

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines.

PubMed

Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao

2016-08-01

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study. Copyright © 2016. Published by Elsevier B.V.

Assessment of cell line competence for studies of pharmacological GPR30 modulation.

PubMed

Sousa, Cátia; Ribeiro, Madalena; Rufino, Ana Teresa; Leitão, Alcino Jorge; Mendes, Alexandrina Ferreira

2017-04-01

Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1. Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models.

PubMed

Tan, Nguyen; Wong, Maureen; Nannini, Michelle A; Hong, Rebecca; Lee, Leslie B; Price, Stephen; Williams, Karen; Savy, Pierre Pascal; Yue, Peng; Sampath, Deepak; Settleman, Jeffrey; Fairbrother, Wayne J; Belmont, Lisa D

2013-06-01

Although mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non-small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. ©2013 AACR

Enhanced cytotoxic activity of effector T-cells against cholangiocarcinoma by dendritic cells pulsed with pooled mRNA.

PubMed

Junking, Mutita; Grainok, Janya; Thepmalee, Chutamas; Wongkham, Sopit; Yenchitsomanus, Pa-Thai

2017-10-01

Cholangiocarcinoma is a malignancy of bile duct epithelia with an increasing in incidence rate worldwide. Surgery is the only curative treatment, while adjuvant chemotherapy and radiotherapy render poor responses. Cell-based immunotherapy is a potential strategy for cholangiocarcinoma treatment. However, variation of tumor antigens in cholangiocarcinoma leads to the ineffectiveness of cell-based immunotherapy. In this study, we examined the activation of effector T-cells by dendritic cells pulsed with protein lysate or total RNA from cholangiocarcinoma cell lines for their cytolytic activity against cholangiocarcinoma. Broad-spectrum antigen types with respect to RNA antigen sources were obtained from combination of three cholangiocarcinoma cell lines (KKU-213, KKU-100, and KKU-055). Compared with protein lysate-pulsed dendritic cells, total RNA-pulsed dendritic cells induced anti-tumor effector T-cell response with higher killing ability to KKU-100 and KKU-213 cells compared with protein lysate-pulsed dendritic cells. Moreover, pooled messenger RNA from three cholangiocarcinoma cell lines significantly increased the specific killing capacity of activated lymphocytes against KKU-213 cells. These results suggest that activation of anti-tumor effector T-cells against cholangiocarcinoma by RNA-pulsed dendritic cells is more effective than that by protein lysate-pulsed dendritic cells. In addition, pulsing dendritic cells with pooled messenger RNA from multiple cell lines enhanced the efficacy of a cellular immune response against cholangiocarcinoma.

Chemical Ototoxicity of the Fish Inner Ear and Lateral Line.

PubMed

Coffin, Allison B; Ramcharitar, John

2016-01-01

Hair cell-driven mechanosensory systems are crucial for successful execution of a number of behaviors in fishes, and have emerged as good models for exploring questions relevant to human hearing. This review focuses on ototoxic effects in the inner ear and lateral line system of fishes. We specifically examine studies where chemical ototoxins such as aminoglycoside antibiotics have been employed as tools to disable the lateral line. Lateral line ablation results in alterations to feeding behavior and orientation to water current in a variety of species. However, neither behavior is abolished in the presence of additional sensory cues, supporting the hypothesis that many fish behaviors are driven by multisensory integration. Within biomedical research, the larval zebrafish lateral line has become an important model system for understanding signaling mechanisms that contribute to hair cell death and for developing novel pharmacological therapies that protect hair cells from ototoxic damage. Furthermore, given that fishes robustly regenerate damaged hair cells, ototoxin studies in fishes have broadened our understanding of the molecular and genetic events in an innately regenerative system, offering potential targets for mammalian hair cell regeneration. Collectively, studies of fish mechanosensory systems have yielded insight into fish behavior and in mechanisms of hair cell death, protection, and regeneration.

Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

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

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes

PubMed Central

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease. PMID:27391595

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

PubMed

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides

NASA Technical Reports Server (NTRS)

Polig, E.; Jee, W. S.; Kruglikov, I. L.

1992-01-01

Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.

Low-dose gemcitabine induces major histocompatibility complex class I-related chain A/B expression and enhances an antitumor innate immune response in pancreatic cancer.

PubMed

Miyashita, Tomoharu; Miki, Kenji; Kamigaki, Takashi; Makino, Isamu; Nakagawara, Hisatoshi; Tajima, Hidehiro; Takamura, Hiroyuki; Kitagawa, Hirohisa; Fushida, Sachio; Ahmed, Ali K; Duncan, Mark D; Harmon, John W; Ohta, Tetsuo

2017-02-01

We investigated the effect of gemcitabine (GEM), a key drug for pancreatic cancer treatment, on the expression of cell surface MICA/B in pancreatic cancer cells and resulting cytotoxicity of γδ T cells. We assessed the effect of GEM on the upregulation of cell surface MICA/B expression by flow cytometry, utilizing six pancreatic cancer cell lines. MICA and CD16 expressions from resected pancreatic cancer patient specimens, which received neoadjuvant chemotherapy (NAC) with GEM, were analyzed by immunohistochemistry. GEM could increase MICA/B expression on cell surface in pancreatic cancer cell lines (in 2 of 6 cell lines). This effect was most effectively at concentration not affecting cell growth of GEM (0.001 μM), because MICA/B negative population was appeared at concentration at cytostatic and cytotoxic effect to cell growth (0.1 and 10 μM). The cytotoxic activity of γδ T cells against PANC-1 was detected and functions through interactions between NKG2D and MICA/B. However, the enhancement of NKG2D-dependent cytotoxicity with increased MICA/B expression, by GEM treatment, was not observed. In addition, soluble MIC molecules were released from pancreatic cancer cell lines in culture supernatant with GEM treatment. Immunohistochemical staining demonstrated that MICA expression in tumor cells and CD16 positive cells surrounding tumors were significantly higher in the NAC group compared to that of the control group. There was a significant correlation between NAC and MICA expression, as well as NAC and CD16 positive cell expression. The present results indicate that low-dose GEM-induced MICA/B expression enhances innate immune function rather than cytotoxicity in pancreatic cancer. In addition, our result suggests that the inhibition of cleavage and release of MIC molecules from the tumor surface could potentially improve NKG2D-dependent cytotoxicity.

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts.

PubMed

Korohoda, Włodzimierz; Hapek, Anna; Pietrzak, Monika; Ryszawy, Damian; Madeja, Zbigniew

2016-11-01

The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

SWATH™- and iTRAQ-based quantitative proteomic analyses reveal an overexpression and biological relevance of CD109 in advanced NSCLC.

PubMed

Zhang, Fanglin; Lin, Hechun; Gu, Aiqin; Li, Jing; Liu, Lei; Yu, Tao; Cui, Yongqi; Deng, Wei; Yan, Mingxia; Li, Jinjun; Yao, Ming

2014-05-06

To identify cancer-related proteins, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATH™ quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic non-small-cell lung cancer (NSCLC) cell lines. In addition, we compared two groups of pooled serum samples (12 early-stage and 12 late-stage patients) to mine data for candidates screened by iTRAQ-labeled proteomic analysis. A total of 110 proteins and 71 proteins were observed to be significantly differentially expressed in the cell line secretome and NSCLC sera, respectively. Among these proteins, CD109 was found to be highly expressed in both the highly metastatic cell line secretome and the group of late-stage patients. A sandwich ELISA assay also demonstrated an elevation of serum CD109 levels in individual NSCLC patients (n=30) compared with healthy subjects (n=19). Furthermore, CD109 displayed higher expression in lung cancer tissues compared with their matched noncancerous lung tissues (n=72). In addition, the knockdown of CD109 influenced several NSCLC cell bio-functions, for instance, depressing cell growth, affecting cell cycle phases. These phenomena suggest that CD109 plays a critical role in NSCLC progression. We simultaneously applied two quantitative proteomic approaches-iTRAQ-labeling and SWATH™-to analyze the secretome of metastatic cell lines, in order to explore the cancer-associated proteins in conditioned media. In this study, our results indicate that CD109 plays a critical role in non-small-cell lung cancer (NSCLC) progression, and is overexpressed in advanced NSCLC. Copyright © 2014 Elsevier B.V. All rights reserved.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

PubMed Central

2012-01-01

Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

Effect of quantum dots on the biological behavior of the EJ human bladder urothelial carcinoma cell line.

PubMed

Yuan, Run; Yu, Wei-Min; Cheng, Fan; Zhang, Xiao-Bin; Ruan, Yuan; Cao, Zhi-Xiu; Larré, Stéphane

2015-10-01

Quantum dots (QDs) are a type of fluorescent label with applications in biological molecules, cells and in vivo imaging. The current study investigated the effect of QDs on the toxicity, proliferation, migration and invasion of the EJ human bladder cancer cell line in vitro. The cell counting kit‑8 test was used to measure the survival rate of EJ cells following incubation with varying concentrations of QDs. Additionally, the effect of QDs on tumor cell migration and invasion was evaluated using the Transwell chamber assay, and cell proliferation rate was assessed using a hemocytometer. Data from the current study demonstrated no significant differences in survival rate between the experimental and control groups with the conventionally used concentrations (5, 10 and 20 nM) of QD605 (P>0.05). However, with high concentrations of QD605 (40 and 80 nM), significant differences were observed (P<0.001). The survival rate of EJ cells, however, remained at 92.6%. In addition, no significant differences were observed between the EJ cells labeled with transactivator of transcription (TAT)‑QD605 and the unlabeled EJ cells with regard to proliferation, migration and invasion (P>0.05). Thus, the results of the current study indicate that QDs exhibit a certain degree of influence on the activity of the EJ bladder cancer cell line at high concentrations. However, at the concentrations that QDs are conventionally used, there was little impact on the survival of the EJ cells. In addition, the proliferation, migration and invasion abilities of the EJ cells were not affected by TAT‑QDs. Therefore, the peptide‑conjugated QDs have potential to be applied in the imaging and tracking of live cells in vitro and of animals in vivo. Notably, QDs may provide the foundation for a novel, non‑invasive imaging strategy for the early diagnosis of tumors.

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

PubMed Central

Simmons, Daniel T.; Martin, Malcolm A.; Mora, Peter T.; Chang, Chungming

1980-01-01

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences. Images PMID:6247503

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

PubMed

Miller, Jason R; Koren, Sergey; Dilley, Kari A; Puri, Vinita; Brown, David M; Harkins, Derek M; Thibaud-Nissen, Françoise; Rosen, Benjamin; Chen, Xiao-Guang; Tu, Zhijian; Sharakhov, Igor V; Sharakhova, Maria V; Sebra, Robert; Stockwell, Timothy B; Bergman, Nicholas H; Sutton, Granger G; Phillippy, Adam M; Piermarini, Peter M; Shabman, Reed S

2018-03-01

The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Cell origin of pyothorax-associated lymphoma: a lymphoma strongly associated with Epstein-Barr virus infection.

PubMed

Takakuwa, T; Tresnasari, K; Rahadiani, N; Miwa, H; Daibata, M; Aozasa, K

2008-03-01

Pyothorax-associated lymphoma (PAL) is an Epstein-Barr virus (EBV)-associated B cell lymphoma developing in the pleural cavity affected by chronic pyothorax. To clarify the cell origin of PAL, the expression of immunoglobulin heavy (IgH) and light chains in relation to somatic hypermutations (SHMs) of rearranged Ig heavy- and light-chain variable (IgV(H), IgV(L)) genes was examined using cell lines as well as clinical samples. SHMs without ongoing mutations of the IgV(H) gene were found in all PAL cell lines and clinical samples available for sequencing, indicating PAL to be derived from B cells at the postgerminal center (GC) stage of the differentiation process. They could be subdivided into post-GC cells with potentially productive IgV(H) genotypes (Group 1) and with sterile IgV(H) genotypes (Group 2). IgH expression was abrogated in Group 2 as expected and also in two cell lines in Group 1. DNA demethylation experiments with 5-aza-dC induced expression of IgH mRNA and protein in these cell lines. Most PAL cells were derived from crippled post-GC cells, which usually could not survive. Transformation of such B cells through EBV infection might provide a basis for the development of PAL with additional genetic changes.

Relationship between interphasic nucleolar organizer regions and growth rate in two neuroblastoma cell lines.

PubMed Central

Derenzini, M.; Pession, A.; Farabegoli, F.; Trerè, D.; Badiali, M.; Dehan, P.

1989-01-01

The relationship between the quantity of silver-stained interphasic nucleolar organizer regions (NORs) and nuclear synthetic activity, caryotype, and growth rate was studied in two established neuroblastoma cell lines (CHP 212 and HTB 10). Statistical analysis of silver-stained NORs revealed four times as many in CHP 212 cells compared with HTB 10 cells. No difference was observed in the ribosomal RNA synthesis between the two cell lines. The caryotype index was 1.2 for CHP 212 and 1.0 for HTB 10 cells. The number of chromosomes carrying NORs and the quantity of ribosomal genes was found to be the same for the two cell lines. Doubling time of CHP 212 cells was 20 hours compared with 54 hours for HTB 10 cells. In CHP 212 cells bindering of cell duplication by serum deprivation induced a progressive lowering (calculated at 48, 72, and 96 hours) of the quantity of silver-stained interphasic NORs. Recovery of duplication by new serum addition induced, after 24 hours, an increase of the quantity of silver-stained interphasic NORs up to control levels. In the light of available data, these results indicate that the quantity of interphasic NORs is strictly correlated only to the growth rate of the cell. Images Figure 2 Figure 3 Figure 4 PMID:2705511

Development of a New Conditionally Immortalized Human Liver Sinusoidal Endothelial Cells.

PubMed

Zhu, Meiyan; Koibuchi, Akira; Ide, Hideyuki; Morio, Hanae; Shibuya, Minaka; Kamiichi, Atsuko; Tsubota, Akihito; Anzai, Naohiko; Akita, Hidetaka; Chiba, Kan; Furihata, Tomomi

2018-01-01

Liver sinusoidal endothelial cells (LSECs), which are specialized endothelial cells that line liver sinusoids, have been reported to participate in a variety of liver functions, such as blood macromolecule clearance and factor VIII production. In addition, LSECs play crucial roles in liver regeneration following acute liver injury, as well as the development and progression of liver diseases or drug-induced hepatotoxicity. However, the molecular mechanisms underlying their roles remain mostly unknown. Therefore, in order to contribute to the clarification of those mechanisms, herein we report on the development of a new immortalized human LSEC (HLSEC) line. To produce this cell line, two immortalized genes were introduced into the primary HLSECs, which eventually resulted in the establishment of the HLSEC/conditionally immortalized, clone-J (HLSEC/ciJ). Consistent with the two-immortalized gene expression, HLSEC/ciJ showed excellent proliferation activity. Additionally, the results of gene expression analyses showed that several LSEC (as well as pan-endothelial) marker mRNAs and proteins were clearly expressed in HLSEC/ciJ. Furthermore, we found that adherence junction proteins were localized at the cell border in the HLSEC/ciJ monolayer, and that the cells exhibited a tube-like structure formation property. Taken together, the results obtained thus far indicate that we have successfully immortalized HLSECs, resulting in creation of HLSEC/ciJ, a cell line that possesses infinite proliferation ability while retaining possession of at least some HLSEC features. We believe that the HLSEC/ciJ have the potential to provide a valuable and unlimited alternative source of HLSECs for use in liver/LSEC physiology/pathophysiology, pharmacology, and toxicology studies.

The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

PubMed

Youns, Mаhmoud; Abdel Halim Hegazy, Wael

2017-01-01

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways

PubMed Central

Youns, Mаhmoud; Abdel Halim Hegazy, Wael

2017-01-01

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes. PMID:28052097

Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

PubMed

Tian, Shenglan; Chen, Heng; Tan, Wei

2018-05-23

Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of the spray adjuvant on the toxicity effects of a glyphosate formulation.

PubMed

Coalova, Isis; Ríos de Molina, María Del Carmen; Chaufan, Gabriela

2014-10-01

In the present study, the influence of the spray adjuvant on the toxicity effects of a glyphosate formulation was examined in HEp-2 cell line. We determined the median lethal concentration (LC50) of Atanor® (glyphosate formulation), Impacto® (spray adjuvant) and the mixture of both agrochemicals. We also compared the toxicities of the pesticides individually and in mixture and we analyzed the effects on oxidative balance from each treatment. Our results showed that all the agrochemicals assayed induce dose and time-dependent cytotoxicity and that the toxicity of Impacto® with Atanor® (mixture) was additive on HEp-2 cell line. All the agrochemicals assayed produced an increase in catalase activity and glutathione levels, while no effects were observed for superoxide dismutase and glutathione-S-transferase activities. We found an important increase in ROS production in cells treated with Atanor® and mixture. Besides, all the agrochemicals used triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. In conclusion, our results demonstrated that the addition of adjuvant to glyphosate formulation increase the toxicity of the mixture in cell culture. Furthermore, cell culture exposed to agrochemical mixture showed an increased ROS production and antioxidant defenses. Copyright © 2014 Elsevier Ltd. All rights reserved.

High sensitive FBG load cell for icing of overhead transmission lines

NASA Astrophysics Data System (ADS)

Mao, Naiqiang; Ma, Guoming; Li, Chengrong; Li, Yabo; Shi, Cheng; Du, Yue

2017-04-01

Heavy ice coating of overhead transmission lines created the serious threat on the safe operation of power grid. The measurement of conductor icing had been an effective and reliable methods to prevent potential risks, such as conductor breakage, insulator flashover and tower collapse. Because of the advantages of immunity to electromagnetic interference and no demand for power supply in site, the optical load cell has been widely applied in monitoring the ice coating of overhead transmission lines. In this paper, we have adopted the shearing structure with additional grooves as elastic element of load cell to detect the eccentric load. Then, two welding package fiber Bragg gratings (FBGs) were mounted onto the grooves of elastic element with a direction deviation of 90° to eliminate temperature effects on strain measurement without extra FBG. After that, to avoid the occurrence of load cell breakage in heavy load measurement, the protection part has been proposed and added to the elastic element. The results of tension experiments indicate that the resolution of the load cell is 7.78 N in the conventional measuring range (0-10 kN). And in addition, the load cell proposed in this paper also has a good performance in actual experiment in which the load and temperature change simultaneously.

The Effect of Garlic Extract on Expression of INFγ And Inos Genes in Macrophages Infected with Leishmania major

PubMed Central

Gharavi, MJ; Nobakht, M; Khademvatan, SH; Bandani, E; Bakhshayesh, M; Roozbehani, M

2011-01-01

Background The study was aimed to show the effect of molecular mechanism of Aqueous Garlic Extract (AGE) on expression of IFNγ and iNOS genes in Leishmania major. Methods Leishmania major promastigotes (MRHO/IR/75/ER) were added to the in-vitro cultured J774 cell line, the cells were incubated for 72 hours. Various concentrations of garlic extract (9.25, 18.5, 37, 74, 148 mg/ml) were added to the infected cells. MTT assay was applied for cellular proliferation. After 72 hours of incubation, supernatants were collected and total RNA was extracted from the infected cells. The express of IFNγ and iNOS genes were studied by RT-PCR method. Results The colorimetric MTT assay after 3 days of incubation showed cytotoxic effect of garlic extract with an IC50 of 37 mg/ml. In addition, IFNγ and iNOS genes expression by RT-PCR indicated that garlic extract lead to over expression of these genes in J774 cell line infected with L. major. Conclusion Garlic extract exerts cytotoxic effect on infected J774 cell line. In addition, the hypothesis that garlic can improve cellular immunity with raising the expression of IFNγ and of iNOS genes confirmed. PMID:22347300

Spheroid Culture of Head and Neck Cancer Cells Reveals an Important Role of EGFR Signalling in Anchorage Independent Survival.

PubMed

Braunholz, Diana; Saki, Mohammad; Niehr, Franziska; Öztürk, Merve; Borràs Puértolas, Berta; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

2016-01-01

In solid tumours millions of cells are shed into the blood circulation each day. Only a subset of these circulating tumour cells (CTCs) survive, many of them presumable because of their potential to form multi-cellular clusters also named spheroids. Tumour cells within these spheroids are protected from anoikis, which allows them to metastasize to distant organs or re-seed at the primary site. We used spheroid cultures of head and neck squamous cell carcinoma (HNSCC) cell lines as a model for such CTC clusters for determining the role of the epidermal growth factor receptor (EGFR) in cluster formation ability and cell survival after detachment from the extra-cellular matrix. The HNSCC cell lines FaDu, SCC-9 and UT-SCC-9 (UT-SCC-9P) as well as its cetuximab (CTX)-resistant sub-clone (UT-SCC-9R) were forced to grow in an anchorage-independent manner by coating culture dishes with the anti-adhesive polymer poly-2-hydroxyethylmethacrylate (poly-HEMA). The extent of apoptosis, clonogenic survival and EGFR signalling under such culture conditions was evaluated. The potential of spheroid formation in suspension culture was found to be positively correlated with the proliferation rate of HNSCC cell lines as well as their basal EGFR expression levels. CTX and gefitinib blocked, whereas the addition of EGFR ligands promoted anchorage-independent cell survival and spheroid formation. Increased spheroid formation and growth were associated with persistent activation of EGFR and its downstream signalling component (MAPK/ERK). Importantly, HNSCC cells derived from spheroid cultures retained their clonogenic potential in the absence of cell-matrix contact. Addition of CTX under these conditions strongly inhibited colony formation in CTX-sensitive cell lines but not their resistant subclones. Altogether, EGFR activation was identified as crucial factor for anchorage-independent survival of HNSCC cells. Targeting EGFR in CTC cluster formation might represent an attractive anti-metastatic treatment approach in HNSCC.

In vitro evaluation of combined temozolomide and radiotherapy using X  rays and high-linear energy transfer radiation for glioblastoma.

PubMed

Barazzuol, Lara; Jena, Raj; Burnet, Neil G; Jeynes, Jonathan C G; Merchant, Michael J; Kirkby, Karen J; Kirkby, Norman F

2012-05-01

High-linear energy transfer radiation offers superior biophysical properties over conventional radiotherapy and may have a great potential for treating radioresistant tumors, such as glioblastoma. However, very little pre-clinical data exists on the effects of high-LET radiation on glioblastoma cell lines and on the concomitant application of chemotherapy. This study investigates the in vitro effects of temozolomide in combination with low-energy protons and α particles. Cell survival, DNA damage and repair, and cell growth were examined in four human glioblastoma cell lines (LN18, T98G, U87 and U373) after treatment with either X rays, protons (LET 12.91 keV/μm), or α particles (LET 99.26 keV/μm) with or without concurrent temozolomide at clinically-relevant doses of 25 and 50 μM. The relative biological effectiveness at 10% survival (RBE(10)) increased as LET increased: 1.17 and 1.06 for protons, and 1.84 and 1.68 for α particles in the LN18 and U87 cell lines, respectively. Temozolomide administration increased cell killing in the O(6)-methylguanine DNA methyltransferase-methylated U87 and U373 cell lines. In contrast, temozolomide provided no therapeutic enhancement in the methylguanine DNA methyltransferase-unmethylated LN18 and T98G cell lines. In addition, the residual number of γ-H2AX foci at 24 h after treatment with radiation and concomitant temozolomide was found to be lower than or equal to that expected by DNA damage with either of the individual treatments. Kinetics of foci disappearance after X-ray and proton irradiation followed similar time courses; whereas, loss of γ-H2AX foci after α particle irradiation occurred at a slower rate than that by low-LET radiation (half-life 12.51-16.87 h). The combination of temozolomide with different radiation types causes additive rather than synergistic cytotoxicity. Nevertheless, particle therapy combined with chemotherapy may offer a promising alternative with the additional benefit of superior biophysical properties. It is also possible that new fractionation schedules could be designed to exploit the change in DNA repair kinetics when MGMT-methylated cells respond to high-LET radiation.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

PubMed

Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

CD30 downregulation, MMAE resistance, and MDR1 upregulation are all associated with resistance to brentuximab vedotin

PubMed Central

Chen, Robert; Hou, Jessie; Newman, Edward; Kim, Young; Donohue, Cecile; Liu, Xueli; Thomas, Sandra H.; Forman, Stephen J.; Kane, Susan E.

2015-01-01

Brentuximab vedotin (BV) is an antibody-drug conjugate that specifically delivers the potent cytotoxic drug MMAE to CD30-positive cells. BV is FDA-approved for treatment of relapsed/refractory Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV resistant disease. We selected for BV-resistant HL (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (HL) exposure to BV. We confirmed drug resistance by MTS assay, and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. Additionally, tissue biopsy samples from 10 HL and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the HL cell line, demonstrated downregulated CD30 expression compared to the parental cell line. In contrast, the HL cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared to the parental line. For both HL and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One HL patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possible modes of BV resistance for HL both in vitro and in patients. PMID:25840583

Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors.

PubMed

Kim, Eunhye; Hwang, Seon-Ung; Yoo, Hyunju; Yoon, Junchul David; Jeon, Yubyeol; Kim, Hyunggee; Jeung, Eui-Bae; Lee, Chang-Kyu; Hyun, Sang-Hwan

2016-03-01

The establishment of porcine embryonic stem cells (ESCs) would have great impact in biomedical studies and preclinical trials through their use in genetic engineering. However, authentic porcine ESCs have not been established until now. In this study, a total of seven putative ESC lines were derived from porcine embryos of various origins, including in vitro fertilization, parthenogenetic activation, and, in particular, induced pluripotent stem (iPS) nuclear transfer (NT) from a donor cell with induced pluripotent stem cells (iPSCs). To characterize these cell lines, several assays including an assessment of intensive alkaline phosphatase activity, karyotyping, embryoid body formation, expression analysis of the pluripotency-associated markers, and the three germ layerassociated markers were performed. Based on quantitative polymerase chain reaction, the expression levels of REX1 and FGFR2 in iPS-NT lines were higher than those of cells of other origins. Additionally, only iPS-NT lines showed multiple aberrant patterns of nuclear foci elucidated by immunofluorescence staining of H3K27me3 as a marker of the state of X chromosome inactivation and a less mature form of mitochondria like naive ESCs, by transmission electron microscopy. Together, these data suggested that established putative porcine ESC lines generally exhibited a primed pluripotent state, like human ESCs. However, iPS-NT lines have especially unique characteristics distinct from other origins because they have more epigenetic instability and naive-like mitochondrial morphology than other putative ESC lines. This is the first study to establish and characterize the iPSC-derived putative ESC lines and compare them with other lines derived from different origins in pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Becerikli, Mustafa; Jacobsen, Frank; Rittig, Andrea

Soft tissue sarcomas (STS) are characterized by co-participation of several epigenetic and genetic events during tumorigenesis. Having bypassed cellular senescence barriers during oncogenic transformation, the factors further affecting growth rate of STS cells remain poorly understood. Therefore, we investigated the role of gene silencing (DNA promoter methylation of LINE-1, PTEN), genetic aberrations (karyotype, KRAS and BRAF mutations) as well as their contribution to the proliferation rate and migratory potential that underlies “initial” and “final” passage sarcoma cells. Three different cell lines were used, SW982 (synovial sarcoma), U2197 (malignant fibrous histiocytoma (MFH)) and HT1080 (fibrosarcoma). Increased proliferative potential of final passagemore » STS cells was not associated with significant differences in methylation (LINE-1, PTEN) and mutation status (KRAS, BRAF), but it was dependent on the amount of chromosomal aberrations. Collectively, our data demonstrate that these fairly differentiated/advanced cancer cell lines have still the potential to gain an additional spontaneous growth benefit without external influences and that maintenance of increased proliferative potential towards longevity of STS cells (having crossed senescence barriers) may be independent of overt epigenetic alterations. -- Highlights: Increased proliferative potential of late passage STS cells was: • Not associated with epigenetic changes (methylation changes at LINE-1, PTEN). • Not associated with mutation status of KRAS, BRAF. • Dependent on presence/absence of chromosomal aberrations.« less

EMT is associated with, but does not drive resistance to ALK inhibitors among EML4-ALK non-small cell lung cancer.

PubMed

Gower, Arjan; Hsu, Wei-Hsun; Hsu, Shuo-Tse; Wang, Yisong; Giaccone, Giuseppe

2016-04-01

ALK gene fusion occurs in approximately 3-7% of non-small cell lung cancer (NSCLC). For patients with ALK positive NCSLC, crizotinib and ceritinib are FDA approved ALK inhibitors, however, patients inevitably acquire resistance to such therapies typically within one to two years. Interrogation of in vitro ALK-positive NSCLC cell line models of acquired resistance to first and second-generation ALK inhibitors revealed acquired epithelial-to-mesenchymal transition (EMT) mechanisms. Here we demonstrated that knockdown of upregulated mesenchymal markers in acquired resistant lines decreased the invasive and migratory capabilities of the cells, however, it did not restore sensitivity to ALK inhibitors. Removing drug for 5 weeks from H3122 cell line that acquired resistance to ceritinib restored its sensitivity to ceritinib. In addition, HSP90 inhibitors ganetespib and 17-AAG were potent in inducing cell death in cell lines resistant to crizotinib and ceritinib. Taken together, EMT does not drive resistance to ALK inhibitors and HSP90 inhibition demonstrates more efficacy when further ALK inhibition may not. This study warrants more exploration of HSP90 inhibitors for ALK-positive patients who progress on 1st and 2nd line ALK inhibitor therapy. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions

NASA Technical Reports Server (NTRS)

Piao, C. Q.; Hei, T. K.; Hall, E. J. (Principal Investigator)

2001-01-01

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

PubMed Central

Michaelis, M; Rothweiler, F; Agha, B; Barth, S; Voges, Y; Löschmann, N; von Deimling, A; Breitling, R; Wilhelm Doerr, H; Rödel, F; Speidel, D; Cinatl, J

2012-01-01

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells. PMID:22476102

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.

PubMed

Michaelis, M; Rothweiler, F; Agha, B; Barth, S; Voges, Y; Löschmann, N; von Deimling, A; Breitling, R; Doerr, H Wilhelm; Rödel, F; Speidel, D; Cinatl, J

2012-04-05

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Chemosensitivity of BRCA1-Mutated Ovarian Cancer Cells and Established Cytotoxic Agents.

PubMed

van Haaften, Caroline; van Eendenburg, Jaap; Boot, Arnoud; Corver, Willem E; Haans, Lucien; van Wezel, Tom; Trimbos, J Baptist

2017-10-01

Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8β-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8β-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.

Anti-tumor effects of suberoylanilide hydroxamic acid on Epstein–Barr virus-associated T cell and natural killer cell lymphoma

PubMed Central

Siddiquey, Mohammed NA; Nakagawa, Hikaru; Iwata, Seiko; Kanazawa, Tetsuhiro; Suzuki, Michio; Imadome, Ken-Ichi; Fujiwara, Shigeyoshi; Goshima, Fumi; Murata, Takayuki; Kimura, Hiroshi

2014-01-01

The ubiquitous Epstein–Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells and is associated with various lymphoid malignancies. Recent studies have reported that histone deacetylase (HDAC) inhibitors exert anticancer effects against various tumor cells. In the present study, we have evaluated both the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on EBV-positive and EBV-negative T and NK lymphoma cells. Several EBV-positive and EBV-negative T and NK cell lines were treated with various concentrations of SAHA. SAHA suppressed the proliferation of T and NK cell lines, although no significant difference was observed between EBV-positive and EBV-negative cell lines. SAHA induced apoptosis and/or cell cycle arrest in several T and NK cell lines. In addition, SAHA increased the expression of EBV-lytic genes and decreased the expression of EBV-latent genes. Next, EBV-positive NK cell lymphoma cells were subcutaneously inoculated into severely immunodeficient NOD/Shi-scid/IL-2Rγnull mice, and then SAHA was administered intraperitoneally. SAHA inhibited tumor progression and metastasis in the murine xenograft model. SAHA displayed a marked suppressive effect against EBV-associated T and NK cell lymphomas through either induction of apoptosis or cell cycle arrest, and may represent an alternative treatment option. PMID:24712440

Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells.

PubMed

Morley, Karen L; Ferguson, Peter J; Koropatnick, James

2007-06-18

Tangeretin and nobiletin are citrus flavonoids that are among the most effective at inhibiting cancer cell growth in vitro and in vivo. The antiproliferative activity of tangeretin and nobiletin was investigated in human breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29. Both flavonoids inhibited proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at G1 in all three cell lines. At concentrations that resulted in significant inhibition of proliferation and cell cycle arrest, neither flavonoid induced apoptosis or cell death in any of the tumor cell lines. To test the ability of arrested cells to recover, cells that were incubated with tangeretin and nobiletin for 4 days were then cultured in flavonoid-free medium for an additional 4 days. Cells resumed proliferation similar to untreated control within a day of flavonoid removal. Cell cycle distribution was similar to that of control within 4 days of flavonoid removal. These data indicate that, in these cell lines at concentrations that inhibit proliferation up to 80% over 4 days, tangeretin and nobiletin are cytostatic and significantly suppress proliferation by cell cycle arrest without apoptosis. Such an agent could be expected to spare normal tissues from toxic side effects. Thus, tangeretin and nobiletin could be effective cytostatic anticancer agents. Inhibition of proliferation of human cancers without inducing cell death may be advantageous in treating tumors as it would restrict proliferation in a manner less likely to induce cytotoxicity and death in normal, non-tumor tissues.

Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

PubMed

Sova, Matej; Žižak, Željko; Stanković, Jelena A Antic; Prijatelj, Matevž; Turk, Samo; Juranić, Zorica D; Mlinarič-Raščan, Irena; Gobec, Stanislav

2013-08-01

Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

Effects of phenformin on the proliferation of human tumor cell lines.

PubMed

Caraci, Filippo; Chisari, Mariangela; Frasca, Giuseppina; Chiechio, Santina; Salomone, Salvatore; Pinto, Antonio; Sortino, Maria Angela; Bianchi, Alfredo

2003-12-19

Phenformin is a biguanide that has been largely used in the past for its anti-diabetic activity. A large body of evidence suggests additional effects of phenformin including antitumoral activity in different animal models in vivo. Thus, the present study has been conducted in order to elucidate possible mechanisms involved in the antitumoral effects of phenformin. In various tumoral cell lines (SH-SY5Y neuroblastoma and LNCaP prostate adenocarcinoma cells), increasing concentrations of phenformin (50-500 microM) induced a concentration-dependent inhibition of cell proliferation. This effect was not dependent on the ability of the drug to reduce glucose levels and was accompanied by induction of apoptotic cell death as measured by cytofluorometric analysis. In addition, a short-time incubation of SH-SY5Y cells with phenformin induced enhanced and transient expression of the cell cycle inhibitor p21 suggesting that phenformin causes inhibition of cell cycle progression prior to induction of apoptosis. These results demonstrate an activity at the cellular level of phenformin that supports its antitumoral effect observed in vivo.

Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines.

PubMed

Gajski, Goran; Čimbora-Zovko, Tamara; Rak, Sanjica; Rožman, Marko; Osmak, Maja; Garaj-Vrhovac, Vera

2014-12-01

In the present study, we investigated the possible combined anticancer ability of bee venom (BV) and cisplatin towards two pairs of tumour cell lines: parental cervical carcinoma HeLa cells and their cisplatin-resistant HeLa CK subline,as well as laryngeal carcinoma HEp-2 cells and their cisplatin-resistant CK2 subline. Additionally, we identified several peptides of BV in the BV sample used in the course of the study and determined the exact concentration of MEL. BV applied alone in concentrations of 30 to 60 μg ml(–1) displayed dose-dependent cytotoxicity against all cell lines tested. Cisplatin-resistant cervical carcinoma cells were more sensitive to BV than their parental cell lines (IC(50) values were 52.50 μg ml(–1) for HeLa vs.47.64 μg ml(–1) for HeLa CK cells), whereas opposite results were obtained for cisplatin-resistant laryngeal carcinoma cells (IC(50) values were 51.98 μg ml(–1) for HEp-2 vs. > 60.00 μg ml(–1) for CK2 cells). Treatment with BV alone induced a necrotic type of cell death, as shown by characteristic morphological features, fast staining with ethidium-bromide and a lack of cleavage of apoptotic marker poly (ADP-ribose) polymerase (PARP) on Western blot. Combined treatment of BV and cisplatin induced an additive and/or weak synergistic effect towards tested cell lines, suggesting that BV could enhance the killing effect of selected cells when combined with cisplatin. Therefore, a greater anticancer effect could be triggered if BV was used in the course of chemotherapy. Our results suggest that combined treatment with BV could be useful from the point of minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance.

A new PDAC mouse model originated from iPSCs-converted pancreatic cancer stem cells (CSCcm)

PubMed Central

Calle, Anna Sanchez; Nair, Neha; Oo, Aung KoKo; Prieto-Vila, Marta; Koga, Megumi; Khayrani, Apriliana Cahya; Hussein, Maram; Hurley, Laura; Vaidyanath, Arun; Seno, Akimasa; Iwasaki, Yoshiaki; Calle, Malu; Kasai, Tomonari; Seno, Masaharu

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the most representative form of pancreatic cancers. PDAC solid tumours are constituted of heterogeneous populations of cells including cancer stem cells (CSCs), differentiated cancer cells, desmoplastic stroma and immune cells. The identification and consequent isolation of pancreatic CSCs facilitated the generation of genetically engineered murine models. Nonetheless, the current models may not be representative for the spontaneous tumour occurrence. In the present study, we show the generation of a novel pancreatic iPSC-converted cancer stem cell lines (CSCcm) as a cutting-edge model for the study of PDAC. The CSCcm lines were achieved only by the influence of pancreatic cancer cell lines conditioned medium and were not subjected to any genetic manipulation. The xenografts tumours from CSCcm lines displayed histopathological features of ADM, PanIN and PDAC lesions. Further molecular characterization from RNA-sequencing analysis highlighted primary culture cell lines (1st CSCcm) as potential candidates to represent the pancreatic CSCs and indicated the establishment of the pancreatic cancer molecular pattern in their subsequent progenies 2nd CSCcm and 3rd CSCcm. In addition, preliminary RNA-seq SNPs analysis showed that the distinct CSCcm lines did not harbour single point mutations for the oncogene Kras codon 12 or 13. Therefore, PDAC-CSCcm model may provide new insights about the actual occurrence of the pancreatic cancer leading to develop different approaches to target CSCs and abrogate the progression of this fatidic disease. PMID:28042501

Characterization of CD133+ parenchymal cells in the liver: histology and culture.

PubMed

Yoshikawa, Seiichi; Zen, Yoh; Fujii, Takahiko; Sato, Yasunori; Ohta, Tetsuo; Aoyagi, Yutaka; Nakanuma, Yasuni

2009-10-21

To reveal the characteristics of CD133(+) cells in the liver. This study examined the histological characteristics of CD133(+) cells in non-neoplastic and neoplastic liver tissues by immunostaining, and also analyzed the biological characteristics of CD133(+) cells derived from human hepatocellular carcinoma (HCC) or cholangiocarcinoma cell lines. Immunostaining revealed constant expression of CD133 in non-neoplastic and neoplastic biliary epithelium, and these cells had the immunophenotype CD133(+)/CK19(+)/HepPar-1(-). A small number of CD133(+)/CK19(-)/HepPar-1(+) cells were also identified in HCC and combined hepatocellular and cholangiocarcinoma. In addition, small ductal structures, resembling the canal of Hering, partly surrounded by hepatocytes were positive for CD133. CD133 expression was observed in three HCC (HuH7, PLC5 and HepG2) and two cholangiocarcinoma cell lines (HuCCT1 and CCKS1). Fluorescence-activated cell sorting (FACS) revealed that CD133(+) and CD133(-) cells derived from HuH7 and HuCCT1 cells similarly produced CD133(+) and CD133(-) cells during subculture. To examine the relationship between CD133(+) cells and the side population (SP) phenotype, FACS was performed using Hoechst 33342 and a monoclonal antibody against CD133. The ratios of CD133(+)/CD133(-) cells were almost identical in the SP and non-SP in HuH7. In addition, four different cellular populations (SP/CD133(+), SP/CD133(-), non-SP/CD133(+), and non-SP/CD133(-)) could similarly produce CD133(+) and CD133(-) cells during subculture. This study revealed that CD133 could be a biliary and progenitor cell marker in vivo. However, CD133 alone is not sufficient to detect tumor-initiating cells in cell lines.

The Antitumor Effect of C-terminus of Hsp70-Interacting Protein via Degradation of c-Met in Small Cell Lung Cancer.

PubMed

Cho, Sung Ho; Kim, Jong In; Kim, Hyun Su; Park, Sung Dal; Jang, Kang Won

2017-06-01

The mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion. To evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays. We found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines. CHIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.

Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines

PubMed Central

Al-Asmari, Abdulrahman Khazim; Riyasdeen, Anvarbatcha; Al-Shahrani, Mohammad Hamed; Islam, Mozaffarul

2016-01-01

Snake venom possesses various kinds of proteins and neurotoxic polypeptides, which can negatively interfere with the neurotransmitter signaling cascade. This phenomenon occurs mainly due to the blocking of ion channels in the body system. Envenomation prevents or severely interrupts nerve impulses from being transmitted, inhibition of adenosine triphosphate synthesis, and proper functioning of the cardiac muscles. However, some beneficial properties of venoms have also been reported. The aim of this study was to examine the snake venom as an anticancer agent due to its inhibitory effects on cancer progression such as cell motility, cell invasion, and colony formation. In this study, the effect of venoms on phenotypic changes and the change on molecular level in colorectal and breast cancer cell lines were examined. A reduction of 60%–90% in cell motility, colony formation, and cell invasion was observed when these cell lines were treated with different concentrations of snake venom. In addition, the increase in oxidative stress that results in an increase in the number of apoptotic cancer cells was significantly higher in the venom-treated cell lines. Further analysis showed that there was a decrease in the expression of pro-inflammatory cytokines and signaling proteins, strongly suggesting a promising role for snake venom against breast and colorectal cancer cell progression. In conclusion, the snake venoms used in this study showed significant anticancer properties against colorectal and breast cancer cell lines. PMID:27799796

FGFR4 Role in Epithelial-Mesenchymal Transition and Its Therapeutic Value in Colorectal Cancer

PubMed Central

Torres, Sofía; Hernández-Varas, Pablo; Teixidó, Joaquín; Bonilla, Félix; de Herreros, Antonio Garcia; Casal, J. Ignacio

2013-01-01

Fibroblast growth factor receptor 4 (FGFR4) is vital in early development and tissue repair. FGFR4 expression levels are very restricted in adult tissues, except in several solid tumors including colorectal cancer, which showed overexpression of FGFR4. Here, FGFR4 mutation analysis discarded the presence of activating mutations, other than Arg388, in different colorectal cancer cell lines and tumoral samples. Stable shRNA FGFR4-silencing in SW480 and SW48 cell lines resulted in a significant decrease in cell proliferation, adhesion, cell migration and invasion. This decrease in the tumorigenic and invasive capabilities of colorectal cancer cells was accompanied by a decrease of Snail, Twist and TGFβ gene expression levels and an increase of E-cadherin, causing a reversion to a more epithelial phenotype, in three different cell lines. In addition, FGFR4-signaling activated the oncogenic SRC, ERK1/2 and AKT pathways in colon cancer cells and promoted an increase in cell survival. The relevance of FGFR4 in tumor growth was supported by two different strategies. Kinase inhibitors abrogated FGFR4-related cell growth and signaling pathways at the same extent than FGFR4-silenced cells. Specific FGFR4-targeting using antibodies provoked a similar reduction in cell growth. Moreover, FGFR4 knock-down cells displayed a reduced capacity for in vivo tumor formation and angiogenesis in nude mice. Collectively, our data support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer. In addition, FGFR4 targeting demonstrated its applicability for colorectal cancer therapy. PMID:23696849

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

PubMed

Seiler, C; Nicolson, T

1999-11-15

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

The influence of biomechanical properties and cannabinoids on tumor invasion.

PubMed

Hohmann, Tim; Grabiec, Urszula; Ghadban, Chalid; Feese, Kerstin; Dehghani, Faramarz

2017-01-02

Cannabinoids are known to have an anti-tumorous effect, but the underlying mechanisms are only sparsely understood. Mechanical characteristics of tumor cells represent a promising marker to distinguish between tumor cells and the healthy tissue. We tested the hypothesis whether cannabinoids influence the tumor cell specific mechanical and migratory properties and if these factors are a prognostic marker for the invasiveness of tumor cells. 3 different glioblastoma cell lines were treated with cannabinoids and changes of mechanical and migratory properties of single cells were measured using atomic force microscopy and time lapse imaging. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. We found that cannabinoids are capable of influencing migratory and mechanical properties in a cell line specific manner. A network analysis revealed a correlation between a "generalized stiffness" and the invasiveness for all tumor cell lines after 3 and 4 d of invasion time: r 3d = -0.88 [-0.52;-0.97]; r 4d = -0.90 [-0.59;-0.98]. Here we could show that a "generalized stiffness" is a profound marker for the invasiveness of a tumor cell population in our model and thus might be of high clinical relevance for drug testing. Additionally cannabinoids were shown to be of potential use for therapeutic approaches of glioblastoma.

Toxicity of allyl esters in insect cell lines and in Spodoptera littoralis larvae.

PubMed

Giner, Marta; Avilla, Jesús; Balcells, Mercè; Caccia, Silvia; Smagghe, Guy

2012-01-01

We investigated the effects of five allyl esters, two aromatic (allyl cinnamate and allyl 2-furoate) and three aliphatic (allyl hexanoate, allyl heptanoate, and allyl octanoate) in established insect cell lines derived from different species and tissues. We studied embryonic cells of the fruit fly Drosophila melanogaster (S2) (Diptera) and the beet armyworm Spodoptera exigua (Se4) (Lepidoptera), fat body cells of the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Coleoptera), ovarian cells of the silkmoth Bombyx mori (Bm5), and midgut cells of the spruce budworm Choristoneura fumiferana (CF203) (Lepidoptera). Cytotoxicity was determined with use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue. In addition, we tested the entomotoxic action of allyl cinnamate against the cotton leafworm Spodoptera littoralis .The median (50%) cytotoxic concentrations (EC₅₀s) of the five allyl esters in the MTT bioassays ranged between 0.25 and 27 mM with significant differences among allyl esters (P = 0.0012), cell lines (P < 0.0001), and the allyl ester-cell line interaction (P < 0.0001). Allyl cinnamate was the most active product, and CF203 the most sensitive cell line. In the trypan blue bioassays, cytotoxicity was produced rapidly and followed the same trend observed in the MTT bioassay. In first instars of S. littoralis, allyl cinnamate killed all larvae at 0.25% in the diet after 1 day, while this happened in third instars after 5 days. The LC₅₀ in first instars was 0.08%. In addition, larval weight gain was reduced (P < 0.05) after 1 day of feeding on diet with 0.05%. In conclusion, the data provide evidence of the significant but differential cytotoxicity among allyl esters in insect cells of different species and tissues. Midgut cells show high sensitivity, indicating the insect midgut as a primary target tissue. Allyl cinnamate caused rapid toxic effects in S. littoralis larvae at low concentrations, suggesting further potential for use in pest control. © 2011 Wiley Periodicals, Inc.

Synthesis of a high molecular weight thyroglobulin dimer by two ovine thyroid cell lines: the OVNIS.

PubMed

Hovsépian, S; Aouani, A; Fayet, G

1986-05-01

The OVNIS 6H and 5H thyroid cells, 2 permanent cell lines isolated 3 years ago from ovine tissue, synthesize a high molecular weight glycosylated protein, immunologically related to ovine thyroglobulin, which is similar to the prothyroid hormone dimer (17-19) S: thyroglobulin. Using sucrose gradient centrifugation and cell labelling with [14C]Leu or [3H]GlNH2, radioactivity was observed in proteins purified from cell layers and from cell culture media. Addition of thyrotropin to or removal from the media resulted respectively in an increase (+773%) or decrease (-1090%) of the total radioactivity detected in the (17-19)S thyroglobulin fraction. Estimation of thyroglobulin by RIA gave similar though less pronounced effects. These experiments prove (1) that thyroglobulin is still expressed in these OVNIS thyroid cell lines even after 3 years of permanent culture, (2) that TSH modulates the level of this protein through a TSH-receptor functional system.

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53

PubMed Central

York, D.; Withers, S. S.; Watson, K. D.; Seo, K. W.; Rebhun, R. B.

2016-01-01

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity. PMID:27333821

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

PubMed

York, D; Withers, S S; Watson, K D; Seo, K W; Rebhun, R B

2017-09-01

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity. © 2016 John Wiley & Sons Ltd.

Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells.

PubMed

Wiest, Nathaniel E; Tomkinson, Alan E

2017-01-01

The isolation of proteins on nascent DNA (iPOND) technique developed by the Cortez laboratory allows a previously unparalleled ability to examine proteins associated with replicating and newly synthesized DNA in mammalian cells. Both the original, formaldehyde-based iPOND technique and a more recent derivative, accelerated native iPOND (aniPOND), have mostly been performed in adherent cell lines. Here, we describe modifications to both protocols for use with suspension cell lines. These include cell culture, pulse, and chase conditions that optimize sample recovery in both protocols using suspension cells and several key improvements to the published aniPOND technique that reduce sample loss, increase signal to noise, and maximize sample recovery. Additionally, we directly and quantitatively compare the iPOND and aniPOND protocols to test the strengths and limitations of both. Finally, we present a detailed protocol to perform the optimized aniPOND protocol in suspension cell lines. © 2017 Elsevier Inc. All rights reserved.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Ultrastructural characterization of the new NG97ht human-derived glioma cell line using two different electron microscopy technical procedures.

PubMed

Machado, Camila Maria Longo; Zorzeto, Tatiane Queiroz; Bianco, Juares E Romero; Rosa, Renata Giardini; Genari, Selma Candelaria; Joazeiro, Paulo Pinto; Verinaud, Liana

2009-04-01

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

PubMed

Vicente, Carolina Meloni; Ricci, Ritchelli; Nader, Helena Bonciani; Toma, Leny

2013-05-25

The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.

PubMed

Gomez-Martinez, Marta; Schmitz, Debora; Hergovich, Alexander

2013-03-05

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon(1), to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells(2,3). In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here. Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells(4), vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs(5-11). However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.(12) modified the shRNA expression vector pSUPER(5) by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest. Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome(13,14) and apoptosis signaling(15,16). In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

SGK is a primary glucocorticoid-induced gene in the human.

PubMed

Náray-Fejes-Tóth, A; Fejes-Tóth, G; Volk, K A; Stokes, J B

2000-12-01

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro

PubMed Central

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-01-01

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC. PMID:29371987

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro.

PubMed

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-12-26

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC.

An in vitro cytotoxic approach to assess the toxicity of heavy metals and their binary mixtures on hippocampal HT-22 cell line.

PubMed

Karri, Venkatanaidu; Kumar, Vikas; Ramos, David; Oliveira, Eliandre; Schuhmacher, Marta

2018-01-05

Humans are exposed to a cocktail of heavy metal toxicants in the environment. Though heavy metals are deleterious, there is a paucity of information on the toxicity of mixtures. In this study, four common neurotoxicity heavy metals lead (Pb) cadmium (Cd), arsenic (As), and methylmercury (MeHg) were exposed individually and as mixtures to HT-22 cell line for 8days. The study established that low dose exposures induced toxicity to the HT-22 cell line during 8days. The results indicates potency dependent response, the toxicity of single metals on the HT-22 cells; MeHg > As > Cd > Pb. The cytotoxicity data of single metals were used to determine the mixtures interaction profile by using the dose additivity and effect additivity method. Metal mixtures showed higher toxicities compared to individual metals. Synergistic, antagonistic or additive effects of the toxicity were observed in different mixtures in low dose exposure. The interactive responses of mixtures depend on the co-exposure metal and their respective concentration. We concluded that the combined effects should be considered in the risk assessment of heavy metal co-exposure and potency. In future, comprehensive mechanistic based investigations needed for understanding the real interactive mixtures effects at molecular level. Copyright © 2017 Elsevier B.V. All rights reserved.

The taccalonolides: microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms.

PubMed

Risinger, April L; Jackson, Evelyn M; Polin, Lisa A; Helms, Gregory L; LeBoeuf, Desiree A; Joe, Patrick A; Hopper-Borge, Elizabeth; Ludueña, Richard F; Kruh, Gary D; Mooberry, Susan L

2008-11-01

The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the betaIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the betaIII isotype of tubulin was generated to evaluate the effect of betaIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the betaIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the betaIII-tubulin-overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emergedmore » cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.« less

Antitumor effects of Marginisporum crassissimum (Rhodophyceae), a marine red alga.

PubMed

Hiroishi, S; Sugie, K; Yoshida, T; Morimoto, J; Taniguchi, Y; Imai, S; Kurebayashi, J

2001-06-26

Marginisporum crassissimum (Yendo) Ganesan, a marine red alga found in the ordinal coastal sea around Japan, revealed antitumor (antimetastatic) effects in vitro and in vivo. In in vitro experiments, extracts of this alga inhibited not only the growth of several tumor cell lines, such as B16-BL6 (a mouse melanoma cell line), JYG-B (a mouse mammary carcinoma cell line) and KPL-1 (a human mammary carcinoma cell line), but also invasion of B16-BL6 cells in a culture system. In in vivo experiments, the lung metastasis of B16-BL6 cells inoculated to the tail vein of B57BL/6J mice was inhibited by intraperitoneal administration of an extract from the alga. In addition, life prolongation of B57BL/6J mice inoculated with B16-BL6 cells was also observed by the intraperitoneal administration of the extract. An effective substance showing B16-BL6 growth inhibition in vitro was partially purified by filtration and hydrophobic column chromatography, and was revealed to be sensitive to trypsin-digestion and heat-treatment. The molecular weight of the substance was greater than 100 kDa. This is the first study demonstrating antitumor (antimetastatic) effects of M. crassissimum.

Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

PubMed

Singh, Mahipal; Sharma, Anil K

2011-02-01

Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

CCL17 and CCL22/CCR4 signaling is a strong candidate for novel targeted therapy against nasal natural killer/T-cell lymphoma

PubMed Central

Kumai, Takumi; Kobayashi, Hiroya; Komabayashi, Yuki; Ueda, Seigo; Kishibe, Kan; Ohkuri, Takayuki; Takahara, Miki; Celis, Esteban; Harabuchi, Yasuaki

2015-01-01

Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein–Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb. PMID:25754123

Anticancer effects of resveratrol in canine hemangiosarcoma cell lines.

PubMed

Carlson, A; Alderete, K S; Grant, M K O; Seelig, D M; Sharkey, L C; Zordoky, B N M

2018-06-01

Hemangiosarcoma (HSA) is a highly malignant tumour with aggressive biological behaviour. HSAs are more common in dogs than other domestic animals. The median survival time of dogs with HSA remains short, even with chemotherapy and surgery. Therefore, there is a critical need to improve the adjuvant chemotherapeutic regimens to improve clinical outcomes in dogs with HSA. Resveratrol has been shown to possess strong anti-proliferative and/or pro-apoptotic properties in human cancer cell lines. Nevertheless, the potential anticancer effects of resveratrol have not been reported in canine HSAs. The objective of this study is to determine the growth inhibitory effects of resveratrol in HSA cells when used alone or in combination with doxorubicin, a commonly used chemotherapeutic agent. Frog and DD-1 canine HSA cell lines were treated with varying concentrations of resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay. The expression of apoptotic proteins, activation of p38 mitogen-activated protein kinase (MAPK), AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) were assessed by western blotting. Similar to human cancer cell lines, resveratrol markedly inhibited the growth and induced apoptosis in both HSA cell lines. Mechanistically, resveratrol activated p38 MAPK, but did not affect the AMPK or the ERK1/2 pathways. Additional experiments showed that resveratrol augmented the growth-inhibitory and apoptotic effects of doxorubicin in both HSA cell lines. These findings suggest that resveratrol has pro-apoptotic effects in canine HSA cells; therefore, its use as a potential adjunct therapy in canine HSA patients warrants further investigation. © 2017 John Wiley & Sons Ltd.

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

Silencing of high-mobility group box 2 (HMGB2) modulates cisplatin and 5-fluorouracil sensitivity in head and neck squamous cell carcinoma.

PubMed

Syed, Nazia; Chavan, Sandip; Sahasrabuddhe, Nandini A; Renuse, Santosh; Sathe, Gajanan; Nanjappa, Vishalakshi; Radhakrishnan, Aneesha; Raja, Remya; Pinto, Sneha M; Srinivasan, Anand; Prasad, T S Keshava; Srikumar, Kotteazeth; Gowda, Harsha; Santosh, Vani; Sidransky, David; Califano, Joseph A; Pandey, Akhilesh; Chatterjee, Aditi

2015-01-01

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

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

2009-05-01

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

A reevaluation of CD22 expression in human lung cancer.

PubMed

Pop, Laurentiu M; Barman, Stephen; Shao, Chunli; Poe, Jonathan C; Venturi, Guglielmo M; Shelton, John M; Pop, Iliodora V; Gerber, David E; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I; Richardson, James A; Minna, John D; Tedder, Thomas F; Vitetta, Ellen S

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins.

Identification of liver cancer-specific aptamers using whole live cells.

PubMed

Shangguan, Dihua; Meng, Ling; Cao, Zehui Charles; Xiao, Zeyu; Fang, Xiaohong; Li, Ying; Cardona, Diana; Witek, Rafal P; Liu, Chen; Tan, Weihong

2008-02-01

Liver cancer is the third most deadly cancers in the world. Unfortunately, there is no effective treatment. One of the major problems is that most cancers are diagnosed in the later stage, when surgical resection is not feasible. Thus, accurate early diagnosis would significantly improve the clinical outcome of liver cancer. Currently, there are no effective molecular probes to recognize biomarkers that are specific for liver cancer. The objective of our current study is to identify liver cancer cell-specific molecular probes that could be used for liver cancer recognition and diagnosis. We applied a newly developed cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) method for the generation of molecular probes for specific recognition of liver cancer cells. The cell-SELEX uses whole live cells as targets to select aptamers (designed DNA/RNA) for cell recognition. In generating aptamers for liver cancer recognition, two liver cell lines were used: a liver cancer cell line BNL 1ME A.7R.1 (MEAR) and a noncancer cell line, BNL CL.2 (BNL). Both cell lines were originally derived from Balb/cJ mice. Through multiple rounds of selection using BNL as a control, we have identified a panel of aptamers that specifically recognize the cancer cell line MEAR with Kd in the nanomolar range. We have also demonstrated that some of the selective aptamers could specifically bind liver cancer cells in a mouse model. There are two major new results (compared with our reported cell-SELEX methodology) in addition to the generation of aptamers specifically for liver cancer. The first one is that our current study demonstrates that cell-based aptamer selection can select specific aptamers for multiple cell lines, even for two cell lines with minor differences (MEAR cell is derived from BNL by chemical inducement); and the second result is that cell-SELEX can be used for adhesive cells and thus open the door for solid tumor selection and investigation. The newly generated cancer-specific aptamers hold great promise as molecular probes for cancer early diagnosis and basic mechanism studies.

Growth and apoptosis of human natural killer cell neoplasms: role of interleukin-2/15 signaling.

PubMed

Yamasaki, Satoshi; Maeda, Motoi; Ohshima, Koichi; Kikuchi, Masahiro; Otsuka, Teruhisa; Harada, Mine

2004-10-01

Interleukin (IL)-15 plays an important role in the survival of human natural killer (NK) cells. We investigated IL-2/15 signaling in NK cell neoplasms from five patients and in five cell lines (NK-92, KHYG-1, SNK-6, HANK1 and MOTN-1) compared to mature peripheral NK cells from 10 healthy subjects. Apoptosis of NK cell lines was prevented by addition of IL-15 in vitro. Blocking IL-2/15Rbeta on IL-2-stimulated NK-92 cells resulted in reduced expression of Bcl-X(L) and phosphorylated Stat5, which paralleled early apoptosis without altering Bcl-2 expression. These data add IL-2/15Rbeta to the list of factors important for the survival of NK cell neoplasms.

Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model

PubMed Central

Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E.; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Escobar, Hugo Murua

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc −/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies. PMID:22761949

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

PubMed

Rütgen, Barbara C; Willenbrock, Saskia; Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Murua Escobar, Hugo

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death.

PubMed

Britain, Colleen M; Holdbrooks, Andrew T; Anderson, Joshua C; Willey, Christopher D; Bellis, Susan L

2018-02-05

The ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated. Using the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were treated with gefitinib, an EGFR inhibitor widely used for cancer therapy. These studies showed that ST6Gal-I promotes resistance to gefitinib-mediated apoptosis, as measured by caspase activity assays. Results herein indicate that ST6Gal-I promotes EGFR activation and protects against gefitinib-mediated cell death. Establishing the tumor-associated ST6Gal-I sialyltransferase as a regulator of EGFR provides novel insight into the role of glycosylation in growth factor signaling and chemoresistance.

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

Deviation from additivity in mixture toxicity: relevance of nonlinear dose-response relationships and cell line differences in genotoxicity assays with combinations of chemical mutagens and gamma-radiation.

PubMed

Lutz, Werner K; Vamvakas, Spyros; Kopp-Schneider, Annette; Schlatter, Josef; Stopper, Helga

2002-12-01

Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore not be generalized. Regarding the testing of mixtures for deviation from additive toxicity, the suggested experimental protocol is easily followed by toxicologists.

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

Morphology, cell viability, karyotype, expression of surface markers and plasticity of three human primary cell line cultures before and after the cryostorage in LN2 and GN2.

PubMed

Del Pino, Alberto; Ligero, Gertrudis; López, María B; Navarro, Héctor; Carrillo, Jose A; Pantoll, Siobhan C; Díaz de la Guardia, Rafael

2015-02-01

Primary cell line cultures from human skin biopsies, adipose tissue and tumor tissue are valuable samples for research and therapy. In this regard, their derivation, culture, storage, transport and thawing are important steps to be studied. Towards this end, we wanted to establish the derivation, and identify the culture characteristics and the loss of viability of three human primary cell line cultures (human adult dermal fibroblasts (hADFs), human adult mesenchymal stem cells (hMSCs), and primary culture of tumor cells from lung adenocarcinoma (PCTCLA)). Compared to fresh hADFs, hMSCs and PCTCLA, thawed cells stored in a cryogenic Dewar tanks with liquid nitrogen (LN2), displayed 98.20% ± 0.99, 95.40% ± 1.41 and 93.31% ± 3.83 of cell viability, respectively. Thawed cells stored in a Dry Vapor Shipper container with gas phase (GN2), for 20 days, in addition displayed 4.61% ± 2.78, 3.70% ± 4.09 and 9.13% ± 3.51 of average loss of cells viability, respectively, showing strong correlation between the loss of viability in hADFs and the number of post-freezing days in the Dry Vapor Shipper. No significant changes in morphological characteristics or in the expression of surface markers (being hADFs, hMSCs and PCTCLA characterized by positive markers CD73+; CD90+; CD105+; and negative markers CD14-; CD20-; CD34-; and CD45-; n=2) were found. Chromosome abnormalities in the karyotype were not found. In addition, under the right conditions hMSCs were differentiated into adipogenic, osteogenic and chondrogenic lineages in vitro. In this paper, we have shown the characteristics of three human primary cell line cultures when they are stored in LN2 and GN2. Copyright © 2014 Elsevier Inc. All rights reserved.

The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line

PubMed Central

Osada, Naoki; Kohara, Arihiro; Yamaji, Toshiyuki; Hirayama, Noriko; Kasai, Fumio; Sekizuka, Tsuyoshi; Kuroda, Makoto; Hanada, Kentaro

2014-01-01

Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells. PMID:25267831

Silencing of Urothelial Carcinoma Associated 1 Inhibits the Proliferation and Migration of Medulloblastoma Cells.

PubMed

Zhengyuan, Xie; Hu, Xiao; Qiang, Wang; Nanxiang, Li; Junbin, Cai; Wangming, Zhang

2017-09-16

BACKGROUND UCA1 is a long non-coding RNA that has been found to be aberrantly upregulated in various cancers. The aim of this study was to determine the expression level and function of UCA1 in medulloblastoma, the most common malignant brain tumor during childhood. MATERIAL AND METHODS Real-time PCR was used to detect the expression of UCA1 in medulloblastoma specimens and cell lines. Lentiviral-mediated expression of a short hairpin RNA (shRNA) targeting UCA1 or a negative control shRNA was also achieved with the medulloblastoma cell line, Daoy. Cell proliferation and cell cycle progression were subsequently characterized with cell counting kit (CCK)-8 and flow cytometry. Cell migration was examined in wound healing and Transwell migration assays. RESULTS Levels of UCA1 mRNA were higher in the medulloblastoma specimens (p<0.05) and cell lines (p<0.05) compared to the corresponding nontumor adjacent tissue specimens and a glioblastoma cell line, respectively. For the Daoy cells with silenced UCA1, their proliferation was reduced by 30% compared to the Daoy cells expressing a negative control shRNA (p=0.017). Cell cycle arrest in the G0/G1 phase, resulting in a decreased number of cells in the S phase, as well as reduced cell migration in both wound scratch healing (p=0.001) and Transwell migration assays (p=0.021) were also observed for the Daoy cells with silenced UCA1. CONCLUSIONS UCA1 was highly expressed in part of medulloblastoma specimens and cell lines examined. In addition, knockdown of UCA1 significantly inhibited the proliferation and migration of medulloblastoma cells in vitro.

Liposomal nanoparticles as a drug delivery vehicle against osteosarcoma

NASA Astrophysics Data System (ADS)

Dhule, Santosh Subhashrao

The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-gamma-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded gamma-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. The second part of this study examines the anti-tumor potential of curcumin and C6 ceramide (C6) against osteosarcoma cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with systems with curcumin alone. Interestingly, C6-curcumin liposomes were found to be less toxic on untransformed human cells in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G 2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. Using pegylated liposomes to increase the plasma half-life and tagging with folate for targeted delivery in vivo, a significant reduction in tumor size was observed with C6-curcumin-folate liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy.

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment

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

Kim, Jong-Hyuk, E-mail: jhkim@umn.edu; Masonic Cancer Center, University of Minnesota, Minneapolis, MN; Frantz, Aric M.

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggestingmore » that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. - Highlights: • IL-8 is expressed in canine hemangiosarcoma tumor samples and cell lines. • IL-8 transduces a relevant biological signal in canine hemangiosarcoma cells. • IL-8 gene signature is associated with reactive tumor microenvironments. • IL-8 potentiates tumor cell survival and engraftment into host tissues. • Canine hemangiosarcoma provides a unique comparative model for IL-8 studies.« less

Dexamethasone treatment promotes Bcl-2 dependence in multiple myeloma resulting in sensitivity to venetoclax.

PubMed

Matulis, S M; Gupta, V A; Nooka, A K; Hollen, H V; Kaufman, J L; Lonial, S; Boise, L H

2016-05-01

Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. The results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in four of the five cell lines tested. The most striking results were seen with dexamethasone (Dex). Co-treatment of human myeloma cell lines and primary patient samples, with Dex and venetoclax, significantly increased cell death over venetoclax alone in four of the five cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of Dex. This results in alterations in Bim binding to anti-apoptotic proteins. Dex shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim-binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with Dex could be an effective therapy for a broader range of patients than would be predicted by single-agent activity.

Dexamethasone treatment promotes Bcl-2-dependence in multiple myeloma resulting in sensitivity to Venetoclax

PubMed Central

Matulis, Shannon M.; Gupta, Vikas A.; Nooka, Ajay K.; Von Hollen, Hayley; Kaufman, Jonathan L.; Lonial, Sagar; Boise, Lawrence H.

2015-01-01

Venetoclax (ABT-199), a specific inhibitor of the anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. Results suggest that venetoclax is only active in a small cohort of patients therefore we wanted to determine its efficacy when used in combination. Combining venetoclax with melphalan or carfilzomib produced additive or better cell death in 4 of the 5 cell lines tested. The most striking results were seen with dexamethasone. Co-treatment of human myeloma cell lines and primary patient samples, with dexamethasone and venetoclax significantly increased cell death over venetoclax alone in 4 of the 5 cell lines, and in all patient samples tested. The mechanism by which this occurs is an increase in the expression of both Bcl-2 and Bim upon addition of dexamethasone. This results in alterations in Bim binding to anti-apoptotic proteins. Dexamethasone shifts Bim binding towards Bcl-2 resulting in increased sensitivity to venetoclax. These data suggest that knowledge of drug-induced alterations of Bim binding patterns may help inform better combination drug regimens. Furthermore, the data indicate combining this novel therapeutic with dexamethasone could be an effective therapy for a broader range of patients than would be predicted by single agent activity. PMID:26707935

Release of copper-amended particles from micronized copper-pressure-treated wood during mechanical abrasion.

PubMed

Civardi, Chiara; Schlagenhauf, Lukas; Kaiser, Jean-Pierre; Hirsch, Cordula; Mucchino, Claudio; Wichser, Adrian; Wick, Peter; Schwarze, Francis W M R

2016-11-28

We investigated the particles released due to abrasion of wood surfaces pressure-treated with micronized copper azole (MCA) wood preservative and we gathered preliminary data on its in vitro cytotoxicity for lung cells. The data were compared with particles released after abrasion of untreated, water (0% MCA)-pressure-treated, chromated copper (CC)-pressure-treated wood, and varnished wood. Size, morphology, and composition of the released particles were analyzed. Our results indicate that the abrasion of MCA-pressure-treated wood does not cause an additional release of nanoparticles from the unreacted copper (Cu) carbonate nanoparticles from of the MCA formulation. However, a small amount of released Cu was detected in the nanosized fraction of wood dust, which could penetrate the deep lungs. The acute cytotoxicity studies were performed on a human lung epithelial cell line and human macrophages derived from a monocytic cell line. These cell types are likely to encounter the released wood particles after inhalation. Our findings indicate that under the experimental conditions chosen, MCA does not pose a specific additional nano-risk, i.e. there is no additional release of nanoparticles and no specific nano-toxicity for lung epithelial cells and macrophages.

Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress

PubMed Central

Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Zenkoh, Junko; Moritake, Takashi; Tsuboi, Koji

2013-01-01

Background Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or γ-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stress–related genes was analyzed by immunoblotting. Results Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and γ-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. PMID:23658321

Potentiation of antiproliferative drug activity by lonidamine in hepatocellular carcinoma cells.

PubMed

Ricotti, L; Tesei, A; De Paola, F; Milandri, C; Amadori, D; Frassineti, G L; Ulivi, P; Zoli, W

2003-10-01

The ability of lonidamine (LND), a derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of anticancer drugs was investigated in two human hepatocarcinoma (HCC) cell lines. The cytotoxicity of drugs used singly, in association or in sequence was evaluated using the Sulforhodamine B (SRB) assay. LND did not appreciably potentiate the effect of antitumor drugs when given before or simultaneously, in either cell line. Conversely, a synergistic interaction was observed in both cell lines when LND was given after conventional drugs. LND produced a moderate decrease in S-phase cell fraction and did not induce apoptosis. Conversely, paclitaxel (TAX) induced an important block in G2 and an increase in apoptosis. Following a 48-h TAX wash out, a progressive passage of cells from G2 to M phase was observed with a corresponding increase in apoptotic cells. Post-treatment with LND increased the cytotoxicity of some antitumor drugs, especially TAX, in hepatocarcinoma cells, possibly by preventing, as an energolytic drug, cell damage repair or by producing an additional effect on microtubule stabilization.

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2

PubMed Central

Deng, Yingjun; Li, Xin; Feng, Jinxin; Zhang, Xiangliang

2018-01-01

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs. PMID:29559564

MHC class II expression in lung cancer.

PubMed

He, Yayi; Rozeboom, Leslie; Rivard, Christopher J; Ellison, Kim; Dziadziuszko, Rafal; Yu, Hui; Zhou, Caicun; Hirsch, Fred R

2017-10-01

Immunotherapy is an exciting development in lung cancer research. In this study we described major histocompatibility complex (MHC) Class II protein expression in lung cancer cell lines and patient tissues. We studied MHC Class II (DP, DQ, DR) (CR3/43, Abcam) protein expression in 55 non-small cell lung cancer (NSCLC) cell lines, 42 small cell lung cancer (SCLC) cell lines and 278 lung cancer patient tissues by immunohistochemistry (IHC). Seven (12.7%) NSCLC cell lines were positive for MHC Class II. No SCLC cell lines were found to be MHC Class II positive. We assessed 139 lung cancer samples available in the Hirsch Lab for MHC Class II. There was no positive MHC Class II staining on SCLC tumor cells. MHC Class II expression on TILs in SCLC was significantly lower than that on TILs in NSCLC (P＜0.001). MHC Class II was also assessed in an additional 139 NSCLC tumor tissues from Medical University of Gdansk, Poland. Patients with positive staining of MHC Class II on TILs had longer regression-free survival (RFS) and overall survival (OS) than those whose TILs were MHC Class II negative (2.980 years, 95% CI 1.628-4.332 vs. 1.050 years, 95% CI 0.556-1.554, P=0.028) (3.230 years, 95% CI 2.617-3.843 vs. 1.390 years, 95% CI 0.629-2.151, P=0.014). MHC Class II was expressed both in NSCLC cell lines and tissues. However, MHC Class II was not detected in SCLC cell lines or tissue tumor cells. MHC Class II expression was lower on SCLC TILs than on NSCLC TILs. Loss of expression of MHC Class II on SCLC tumor cells and reduced expression on SCLC TILs may be a means of escaping anti-cancer immunity. Higher MHC Class II expression on TILs was correlated with better prognosis in patients with NSCLC. Copyright © 2017. Published by Elsevier B.V.

Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

NASA Technical Reports Server (NTRS)

Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

2000-01-01

We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

Interleukin-8 Promotes Canine Hemangiosarcoma Growth by Regulating the Tumor Microenvironment

PubMed Central

Kim, Jong-Hyuk; Frantz, Aric M.; Anderson, Katie L.; Graef, Ashley J.; Scott, Milcah C.; Robinson, Sally; Sharkey, Leslie C.; O’Brien, Timothy D.; Dickerson, Erin B.; Modiano, Jaime F.

2014-01-01

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. PMID:24582862

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment.

PubMed

Kim, Jong-Hyuk; Frantz, Aric M; Anderson, Katie L; Graef, Ashley J; Scott, Milcah C; Robinson, Sally; Sharkey, Leslie C; O'Brien, Timothy D; Dickerson, Erin B; Modiano, Jaime F

2014-04-15

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into "IL-8 high" and "IL-8 low" groups. Genome-wide gene expression profiling showed that samples in the "IL-8 high" tumor group were enriched for genes associated with a "reactive microenvironment," including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. Copyright © 2014 Elsevier Inc. All rights reserved.

Integrated QSAR study for inhibitors of hedgehog signal pathway against multiple cell lines:a collaborative filtering method

PubMed Central

2012-01-01

Background The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. Results In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Conclusions Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1. PMID:22849868

Integrated QSAR study for inhibitors of Hedgehog Signal Pathway against multiple cell lines:a collaborative filtering method.

PubMed

Gao, Jun; Che, Dongsheng; Zheng, Vincent W; Zhu, Ruixin; Liu, Qi

2012-07-31

The Hedgehog Signaling Pathway is one of signaling pathways that are very important to embryonic development. The participation of inhibitors in the Hedgehog Signal Pathway can control cell growth and death, and searching novel inhibitors to the functioning of the pathway are in a great demand. As the matter of fact, effective inhibitors could provide efficient therapies for a wide range of malignancies, and targeting such pathway in cells represents a promising new paradigm for cell growth and death control. Current research mainly focuses on the syntheses of the inhibitors of cyclopamine derivatives, which bind specifically to the Smo protein, and can be used for cancer therapy. While quantitatively structure-activity relationship (QSAR) studies have been performed for these compounds among different cell lines, none of them have achieved acceptable results in the prediction of activity values of new compounds. In this study, we proposed a novel collaborative QSAR model for inhibitors of the Hedgehog Signaling Pathway by integration the information from multiple cell lines. Such a model is expected to substantially improve the QSAR ability from single cell lines, and provide useful clues in developing clinically effective inhibitors and modifications of parent lead compounds for target on the Hedgehog Signaling Pathway. In this study, we have presented: (1) a collaborative QSAR model, which is used to integrate information among multiple cell lines to boost the QSAR results, rather than only a single cell line QSAR modeling. Our experiments have shown that the performance of our model is significantly better than single cell line QSAR methods; and (2) an efficient feature selection strategy under such collaborative environment, which can derive the commonly important features related to the entire given cell lines, while simultaneously showing their specific contributions to a specific cell-line. Based on feature selection results, we have proposed several possible chemical modifications to improve the inhibitor affinity towards multiple targets in the Hedgehog Signaling Pathway. Our model with the feature selection strategy presented here is efficient, robust, and flexible, and can be easily extended to model large-scale multiple cell line/QSAR data. The data and scripts for collaborative QSAR modeling are available in the Additional file 1.

Functional significance of CD105-positive cells in papillary renal cell carcinoma.

PubMed

Matak, Damian; Brodaczewska, Klaudia K; Szczylik, Cezary; Koch, Irena; Myszczyszyn, Adam; Lipiec, Monika; Lewicki, Slawomir; Szymanski, Lukasz; Zdanowski, Robert; Czarnecka, Anna M

2017-01-05

CD105 was postulated as a renal cell carcinoma (RCC) stem cell marker, and CD133 as a putative RCC progenitor. Hypoxia, a natural microenvironment that prevails in tumors, was also incorporated into the study, especially in terms of the promotion of hypothetical stem-like cell properties. Within this study, we verify the existence of CD105+ and CD133+ populations in selected papillary subtype RCC (pRCC) cell lines. Both populations were analyzed for correlation with stem-like cell properties, such as stemness gene expression, and sphere and colony formation. For the preliminary analysis, several RCC cell lines were chosen (786-O, SMKT-R2, Caki-2, 796-P, ACHN, RCC6) and the control was human kidney cancer stem cells (HKCSC) and renal cells of embryonic origin (ASE-5063). Four cell lines were chosen for further investigation: Caki-2 (one of the highest numbers of CD105+ cells; primary origin), ACHN (a low number of CD105+ cells; metastatic origin), HKCSC (putative positive control), and ASE-5063 (additional control). In 769-P and RCC6, we could not detect a CD105+ population. Hypoxia variously affects pRCC cell growth, and mainly diminishes the stem-like properties of cells. Furthermore, we could not observe the correlation of CD105 and/or CD133 expression with the enhancement of stem-like properties. Based on this analysis, CD105/CD133 cannot be validated as cancer stem cell markers of pRCC cell lines.

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

PubMed

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

2016-01-26

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

Cytotoxic constituents of propolis from Myanmar and their structure-activity relationship.

PubMed

Li, Feng; Awale, Suresh; Tezuka, Yasuhiro; Kadota, Shigetoshi

2009-12-01

Thirteen cycloartane-type tritepenes (1-13) and four prenylated flavanones (14-17) isolated from propolis collected in Myanmar, were evaluated for their cytotoxic activity against a panel of six different cancer cell lines; three murine cancer cell lines (colon 26-L5 carcinoma, B16-BL6 melanoma, and Lewis lung carcinoma) and three human cancer cell lines (lung A549 adenocarcinoma, cervix HeLa adenocarcinoma and HT-1080 fibrosarcoma). Among them, a cycloartane-type triterpene, 3alpha,27-dihydroxycycloart-24E-en-26-oic acid (3), showed the most potent cytotoxicity against B16-BL6 cells with an IC(50) value of 5.91 microM, comparable to those of positive controls, doxorubicin (IC(50), 5.66 microM) and 5-fluorouracil (IC(50), 4.88 microM). In addition, (2S)-5,7-dihydroxy-4'-methoxy-8,3'-diprenylflavanone (14) exhibited strong cytotoxicity against all the tested cancer cell lines with the IC(50) values ranging from 14.0 to 26.4 microM. Based on the observed results, the structure-activity relationships are discussed.

Expression of extracellular calcium (Ca2 + o)-sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2

NASA Technical Reports Server (NTRS)

Yamaguchi, T.; Kifor, O.; Chattopadhyay, N.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

1998-01-01

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2 + o) homeostasis in parathyroid gland and kidney. More recent data have suggested the presence of this receptor in additional tissues, such as brain, intestine and skin. In this study, we examined the expression of the CaR in the rat and human osteosarcoma cell lines, UMR-106 and SAOS-2, respectively, which possess osteoblast-like characteristics. Both immunocytochemistry and Western blot analysis, using a polyclonal antiserum specific for the CaR, detected CaR protein in UMR-106 and SAOS-2 cells. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in each cell line. Therefore, taken together, our data strongly suggest that the osteoblast-like cell lines, UMR-106 and SAOS-2, possess both CaR protein and mRNA very similar if not identical to those in parathyroid and kidney.

Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.

PubMed

Cocks, Graham; Romanyuk, Nataliya; Amemori, Takashi; Jendelova, Pavla; Forostyak, Oksana; Jeffries, Aaron R; Perfect, Leo; Thuret, Sandrine; Dayanithi, Govindan; Sykova, Eva; Price, Jack

2013-06-07

The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues. Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity. We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca(2+) channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity. We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.

Use of HL cells for improved isolation and passage of Chlamydia pneumoniae.

PubMed Central

Cles, L D; Stamm, W E

1990-01-01

We compared growth of the recently discovered respiratory pathogen Chlamydia pneumoniae in McCoy, HeLa 229, BHK-21, and HL cells. When cells were not pretreated with DEAE-dextran, HL cells had significantly higher mean numbers of inclusion-forming units (IFUs) on initial inoculation than the other cell lines. When cells were pretreated with DEAE-dextran, HeLa 229 and HL cells had equivalent mean numbers of IFUs on initial inoculation. HL cells had strikingly higher mean numbers of IFUs in passage than HeLa 229, BHK-21, or McCoy cells. In addition, HL cells did not require pretreatment with DEAE-dextran and could be used from 2 to 4 days after seeding. We conclude that HL cells are an excellent cell culture system for laboratory propagation of C. pneumoniae and may be a more sensitive cell line for initial isolation. PMID:2191013

Pharmacological relevance of endoxifen in a laboratory simulation of breast cancer in postmenopausal patients.

PubMed

Maximov, Philipp Y; McDaniel, Russell E; Fernandes, Daphne J; Bhatta, Puspanjali; Korostyshevskiy, Valeriy R; Curpan, Ramona F; Jordan, V Craig

2014-10-01

Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Student's t test. All statistical tests were two-sided. Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

PubMed Central

Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

2016-01-01

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID:27977675

Activity of Saponins from Medicago species Against HeLa and MCF-7 Cell Lines and their Capacity to Potentiate Cisplatin Effect.

PubMed

Avato, Pinarosa; Migoni, Danilo; Argentieri, Mariapia; Fanizzi, Francesco P; Tava, Aldo

2017-11-24

Saponins from Medicago species display several biological activities, among them apoptotic effects against plant cells have been evidenced. In contrast, their cytotoxic and antitumor activity against animal cells have not been studied in great details. To explore the cytotoxic properties of saponin from Medicago species against animal cells and their effect in combination with the antitumoral drug cisplatin. Cytotoxic activity of saponin mixtures from M. arabica (tops and roots), M. arborea (tops) and M. sativa (tops, roots and seeds) and related prosapogenins from M. arborea and M. sativa (tops) against HeLa and MCF-7 cell lines is described. In addition, cytotoxicity of soyasaponin I and purified saponins (1-8) of hederagenin, medicagenic and zanhic acid is also presented. Combination experiments with cisplatin have been also conducted. Saponins from M. arabica tops and roots (mainly monodesmosides of hederagenin and bayogenin) were the most effective to reduce proliferation of HeLa and MCF-7 cell lines. Among the purified saponins, the most cytotoxic was saponin 1, 3-O-ß-D-glucopyranosyl(1→2)-α-L-arabinopyranosyl hederagenin. When saponins, derived prosapogenins and pure saponins were used in combination with cisplatin, they all, to different extent, were able to potentiate cisplatin activity against HeLa cells but not against MCF-7 cell lines. Moreover uptake of cisplatin in these cell lines was significantly reduced. Overall results showed that specific molecular types of saponins (hederagenin glycosides) have potential as anti-cancer agents or as leads for anti-cancer agents. Moreover saponins from Medicago species have evidenced interesting properties to mediate cisplatin effects in tumor cell lines. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PKC signaling is involved in the regulation of progranulin (acrogranin/PC-cell-derived growth factor/granulin-epithelin precursor) protein expression in human ovarian cancer cell lines.

PubMed

Diaz-Cueto, Laura; Arechavaleta-Velasco, Fabian; Diaz-Arizaga, Adriana; Dominguez-Lopez, Pablo; Robles-Flores, Martha

2012-07-01

Overexpression of progranulin (also named acrogranin, PC-cell-derived growth factor, or granulin-epithelin precursor) is associated with ovarian cancer, specifically with cell proliferation, malignancy, chemoresistance, and shortened overall survival. The objective of the current study is to identify the signaling pathways involved in the regulation of progranulin expression in ovarian cancer cell lines. We studied the relation of protein kinase C (PKC), phosphatidylinositol 3-kinase, protein kinase A, P38, extracellular signal-regulated kinase, and Akt pathways on the modulation of progranulin expression levels in NIH-OVCAR-3 and SK-OV-3 ovarian cancer cell lines. The different pathways were examined using pharmacological inhibitors (calphostin C, LY294002, H89, SB203580, PD98059, and Akt Inhibitor), and mRNA and protein progranulin expression were analyzed by reverse transcriptase polymerase chain reaction and Western blot techniques, respectively. Inhibition of PKC signal transduction pathway by calphostin C decreased in a dose-dependent manner protein but not mRNA levels of progranulin in both ovarian cancer cell lines. LY294002 but not wortmannin, which are phosphatidylinositol 3-kinase inhibitors, also diminished the expression of progranulin in both cell lines. In addition, LY294002 treatment produced a significant reduction in cell viability. Inhibition of protein kinase A, P38, extracellular signal-regulated kinase, and Akt did not affect progranulin protein expression. These results suggest that the PKC signaling is involved in the regulation of progranulin protein expression in 2 different ovarian cancer cell lines. Inhibiting these intracellular signal transduction pathways may provide a future therapeutic target for hindering the cellular proliferation and invasion in ovarian cancer produced by progranulin.

USC-HN2, A NEW MODEL CELL LINE FOR RECURRENT ORAL CAVITY SQUAMOUS CELL CARCINOMA WITH IMMUNOSUPPRESSIVE CHARACTERISTICS

PubMed Central

Russell, Sarah M; Lechner, Melissa G; Gong, Lucy; Megiel, Carolina; Liebertz, Daniel J.; Masood, Rizwan; Correa, Adrian J; Han, Jing; Puri, Raj K; Sinha, Uttam K; Epstein, Alan L

2011-01-01

Objectives Head and neck squamous cell carcinomas (HNSCC) are common and aggressive tumors that have not seen an improvement in survival rates in decades. These tumors are believed to evade the immune system through a variety of mechanisms and are therefore highly immune modulatory. In order to elucidate their interaction with the immune system and develop new therapies targeting immune escape, new pre-clinical models are needed. Materials and Methods A novel human cell line, USC-HN2, was established from a patient biopsy specimen of invasive, recurrent buccal HNSCC and characterized by morphology, heterotransplantation, cytogenetics, phenotype, gene expression and immune modulation studies and compared to a similar HNSCC cell line; SCCL-MT1. Results and Conclusion Characterization studies confirmed the HNSCC origin of USC-HN2 and demonstrated a phenotype similar to the original tumor and typical of aggressive oral cavity HNSCC (EGFR+CD44v6+FABP5+Keratin+ and HPV−). Gene and protein expression studies revealed USC-HN2 to have highly immune-modulatory cytokine production (IL-1β, IL-6, IL-8, GM-CSF, and VEGF) and strong regulatory T and myeloid derived suppressor cell (MDSC) induction capacity in vitro. Of note, both USC-HN2 and SCCL-MT1 were found to have a more robust cytokine profile and MDSC induction capacity when compared to 7 previously established HNSCC cell lines. Additionally, microarray gene expression profiling of both cell lines demonstrate up-regulation of antigen presenting genes. Because USC-HN2 is therefore highly immunogenic, it also induces strong immune suppression to evade immunologic destruction. Based upon these results, both cell lines provide an excellent model for the development of new suppressor cell-targeted immunotherapies. PMID:21719345

USC-HN2, a new model cell line for recurrent oral cavity squamous cell carcinoma with immunosuppressive characteristics.

PubMed

Russell, Sarah M; Lechner, Melissa G; Gong, Lucy; Megiel, Carolina; Liebertz, Daniel J; Masood, Rizwan; Correa, Adrian J; Han, Jing; Puri, Raj K; Sinha, Uttam K; Epstein, Alan L

2011-09-01

Head and neck squamous cell carcinomas (HNSCC) are common and aggressive tumors that have not seen an improvement in survival rates in decades. These tumors are believed to evade the immune system through a variety of mechanisms and are therefore highly immune modulatory. In order to elucidate their interaction with the immune system and develop new therapies targeting immune escape, new pre-clinical models are needed. A novel human cell line, USC-HN2, was established from a patient biopsy specimen of invasive, recurrent buccal HNSCC and characterized by morphology, heterotransplantation, cytogenetics, phenotype, gene expression, and immune modulation studies and compared to a similar HNSCC cell line; SCCL-MT1. Characterization studies confirmed the HNSCC origin of USC-HN2 and demonstrated a phenotype similar to the original tumor and typical of aggressive oral cavity HNSCC (EGFR(+)CD44v6(+)FABP5(+)Keratin(+) and HPV(-)). Gene and protein expression studies revealed USC-HN2 to have highly immune-modulatory cytokine production (IL-1β, IL-6, IL-8, GM-CSF, and VEGF) and strong regulatory T and myeloid derived suppressor cell (MDSC) induction capacity in vitro. Of note, both USC-HN2 and SCCL-MT1 were found to have a more robust cytokine profile and MDSC induction capacity when compared to seven previously established HNSCC cell lines. Additionally, microarray gene expression profiling of both cell lines demonstrate up-regulation of antigen presenting genes. Because USC-HN2 is therefore highly immunogenic, it also induces strong immune suppression to evade immunologic destruction. Based upon these results, both cell lines provide an excellent model for the development of new suppressor cell-targeted immunotherapies. Copyright © 2011 Elsevier Ltd. All rights reserved.

High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

PubMed

Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

2012-07-01

In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer.

PubMed

Griffith, M; Mwenifumbo, J C; Cheung, P Y; Paul, J E; Pugh, T J; Tang, M J; Chittaranjan, S; Morin, R D; Asano, J K; Ally, A A; Miao, L; Lee, A; Chan, S Y; Taylor, G; Severson, T; Hou, Y-C; Griffith, O L; Cheng, G S W; Novik, K; Moore, R; Luk, M; Owen, D; Brown, C J; Morin, G B; Gill, S; Tai, I T; Marra, M A

2013-04-01

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases

PubMed Central

Harvey, P; Clark, I M; Jaurand, M-C; Warn, R M; Edwards, D R

2000-01-01

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels. © 2000 Cancer Research Campaign PMID:11027427

EphA3 maintains radioresistance in head and neck cancers through epithelial mesenchymal transition.

PubMed

Kim, Song Hee; Lee, Won Hyeok; Kim, Seong Who; Je, Hyoung Uk; Lee, Jong Cheol; Chang, Hyo Won; Kim, Young Min; Kim, Kyungbin; Kim, Sang Yoon; Han, Myung Woul

2018-07-01

Radiotherapy is a well-established therapeutic modality used in the treatment of many cancers. However, radioresistance remains a serious obstacle to successful treatment. Radioresistance can cause local recurrence and distant metastases in some patients after radiation treatment. Thus, many studies have attempted to identify effective radiosensitizers. Eph receptor functions contribute to tumor development, modulating cell-cell adhesion, invasion, neo-angiogenesis, tumor growth and metastasis. However, the role of EphA3 in radioresistance remains unclear. In the current study, we established a stable radioresistant head and neck cancer cell line (AMC HN3R cell line) and found that EphA3 was expressed predominantly in the radioresistant head and neck cancer cell line through DNA microarray, real time PCR and Western blotting. Additionally, we found that EphA3 was overexpressed in recurrent laryngeal cancer specimens after radiation therapy. EphA3 mediated the tumor invasiveness and migration in radioresistant head and neck cancer cell lines and epithelial mesenchymal transition- related protein expression. Inhibition of EphA3 enhanced radiosensitivity in the AMC HN 3R cell line in vitro and in vivo study. In conclusion, our results suggest that EphA3 is overexpressed in radioresistant head and neck cancer and plays a crucial role in the development of radioresistance in head and neck cancers by regulating the epithelial mesenchymal transition pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

5-(Furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f), a new synthetic compound, causes human fibrosarcoma HT-1080 cell apoptosis by disrupting tubulin polymerisation and inducing G2/M arrest.

PubMed

Zuo, Daiying; Pang, Lili; Shen, Jiwei; Guan, Qi; Bai, Zhaoshi; Zhang, Huijuan; Li, Yao; Lu, Guodong; Zhang, Weige; Wu, Yingliang

2017-06-01

In the current study, we synthesized a series of new compounds targeting tubulin and tested their anti-proliferative activities. Among these new synthetic com-pounds, 5-(furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f) exhibited significant anti-proliferative activity against different human cancer cell lines including human gastric adenocarcinoma SGC-7901, human non-small cell lung cancer A549, and human fibrosarcoma HT-1080. As a result, 6f was selected to further test the sensitivity to different cancer cell lines including human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, non-small cell lung cancer cell line A549, human liver carcinoma cell line HepG-2, human oral squamous cell carcinoma cell lines KB, SGC-7901 and HT-1080. Among these cell lines, HT-1080 and HeLa are the most sensitive. Therefore, HT-1080 was selected to further explore the properties of anti-proliferative activity and the underlying mechanisms. Our data proved that 6f exhibited strong anti-proliferative effects against HT-1080 cells in a time- and dose-dependent manner. We showed that the growth inhibitory effect of 6f in HT-1080 cells was related with microtubule depolymerisation. Molecular docking studies revealed that 6f interacted and bound efficiently with the colchicine-binding site of tubulin. In addition, 6f treatment induced G2/M cell cycle arrest dose-dependently and subsequently induced cell apoptosis. Western blot study indicated that upregulation of cyclin B1 and p-cdc2 was related with G2/M arrest. 6f-induced cell apoptosis was associated with both mitochondrial and death receptor pathway. In conclusion, our data showed that 6f, among the newly synthetic compounds, exhibited highest anti-proliferative activity by disrupting the microtubule polymerisation, causing G2/M arrest and subsequently inducing cell apoptosis in HT-1080 cells. Hence, 6f is a promising microtubule depolymerising agent for the treatment of various cancers especially human fibrosarcoma.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

PubMed

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

PubMed Central

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-01-01

Background CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. Design and Methods We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d+ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. Results The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d+ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d+ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Conclusions Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d+ hematologic malignancies. PMID:19454494

Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin.

PubMed

Furlong, Suzanne J; Ridgway, Neale D; Hoskin, David W

2008-03-01

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Radiation Response of Cancer Stem-Like Cells From Established Human Cell Lines After Sorting for Surface Markers

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

Al-Assar, Osama; Muschel, Ruth J.; Mantoni, Tine S.

2009-11-15

Purpose: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. Methods and Materials: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. Results: The quantity of CSLC was generally in good agreement withmore » primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. Conclusions: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.« less

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2).

PubMed

Fredericksen, T L; Gilmour, D G

1983-06-01

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.

Expression of Zinc Finger and BTB Domain-containing 7A in Colorectal Carcinoma.

PubMed

Joo, Jin Woo; Kim, Hyun-Soo; Do, Sung-Im; Sung, Ji-Youn

2018-05-01

Previous studies have revealed that zinc finger and BTB domain-containing 7A (ZBTB7A), an important proto-oncogene, plays multiple roles in carcinogenesis and is up-regulated in several human malignancies. However, the expression of ZBTB7A in colorectal carcinoma (CRC) has seldom been documented. In this study, we investigated the differential expression of ZBTB7A in CRC cell lines and tissues. Expression levels of ZBTB7A mRNA and protein were examined in CRC cell lines. ZBTB7A protein expression was also evaluated in tissue samples of normal colonic mucosa, high-grade dysplasia, and CRC using immunohistochemical staining. All CRC cell lines exhibited significantly higher ZBTB7A mRNA expression levels than did normal colonic epithelial cells. The ZBTB7A protein expression levels were clearly higher in the CRC cell lines than in the normal colonic epithelial cells. Consistent with the cell line data, immunostaining revealed that there were significant differences in ZBTB7A protein expression between tissue samples of CRC and normal colonic mucosa (p=0.048) and high-grade dysplasia (p=0.015). In addition, metastatic CRC exhibited significantly higher ZBTB7A protein expression levels than primary CRC (p=0.027). We demonstrated that ZBTB7A expression is up-regulated in CRC cell lines and tissues. Our data suggest that ZBTB7A is involved in the development and progression of CRC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Induction of DNA-strand breaks after X- irradiation in murine bone cells of various differentiation capacities

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Kirchner, S.; Arenz, A.; Baumstark-Khan, C.; Horneck, G.

Bone loss resulting from long-duration space flight is a well known medical risk for space travellers, as a weakened skeleton is more susceptible to bone fractures. In addition to weightlessness the astronaut is also exposed to cosmic ionizing radiation. In order to elucidate changes in bone cell metabolism by ionizing radiation, a ground-based bone cell model has been developed. This model consists of a bunch of immortalized murine osteocyte, osteoblast and pre-osteoblast cell lines representing discrete stages of differentiation: The osteocyte cell line MLO-Y4 (obtained from L. Bonewald, Kansas City, USA), the osteoblast cell line OCT-1 (obtained from D. Chen, San Antonio, USA), and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 (obtained from ATCC, Manassas, Virginia, USA). Regarding their growth properties, MLO-Y4 cells show the highest growth velocity with a doubling time of 15.8 h. The osteoblast cell line OCT-1 has a doubling time of 27.3 h. The respective values for MC3T3-E1 subclone 24 and S4 are 90.5 h and 51.6 h. To investigate the stage of differentiation, the expression of alkaline phosphatase, of osteocalcin and of E11 was examined. Survival after X-ray exposure was determined using the colony forming ability test. The resulting dose-effect relationships revealed significant differences. The parameter D0 of the survival curves ranges between 1.8 Gy for OCT-1, 1.9 Gy for MLO-Y4, 2.0 Gy for subclone 24 and 2,3 Gy for subclone 4. The quantitative acquisition of DNA-strand breaks was performed by Fluorescent Analysis of DNA-Unwinding (FADU). The results can be correlated with the corresponding survival curve. In conclusion, the cell lines with higher differentiation levels are less sensitive to radiation when compared to the lower differentiated osteoblast cell lines.

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

PubMed

Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

2012-01-01

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

PubMed

Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C

2012-09-01

What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest. © 2012 BJU INTERNATIONAL.

[Using of cell biocomposite material in tissue engineering of the urinary bladder].

PubMed

Glybochko, P V; Olefir, Yu V; Alyaev, Yu G; Butnaru, D V; Bezrukov, E A; Chaplenko, A A; Zharikova, T M

2017-06-01

In a systematic review, to present an overview of the current situation in the field of tissue engineering of urinary bladder related to the use of cell lines pre-cultured on matrices. The selection of eligible publications was conducted according to the method described in the article Glybochko P.V. et al. "Tissue engineering of urinary bladder using acellular matrix." At the final stage, studies investigating the application of matrices with human and animal cell lines were analyzed. Contemporary approaches to using cell-based tissue engineering of the bladder were analyzed, including the formation of 3D structures from several types of cells, cell layers and genetic modification of injected cells. The most commonly used cell lines are urothelial cells, mesenchymal stem cells and fibroblasts. The safety and efficacy of any types of composite cell structures used in the cell-based bladder tissue engineering has not been proven sufficiently to warrant clinical studies of their usefulness. The results of cystoplasty of rat bladder are almost impossible to extrapolate to humans; besides, it is difficult to predict possible side effects. For the transition to clinical trials, additional studies on relevant animal models are needed.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

PubMed Central

Ivanov, Sergey V.; Kuzmin, Igor; Wei, Ming-Hui; Pack, Svetlana; Geil, Laura; Johnson, Bruce E.; Stanbridge, Eric J.; Lerman, Michael I.

1998-01-01

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth. PMID:9770531

Degraded iota-carrageenan can induce apoptosis in human osteosarcoma cells via the Wnt/β-catenin signaling pathway.

PubMed

Jin, Zhe; Han, Ya-Xin; Han, Xiao-Rui

2013-01-01

Osteosarcoma (OS) is a high-grade malignant bone tumor. Therefore, using both in vitro and in vivo assays, the effects of degraded iota-Carrageenan (ι-CGN) on a human osteosarcoma cell line, HOS, were examined. Degraded ι-CGN was observed to induce apoptosis and G(1) phase arrest in HOS cells. Moreover, degraded ι-CGN suppressed tumor growth in established xenograft tumor models. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumors treated with native ι-CGN or PBS. In addition, the formation of intratumoral microvessels was inhibited following treatment with degraded ι-CGN. In Western blot assays, degraded ι-CGN was found to inhibit the Wnt/β-catenin signaling pathway. Overall, these studies demonstrate the antitumor activity of degraded ι-CGN toward the OS cell line, HOS. Moreover, valuable insight into the mechanisms mediated by degraded ι-CGN was obtained, potentially leading to the identification of novel treatments for OS. However, additional studies are needed to confirm these results in other cell types, particularly in human umbilical vein endothelial cells.

Small interfering RNA-mediated suppression of serum response factor, E2-promotor binding factor and survivin in non-small cell lung cancer cell lines by non-viral transfection.

PubMed

Walker, Tobias; Nolte, Andrea; Steger, Volker; Makowiecki, Christina; Mustafi, Migdat; Friedel, Godehard; Schlensak, Christian; Wendel, Hans-Peter

2013-03-01

Serum response factor (SRF), E2F1 and survivin are well-known factors involved in a multitude of cancer-related regulation processes. However, to date, no suitable means has been found to apply their potential in the therapy of non-small cell lung cancer (NSCLC). This study deals with questions of small interfering ribonucleic acid (siRNA) transfection efficiency by a non-viral transfection of NSCLC cell-lines and the power of siRNA to transiently influence cell division by specific silencing. Different NSCLC cell lines were cultured under standard conditions and transfected, with specific siRNA targeting SRF, E2F1 and survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for messenger RNA (mRNA) expression levels. Additionally, transfection efficiency was evaluated by flow cytometry. The analysis of cell proliferation was determined with a CASY cell counter 3 days after transfection with SRF or SCR-siRNA. Transfection of the NSCLC cell lines with specific siRNAs against SRF, E2F1 and survivin resulted in a very considerable reduction of the intracellular mRNA concentration. CASY confirmation of cell viability demonstrated an excellent survival of the cell lines treated with non-specific siRNA, in contrast to with application of specific siRNA. This study reports a reliable transfectability of NSCLC-cell lines by siRNA, initially in a non-viral manner, and a reproducible knockdown of the focussed targets, consequently leading to the death of the tumour cells. This constitutes a strong candidate for a new assessment strategy in the therapy of non-small cell lung cancer.

Characterization of LHY-821, a novel moderately differentiated endometrial carcinoma cell line.

PubMed

Hu, Qian; Yu, Li; Chen, Rui; Zhang, Yan; Xie, Ya; Liao, Qinping

2012-08-01

Endometrial cancer is a major problem for women but only a small number of comprehensively characterized cell models are available for studies. Here, we established a new cell line derived from a Stage IIIc(1) Grade 2 endometrial adenocarcinoma. The cell line, designated LHY-821, was characterized using growth curve, karyotyping, immunohistochemical staining, immunoblotting, drug sensitivity assay, invasion assay, and xenografting in nude mice. LHY-821 has a doubling time of about 46 h and a colony-forming efficiency of approximately 71 %. These cells expresse high levels of progesterone receptor but not estrogen receptor and are sensitive to medroxyprogesterone acetate (MPA). LHY-821 also expresses pan-cytokeratin, PTEN, p53, β-catenin, IGF-1, and IGF-2. In addition, karyotype analysis revealed that LHY-821 possessed a near diploid karyotype including 6q-, 10p-, Xq-, 13q+, 17p+, and Triplo-12. LHY-821 showed highly tumorigenicity in nude mice (100 %) and weak invasiveness. Chemosensitivity tests showed that LHY-821 was sensitive to both carboplatin and paclitaxel. LHY-821 is an immortalized cell line which had survived more than 80 serial passages; it may provide a novel tool to study the molecular mechanism and potential treatment for endometrial cancer.

SGK2 promotes hepatocellular carcinoma progression and mediates GSK-3β/β-catenin signaling in HCC cells.

PubMed

Liu, Junying; Zhang, Guangdong; Lv, Yanping; Zhang, Xiaoyang; Ying, Cui; Yang, Suocheng; Kong, Xin; Yu, Yanzhang

2017-06-01

The phosphoinositide 3-kinase pathway is one of the most commonly altered pathways in human cancers. The serum/glucocorticoid-regulated kinase (SGK) family of serine/threonine kinases consists of three isoforms, SGK1, SGK2, and SGK3. This family of kinases is highly homologous to the AKT kinase family, sharing similar upstream activators and downstream targets. Few studies have investigated the role of SGK2 in hepatocellular carcinoma. Here, we report that SGK2 expression levels were upregulated in hepatocellular carcinoma tissues and human hepatoma cell lines compared to the adjacent normal liver tissues and a normal hepatocyte line, respectively. We found that downregulated SGK2 inhibits cell migration and invasive potential of hepatocellular carcinoma cell lines (SMMC-7721 and Huh-7).We also found that downregulated SGK2 suppressed the expression level of unphosphorylated (activated) glycogen synthase kinase 3 beta. In addition, SGK2 downregulation decreased the dephosphorylation (activation) of β-catenin by preventing its proteasomal degradation in the hepatocellular carcinoma cell lines. These findings suggest that SGK2 promotes hepatocellular carcinoma progression and mediates glycogen synthase kinase 3 beta/β-catenin signaling in hepatocellular carcinoma cells.

Extracellular Hsp90 and TGFβ regulate adhesion, migration and anchorage independent growth in a paired colon cancer cell line model.

PubMed

de la Mare, Jo-Anne; Jurgens, Tamarin; Edkins, Adrienne L

2017-03-16

Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-β pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-β pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown. The paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and its lymph node metastasis, respectively, were used as an in vitro model to study key cellular processes required for metastasis. The status of the TGF-β pathway was examined in these cells using ELISA, flow cytometry, western blot analysis and confocal microscopy. Furthermore, the effect of addition or inhibition of the TGF-β pathway and Hsp90 on adhesion, migration and anchorage-independent growth, was determined in the cell lines. When comparing the canonical TGF-β1 pathway in the genetically paired cell lines our data suggests that this pathway may be constitutively active in the SW620 metastasis-derived cell line and not the SW480 primary tumour-derived line. In addition, we report that, when present in combination, TGF-β1 and Hsp90β stimulate anchorage-independent growth, reduce adhesion and stimulate migration. This effect is potentiated by inhibition of the TGF-β1 receptor and occurs via an alternate TGF-β1 pathway, mediated by αvβ6 integrin. Interestingly, in the SW620 cells, activation of this alternate TGF-β1 signalling machinery does not appear to require inhibition of the canonical TGF-β1 receptor, which would allow them to respond more effectively to the pro-metastasis stimulus of a combination of Hsp90β and TGF-β1 and this could account for the increased migratory capacity of these cells. In this study we report an apparent synergy between TGF-β1 and Hsp90β in stimulating migratory behaviour of colon cancer cells when signalling occurs via αvβ6 integrin as opposed to the canonical TGF-β1 pathway.

Process development for a recombinant Chinese hamster ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system.

PubMed

Huang, Edwin P; Marquis, Christopher P; Gray, Peter P

2004-11-20

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 microM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 10(7) cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (c) 2004 Wiley Periodicals, Inc

Synergistic effects of the sesquiterpene lactone, EPD, with cisplatin and paclitaxel in ovarian cancer cells.

PubMed

van Haaften, Caroline; Boot, Arnoud; Corver, Willem E; van Eendenburg, Jaap D H; Trimbos, Baptist J M Z; van Wezel, Tom

2015-04-25

Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.

Mammary Gland Tumor Development in Transgenic Mice Overexpressing Different Isoforms of the CDP/Cux Transcription Factor

DTIC Science & Technology

2008-03-01

were found to be overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas , suggesting that these proteins play a...1-4). In addition, short CUX1 isoforms were found to be overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas ...alternative mRNA. The p110 and p75 isoforms are overexpressed in different types of cancers, such as in leiomyomas and breast cancers. In tissue culture

Mammary Gland Tumor Development in Transgenic Mice Overexpressing Different Isoforms of the CDP/Cux Transcription Factor

DTIC Science & Technology

2007-03-01

overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas , suggesting that these proteins play a key role in tumor...isoforms were found to be overexpressed in breast cancer cell lines, in human breast tumors and in uterine leiomyomas , suggesting that these proteins...G1/S transition. In addition, the p110 and p75 isoforms are overexpressed in different types of human cancers, such as in leiomyomas and breast

Novel Immune-Modulating Cellular Vaccine for Prostate Cancer Immunotherapy

DTIC Science & Technology

2015-10-01

the 3-month timeline and details provided in year 1 report. Anti-murine CTLA4 RNA Murine PAP Murine PSMA Murine PAP no signal sequence (mPAP-SS...Murine PSMA no signal sequence (mPSMA-SS) Year 2 (current) report: In addition to what we proposed to do, we wanted to determine if other...PAP, PSCA, PSMA and STEAP in the prostate cell lines TRAMP-C1 and TRAMP-C2. Both are cell lines that have been generated by in vitro propagation of

CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line.

PubMed

Yang, Maozhou; Zhang, Liang; Stevens, Jeff; Gibson, Gary

2014-12-01

The Swarm rat chondrosarcoma (RCS) cell lines derived from a spontaneous neoplasm in a rat spine several decades ago have provided excellent models of chondrosarcoma tumor development. In addition the robust chondrocyte phenotype (expression of a large panel of genes identical to that seen in normal rat cartilage) and the ability to generate cell clones have facilitated their extensive use in the identification of chondrocyte proteins and genes. The clustered regularly interspersed short palindromic repeat (CRISPR) technology employing the RNA-guided nuclease Cas9 has rapidly dominated the genome engineering field as a unique and powerful gene editing tool. We have generated a stable RCS cell line (RCS Cas9) expressing the nuclease Cas9 that enables the editing of any target gene or non-coding RNA by simple transfection with a guide RNA. As proof of principle, stable cell lines with targeted ablation of aggrecan expression (Acan KO) were generated and characterized. The studies show that stable chondrocyte cell lines with targeted genome editing can be quickly generated from RCS Cas9 cells using this system. The Acan KO cell lines also provided a tool for characterizing the response of chondrocytes to aggrecan loss and the role of aggrecan in chondrosarcoma development. Loss of aggrecan expression while not affecting the chondrocyte phenotype resulted in a much firmer attachment of cells to their substrate in culture. Large changes in the expression of several genes were observed in response to the absence of the proteoglycan matrix, including those for several small leucine rich proteoglycans (SLRPs), transcription factors and membrane transporters. Acan KO cells failed to form a substantial chondrosarcoma when injected subcutaneously in nude mice consistent with previous suggestions that the glycosaminoglycan-rich matrix surrounding the chondrosarcoma protects it from destruction by the host immune system. The studies provide new understanding of aggrecan function and the RCS Cas9 cell line is expected to provide a very valuable tool for the study gene function in chondrocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase

PubMed Central

WANG, CHUNHUAI; XIANG, RU; ZHANG, XIANGZHONG; CHEN, YUNXIAN

2015-01-01

Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix-coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti-β1-integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, incubation with blocking anti-β1-integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia. PMID:26004127

Effects of HRAS oncogene on cell cycle progression in a cervical cancer-derived cell line.

PubMed

Córdova-Alarcón, Emilio; Centeno, Federico; Reyes-Esparza, Jorge; García-Carrancá, Alejandro; Garrido, Efraín

2005-01-01

Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes. HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways. We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase. Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells.

The influence of biomechanical properties and cannabinoids on tumor invasion

PubMed Central

Hohmann, Tim; Grabiec, Urszula; Ghadban, Chalid; Feese, Kerstin; Dehghani, Faramarz

2017-01-01

ABSTRACT Background: Cannabinoids are known to have an anti-tumorous effect, but the underlying mechanisms are only sparsely understood. Mechanical characteristics of tumor cells represent a promising marker to distinguish between tumor cells and the healthy tissue. We tested the hypothesis whether cannabinoids influence the tumor cell specific mechanical and migratory properties and if these factors are a prognostic marker for the invasiveness of tumor cells. Methods: 3 different glioblastoma cell lines were treated with cannabinoids and changes of mechanical and migratory properties of single cells were measured using atomic force microscopy and time lapse imaging. The invasiveness of cell lines was determined using a co-culture model with organotypic hippocampal slice cultures. Results: We found that cannabinoids are capable of influencing migratory and mechanical properties in a cell line specific manner. A network analysis revealed a correlation between a “generalized stiffness” and the invasiveness for all tumor cell lines after 3 and 4 d of invasion time: r3d = −0.88 [−0.52;−0.97]; r4d = −0.90 [−0.59;−0.98]. Conclusions: Here we could show that a “generalized stiffness” is a profound marker for the invasiveness of a tumor cell population in our model and thus might be of high clinical relevance for drug testing. Additionally cannabinoids were shown to be of potential use for therapeutic approaches of glioblastoma. PMID:27149140

Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

PubMed

Siebenkäs, Cornelia; Chiappinelli, Katherine B; Guzzetta, Angela A; Sharma, Anup; Jeschke, Jana; Vatapalli, Rajita; Baylin, Stephen B; Ahuja, Nita

2017-01-01

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells.

PubMed

Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios

2016-12-30

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro

PubMed Central

Weingartshofer, Sigrid; Grunt, Thomas W.; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F.

2017-01-01

Background Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Materials and methods Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. Results In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). Conclusion We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status. PMID:28945801

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro.

PubMed

Gschwantler-Kaulich, Daphne; Weingartshofer, Sigrid; Grunt, Thomas W; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F

2017-01-01

Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

Researching glutamate – induced cytotoxicity in different cell lines: a comparative/collective analysis/study

PubMed Central

Kritis, Aristeidis A.; Stamoula, Eleni G.; Paniskaki, Krystallenia A.; Vavilis, Theofanis D.

2015-01-01

Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca2+ levels, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress, and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine (CySS) uptake by reversing the action of the CySS/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione’s reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA, or kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However, in the greatest majority of the cell lines ionotropic glutamate receptors are present, co-existing to CySS/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5, and SCN2.2 cell systems are systematically collected and analyzed. PMID:25852482

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

PubMed

Liu, Weiqiang; Yin, Yifei; Long, Xiaolin; Luo, Yumei; Jiang, Yonghua; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Li, Qing; Chen, Xinjie; Liao, Baoping; Xiao, Guohong; Wang, Weihua; Sun, Xiaofang

2009-04-01

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level.

PubMed

Ricci, Francesca; Berardi, Valerio; Risuleo, Gianfranco

2008-12-31

Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

Peritoneal sarcomatosis: site of origin for the establishment of an in vitro and in vivo cell line model to study therapeutic resistance in dedifferentiated liposarcoma.

PubMed

Mersch, Sabrina; Riemer, Jasmin C; Schlünder, Philipp M; Ghadimi, Markus P; Ashmawy, Hany; Möhlendick, Birte; Topp, Stefan A; Arent, Tanja; Kröpil, Patric; Stoecklein, Nikolas H; Gabbert, Helmut E; Knoefel, Wolfram T; Krieg, Andreas

2016-02-01

Approximately 50-70 % of patients with retroperitoneal or intraabdominal sarcoma develop a relapse after surgical therapy, including peritoneal sarcomatosis, an extremely rare site of metastatic disease which is associated with an extremely poor prognosis. Accordingly, the establishment of a permanent cell line derived from peritoneal sarcomatosis might provide a helpful tool to understand the biological behavior and to develop new therapeutic strategies. Thus, we established and characterized a liposarcoma cell line (Lipo-DUE1) from a peritoneal sarcomatosis that was permanently cultured without showing any morphological changes. Lipo-DUE1 cells exhibited a spindle-shaped morphology and positive staining for S100. Tumorigenicity was demonstrated in vitro by invasion and migration assays and in vivo by using a subcutaneous xenograft mouse model. In addition, aCGH analysis revealed concordant copy number variations on chromosome 12q in the primary tumor, peritoneal sarcomatosis, and Lipo-DUE1 cells that are commonly observed in liposarcoma. Chemotherapeutic sensitivity assays revealed a pronounced drug-resistant phenotype of Lipo-DUE1 cells to conventionally used chemotherapeutic agents. In conclusion, we describe for the first time the establishment and characterization of a liposarcoma cell line derived from a peritoneal sarcomatosis. Hence, in the future, the newly established cell line Lipo-DUE1 might serve as a useful in vitro and in vivo model to investigate the biological behavior of liposarcoma and to assess novel targeted therapies.

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

PubMed

Li, Wenxue; Lee, Michael K

2005-06-01

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

HPV16 early gene E5 specifically reduces miRNA-196a in cervical cancer cells

PubMed Central

Liu, Chanzhen; Lin, Jianfei; Li, Lianqin; Zhang, Yonggang; Chen, Weiling; Cao, Zeyi; Zuo, Huancong; Chen, Chunling; Kee, Kehkooi

2015-01-01

High-risk human papillomavirus (HPV) type 16, which is responsible for greater than 50% of cervical cancer cases, is the most prevalent and lethal HPV type. However, the molecular mechanisms of cervical carcinogenesis remain elusive, particularly the early steps of HPV infection that may transform normal cervical epithelium into a pre-neoplastic state. Here, we report that a group of microRNAs (microRNAs) were aberrantly decreased in HPV16-positive normal cervical tissues, and these groups of microRNAs are further reduced in cervical carcinoma. Among these miRNAs, miR196a expression is the most reduced in HPV16-infected tissues. Interestingly, miR196a expression is low in HPV16-positive cervical cancer cell lines but high in HPV16-negative cervical cancer cell lines. Furthermore, we found that only HPV16 early gene E5 specifically down-regulated miRNA196a in the cervical cancer cell lines. In addition, HoxB8, a known miR196a target gene, is up-regulated in the HPV16 cervical carcinoma cell line but not in HPV18 cervical cancer cell lines. Various doses of miR196a affected cervical cancer cell proliferation and apoptosis. Altogether, these results suggested that HPV16 E5 specifically down-regulates miR196a upon infection of the human cervix and initiates the transformation of normal cervix cells to cervical carcinoma. PMID:25563170

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

PubMed Central

Warenius, H M; Jones, M; Gorman, T; McLeish, R; Seabra, L; Barraclough, R; Rudland, P

2000-01-01

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign PMID:10993658

A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

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

Chang, Junghwa; Jung, Hye Jin; Jeong, Seung Hun

2014-12-12

Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identificationmore » and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.« less

Pro-neurotensin/neuromedin N expression and processing in human colon cancer cell lines.

PubMed

Rovère, C; Barbero, P; Maoret, J J; Laburthe, M; Kitabgi, P

1998-05-08

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.

Relative biological effectiveness in canine osteosarcoma cells irradiated with accelerated charged particles

PubMed Central

Maeda, Junko; Cartwright, Ian M.; Haskins, Jeremy S.; Fujii, Yoshihiro; Fujisawa, Hiroshi; Hirakawa, Hirokazu; Uesaka, Mitsuru; Kitamura, Hisashi; Fujimori, Akira; Thamm, Douglas H.; Kato, Takamitsu A.

2016-01-01

Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/µm carbon ion spread out Bragg peak beams and 200 keV/µm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming as says, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0–1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56–2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51–3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA. PMID:27446477

A Re-evaluation of CD22 Expression by Human Lung Cancer

PubMed Central

Pop, Laurentiu M.; Barman, Stephen; Shao, Chunli; Poe, Jonathan C.; Venturi, Guglielmo M.; Shelton, John M.; Pop, Iliodora V.; Gerber, David E.; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I.; Richardson, James A.; Minna, John D.; Tedder, Thomas F.; Vitetta, Ellen S.

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins. PMID:24395821

Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

PubMed Central

2013-01-01

Background As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype. Methods To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed. Results We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process. Conclusion Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents. PMID:24298994

Combined cetuximab and genistein treatment shows additive anti-cancer effect on oral squamous cell carcinoma.

PubMed

Park, Sung-Jin; Kim, Myung-Jin; Kim, Yu-Kyoung; Kim, Soung-Min; Park, Ju-Yong; Myoung, Hoon

2010-06-01

The purpose of this study was to evaluate the potency of EGFR pathway inhibition achieved by combining cetuximab, an anti-EGFR monoclonal antibody, and genistein, a tyrosine kinase inhibitor, which target extracellular and intracellular domains of the receptor, respectively, in oral squamous cell carcinoma (OSCC) in vitro and in vivo. Two OSCC cell lines, HSC3 and KB, were treated with cetuximab (C, 0-400mug/ml), genistein (G, 0-80muM), or a combination of both at a range of concentrations. Downstream protein expression of EGFR, p-EGFR, and p-Akt were evaluated by Western blot. Cell proliferation and apoptosis indices were calculated to assess anti-cancer effects in vitro. The in vivo effects of cetuximab and genistein on tumor cell growth were examined using an OSCC xenografted nude mouse model and immunohistochemical analyses of proliferation (PCNA) and microvessel density (CD31). Treatment of cells with dual anti-EGFR agents reduced the expressions of p-EGFR, and p-Akt in HSC3 cell line, but there was no significant difference in downregulation between cetuximab alone and in combination with genistein in KB cells. Both HSC3 and KB cells showed a dose-dependent decrease in cell proliferation significantly with single agent treatment and combination (p<0.05). In low concentration, combined cetuximab and genistein therapy resulted in additive growth inhibition and more apoptosis compared to that achieved with single-agent exposure in both cell lines. A combination of cetuximab and genistein significantly inhibited tumor growth and caused a substantial growth delay in in vivo models of both cell lines while each single-agent exposure caused no delay of tumor growth. Immunohistochemical staining with PCNA revealed that the group receiving combined cetuximab and genistein exhibited the lowest number of proliferating cells and microvessel density (p<0.05). Combined therapy with genistein and cetuximab can add the potency of EGFR signaling inhibition. Because not all OSCC cell types appear to respond uniformly, however, selective targeting of distinct molecular pathways is required for effective clinical response. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5

PubMed Central

Yoshida, S; Arakawa, F; Higuchi, F; Ishibashi, Y; Goto, M; Sugita, Y; Nomura, Y; Niino, D; Shimizu, K; Aoki, R; Hashikawa, K; Kimura, Y; Yasuda, K; Tashiro, K; Kuhara, S; Nagata, K; Ohshima, K

2012-01-01

Objectives The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. Methods Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. Results The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. Conclusions Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology. PMID:22401175

Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

PubMed Central

Zhao, Zhenqiang; Ma, Yanlin; Chen, Zhibin; Liu, Qian; Li, Qi; Kong, Deyan; Yuan, Kunxiong; Hu, Lan; Wang, Tan; Chen, Xiaowu; Peng, Yanan; Jiang, Weimin; Yu, Yanhong; Liu, Xinfeng

2016-01-01

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons. PMID:28066186

A novel mechanism of EML4-ALK rearrangement mediated by chromothripsis in a patient-derived cell line.

PubMed

Kodama, Tatsushi; Motoi, Noriko; Ninomiya, Hironori; Sakamoto, Hiroshi; Kitada, Kunio; Tsukaguchi, Toshiyuki; Satoh, Yasuko; Nomura, Kimie; Nagano, Hiroko; Ishii, Nobuya; Terui, Yasuhito; Hatake, Kiyohiko; Ishikawa, Yuichi

2014-11-01

EML4-ALK is a driver oncogene in non-small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

Murine Endogenous Retroviruses Are Detectable in Patient-Derived Xenografts but Not in Patient-Individual Cell Lines of Human Colorectal Cancer.

PubMed

Bock, Stephanie; Mullins, Christina S; Klar, Ernst; Pérot, Philippe; Maletzki, Claudia; Linnebacher, Michael

2018-01-01

Endogenous retroviruses are remnants of retroviral infections. In contrast to their human counterparts, murine endogenous retroviruses (mERV) still can synthesize infectious particles and retrotranspose. Xenotransplanted human cells have occasionally been described to be mERV infected. With genetic engineered mice and patient-derived xenografts (PDXs) on the rise as eminent research tools, we here systematically investigated, if different tumor models harbor mERV infections. Relevant mERV candidates were first preselected by next generation sequencing (NGS) analysis of spontaneous lymphomas triggered by colorectal cancer (CRC) PDX tissue. Two primer systems were designed for each of these candidates (AblMLV, EcoMLV, EndoPP, MLV, and preXMRV) and implemented in an quantitative real-time (RT-qPCR) screen using murine tissues ( n = 11), PDX-tissues ( n = 22), PDX-derived cell lines ( n = 13), and patient-derived tumor cell lines ( n = 14). The expression levels of mERV varied largely both in the PDX samples and in the mouse tissues. No mERV signal was, however, obtained from cDNA or genomic DNA of CRC cell lines. Expression of EcoMLV was higher in PDX than in murine tissues; for EndoPP it was the opposite. These two were thus further investigated in 40 additional PDX. In addition, four patient-derived cell lines free of any mERV expression were subcutaneously injected into immunodeficient mice. Outgrowing cell-derived xenografts barely expressed EndoPP. In contrast, the expression of EcoMLV was even higher than in surrounding mouse tissues. This expression gradually vanished within few passages of re-cultivated cells. In summary, these results strongly imply that: (i) PDX and murine tissues in general are likely to be contaminated by mERV, (ii) mERV are expressed transiently and at low level in fresh PDX-derived cell cultures, and (iii) mERV integration into the genome of human cells is unlikely or at least a very rare event. Thus, mERVs are stowaways present in murine cells, in PDX tissues and early thereof-derived cell cultures. We conclude that further analysis is needed concerning their impact on results obtained from studies performed with PDX but also with murine tumor models.

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

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

Suzuki, Takahiro; Kanamori, Takao; Inouye, Satoshi

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- andmore » 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs. - Highlights: • An imaging method for protein secretion from 3D-cultured cells was established. • The fused protein of insulin to GLase, Insulin-GLase, was used as a reporter. • Synchronous insulin secretion was visualized in rat islets and spheroidal beta cells. • A rat beta cell line stably expressing Insulin-GLase, named iGL, was established. • Effect of an antidiabetic drug on insulin secretion was visualized in iGL cells.« less

Anti-cancer stem cell activity of a hedgehog inhibitor GANT61 in estrogen receptor-positive breast cancer cells.

PubMed

Kurebayashi, Junichi; Koike, Yoshikazu; Ohta, Yusuke; Saitoh, Wataru; Yamashita, Tetsumasa; Kanomata, Naoki; Moriya, Takuya

2017-05-01

Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. It has been suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER-positive breast cancer cells. We studied anti-CSC activity of a non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER-positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti-cell growth and anti-CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER-positive cell lines. E2 increased the expression levels of glioma-associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1-S cell cycle retardation and increased apoptosis. GANT61 decreased the E2-induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2-induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti-cell growth and/or anti-CSC activities in some ER-positive cell lines. In conclusion, the non-canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced anti-cell growth and/or anti-CSC activities of antiestrogens in ER-positive cell lines. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

PubMed Central

Jiménez Pérez, Zuly Elizabeth; Mathiyalagan, Ramya; Markus, Josua; Kim, Yeon-Ju; Kang, Hyun Mi; Abbai, Ragavendran; Seo, Kwang Hoon; Wang, Dandan; Soshnikova, Veronika; Yang, Deok Chun

2017-01-01

There has been a growing interest in the design of environmentally affable and biocompatible nanoparticles among scientists to find novel and safe biomaterials. Panax ginseng Meyer berries have unique phytochemical profile and exhibit beneficial pharmacological activities such as antihyperglycemic, antiobesity, antiaging, and antioxidant properties. A comprehensive study of the biologically active compounds in ginseng berry extract (GBE) and the ability of ginseng berry (GB) as novel material for the biosynthesis of gold nanoparticles (GBAuNPs) and silver nanoparticles (GBAgNPs) was conducted. In addition, the effects of GBAuNPs and GBAgNPs on skin cell lines for further potential biological applications are highlighted. GBAuNPs and GBAgNPs were synthesized using aqueous GBE as a reducing and capping agent. The synthesized nanoparticles were characterized for their size, morphology, and crystallinity. The nanoparticles were evaluated for antioxidant, anti-tyrosinase, antibacterial, and cytotoxicity activities and for morphological changes in human dermal fibroblast and murine melanoma skin cell lines. The phytochemicals contained in GBE effectively reduced and capped gold and silver ions to form GBAuNPs and GBAgNPs. The optimal synthesis conditions (ie, temperature and v/v % of GBE) and kinetics were investigated. Polysaccharides and phenolic compounds present in GBE were suggested to be responsible for stabilization and functionalization of nanoparticles. GBAuNPs and GBAgNPs showed increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals compared to GBE. GBAuNPs and GBAgNPs effectively inhibited mushroom tyrosinase, while GBAgNPs showed antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, GBAuNPs were nontoxic to human dermal fibroblast and murine melanoma cell lines, and GBAgNPs showed cytotoxic effect on murine melanoma cell lines. The current results evidently suggest that GBAgNPs can act as potential agents for antioxidant, anti-tyrosinase, and antibacterial activities. In addition, GBAuNPs can be further developed into mediators in drug delivery and as antioxidant, anti-tyrosinase, and protective skin agents in cosmetic products. Consequently, the study showed the advantages of using nanotechnology and green chemistry to enhance the natural properties of GBs. PMID:28260881

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

PubMed Central

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

Post-mortem re-cloning of a transgenic red fluorescent protein dog

PubMed Central

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

2011-01-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

CD44+CD24+ subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species.

PubMed

Wang, Lei; Li, Pengping; Hu, Wei; Xia, Youyou; Hu, Chenxi; Liu, Liang; Jiang, Xiaodong

2017-08-01

Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44 + and CD24 + in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44 + CD24 + group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44 + CD24 + group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44 + CD24 + group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44 + CD24 + exhibited stem cell properties. The lower level of ROS and apoptosis in CD44 + CD24 + cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.

Bone sialoprotein stimulates focal adhesion-related signaling pathways: role in migration and survival of breast and prostate cancer cells.

PubMed

Gordon, Jonathan A R; Sodek, Jaro; Hunter, Graeme K; Goldberg, Harvey A

2009-08-15

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP-induced migration and tumor survival were examined in breast and prostate cancer cells (MDA-MB-231, Hs578T and PC3). Additionally, the effects of exogenous TGF-beta1 and EGF, cytokines associated with tumor metastasis and present in high-levels in the bone microenvironment, were examined in BSP-expressing cancer cells. Expression of BSP but not an integrin-binding mutant (BSP-KAE) in tumor cell lines resulted in increased levels of alpha(v)-containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP-1-family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP-2, MMP-9, and MMP-14. The BSP-mediated activation of the FAK-associated pathway resulted in increased cancer cell invasion in a Matrigel-coated Boyden-chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF-beta1 to the BSP-expressing cell lines significantly increased ERK phosphorylation, AP-1 activation, MMP-2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF-beta1 and EGF. (c) 2009 Wiley-Liss, Inc.

Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis.

PubMed

Eiro, Noemi; González, Lucía; Martínez-Ordoñez, Anxo; Fernandez-Garcia, Belen; González, Luis O; Cid, Sandra; Dominguez, Francisco; Perez-Fernandez, Román; Vizoso, Francisco J

2018-03-01

It has been reported that stromal cell features may affect the clinical outcome of breast cancer patients. Cancer associated fibroblasts (CAFs) represent one of the most abundant cell types within the breast cancer stroma. Here, we aimed to explore the influence of CAFs on breast cancer gene expression, as well as on invasion and angiogenesis. qRT-PCR was used to evaluate the expression of several cancer progression related genes (S100A4, TGFβ, FGF2, FGF7, PDGFA, PDGFB, VEGFA, IL-6, IL-8, uPA, MMP2, MMP9, MMP11 and TIMP1) in the human breast cancer-derived cell lines MCF-7 and MDA-MB-231, before and after co-culture with CAFs. Stromal mononuclear inflammatory cell (MIC) MMP11 expression was used to stratify primary tumors. In addition, we assessed the in vitro effects of CAFs on both MDA-MB-231 breast cancer cell invasion and endothelial cell (HUVEC) tube formation. We found that the expression levels of most of the genes tested were significantly increased in both breast cancer-derived cell lines after co-culture with CAFs from either MMP11+ or MMP11- MIC tumors. IL-6 and IL-8 showed an increased expression in both cancer-derived cell lines after co-culture with CAFs from MMP11+ MIC tumors. We also found that the invasive and angiogenic capacities of, respectively, MDA-MB-231 and HUVEC cells were increased after co-culture with CAFs, especially those from MMP11+ MIC tumors. Our data indicate that tumor-derived CAFs can induce up-regulation of genes involved in breast cancer progression. Our data additionally indicate that CAFs, especially those derived from MMP11+ MIC tumors, can promote breast cancer cell invasion and angiogenesis.

Efficacy of ribavirin against malignant glioma cell lines: Follow-up study

PubMed Central

Ochiai, Yushi; Sano, Emiko; Okamoto, Yutaka; Yoshimura, Sodai; Makita, Kotaro; Yamamuro, Shun; Ohta, Takashi; Ogino, Akiyoshi; Tadakuma, Hisashi; Ueda, Takuya; Nakayama, Tomohiro; Hara, Hiroyuki; Yoshino, Atsuo; Katayama, Yoichi

2018-01-01

Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 µM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time-lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin. PMID:29251333

Increasing The Genetic Admixture of Available Lines of Human Pluripotent Stem Cells

PubMed Central

Tofoli, Fabiano A.; Dasso, Maximiliano; Morato-Marques, Mariana; Nunes, Kelly; Pereira, Lucas Assis; da Silva, Giselle Siqueira; Fonseca, Simone A. S.; Costas, Roberta Montero; Santos, Hadassa Campos; da Costa Pereira, Alexandre; Lotufo, Paulo A.; Bensenor, Isabela M.; Meyer, Diogo; Pereira, Lygia Veiga

2016-01-01

Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response. PMID:27708369

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Identification of genes expressed in the hermaphrodite germ line of C. elegans using SAGE

PubMed Central

Wang, Xin; Zhao, Yongjun; Wong, Kim; Ehlers, Peter; Kohara, Yuji; Jones, Steven J; Marra, Marco A; Holt, Robert A; Moerman, Donald G; Hansen, Dave

2009-01-01

Background Germ cells must progress through elaborate developmental stages from an undifferentiated germ cell to a fully differentiated gamete. Some of these stages include exiting mitosis and entering meiosis, progressing through the various stages of meiotic prophase, adopting either a male (sperm) or female (oocyte) fate, and completing meiosis. Additionally, many of the factors needed to drive embryogenesis are synthesized in the germ line. To increase our understanding of the genes that might be necessary for the formation and function of the germ line, we have constructed a SAGE library from hand dissected C. elegans hermaphrodite gonads. Results We found that 4699 genes, roughly 21% of all known C. elegans genes, are expressed in the adult hermaphrodite germ line. Ribosomal genes are highly expressed in the germ line; roughly four fold above their expression levels in the soma. We further found that 1063 of the germline-expressed genes have enriched expression in the germ line as compared to the soma. A comparison of these 1063 germline-enriched genes with a similar list of genes prepared using microarrays revealed an overlap of 460 genes, mutually reinforcing the two lists. Additionally, we identified 603 germline-enriched genes, supported by in situ expression data, which were not previously identified. We also found >4 fold enrichment for RNA binding proteins in the germ line as compared to the soma. Conclusion Using multiple technological platforms provides a more complete picture of global gene expression patterns. Genes involved in RNA metabolism are expressed at a significantly higher level in the germ line than the soma, suggesting a stronger reliance on RNA metabolism for control of the expression of genes in the germ line. Additionally, the number and expression level of germ line expressed genes on the X chromosome is lower than expected based on a random distribution. PMID:19426519

Induction of Apoptosis of 2,4′,6-Trihydroxybenzophenone in HT-29 Colon Carcinoma Cell Line

PubMed Central

Lay, Ma Ma; Karsani, Saiful Anuar

2014-01-01

2,4′,6-Trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl. fruits. It was found to inhibit cell proliferation in HT-29 human colon carcinoma cell line but caused little damage to WRL-68 normal human liver and MRC-5 normal human fibroblast lung cell lines. The compound was found to sharply affect the viability of HT-29 cells in a dose- and time-dependent manner. HT-29 cells treated with the compound showed morphological changes under microscopic examination such as cell shrinkage, membrane blebbing, DNA fragmentation, and the occurrence of apoptotic nuclei. The percentage of early apoptotic, late apoptotic, and dead or necrotic cells was determined by flow cytometry using annexin V-FTIC/PI staining. In addition, flow cytometry showed that, when the HT-29 cells were treated with 115 µM of the compound, it resulted in G0/G1 phase arrest in a time-dependent manner. Western blot revealed an upregulation of PUMA, Bak, Bcl-2, and Mcl-1 proteins suggesting that the compound induced apoptosis in HT-29 cells by regulating these proteins. PMID:24579081

Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio.

PubMed

Lin, S-L; Cheng, Y-H; Wen, C-M; Chen, S-N

2013-06-01

A continuous cell line (KF-101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF-101 cell line multiplied abundantly in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF-101 cells contain keratin, junction proteins connexin-43 and occludin, and ectodermal stem-cell marker Pax-6, but not vimentin. Furthermore, the KF-101 cells reacted with anti-human DARPP-32 and anti-human GATA-4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP-32 and GATA-4 antibodies in the KF-101 cells were the suggested phosphatase-1 inhibitor-1 and GATA-3, respectively. In addition, the KF-101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF-101 cells are suitable materials for investigating biological and virological development. © 2013 The Authors. Journal of Fish Biology © 2013 The Fisheries Society of the British Isles.

Development of sulfadiazine-decorated PLGA nanoparticles loaded with 5-fluorouracil and cell viability.

PubMed

Guimarães, Pedro Pires Goulart; Oliveira, Sheila Rodrigues; de Castro Rodrigues, Gabrielle; Gontijo, Savio Morato Lacerda; Lula, Ivana Silva; Cortés, Maria Esperanza; Denadai, Ângelo Márcio Leite; Sinisterra, Rubén Dario

2015-01-08

The aim of this work was to synthesize sulfadiazine-poly(lactide-co-glycolide) (SUL-PLGA) nanoparticles (NPs) for the efficient delivery of 5-fluorouracil to cancer cells. The SUL-PLGA conjugation was assessed using FTIR, 1H-NMR, 13C-NMR, elemental analysis and TG and DTA analysis. The SUL-PLGA NPs were characterized using transmission and scanning electron microscopy and dynamic light scattering. Additionally, the zeta potential, drug content, and in vitro 5-FU release were evaluated. We found that for the SUL-PLGA NPs, Dh = 114.0 nm, ZP = -32.1 mV and the encapsulation efficiency was 49%. The 5-FU was released for up to 7 days from the NPs. Cytotoxicity evaluations of 5-FU-loaded NPs (5-FU-SUL-PLGA and 5-FU-PLGA) on two cancer cell lines (Caco-2, A431) and two normal cell lines (fibroblast, osteoblast) were compared. Higher cytotoxicity of 5-FU-SUL-PLGA NPs were found to both cancer cell lines when compared to normal cell lines, demonstrating that the presence of SUL could significantly enhance the cytotoxicity of the 5-FU-SUL-PLGA NPs when compared with 5-FU-PLGA NPs. Thus, the development of 5-FU-SUL-PLGA NPs to cancer cells is a promising strategy for the 5-FU antitumor formulation in the future.

Osthole inhibits the tumorigenesis of hepatocellular carcinoma cells.

PubMed

Lin, Zhi-Kun; Liu, Jia; Jiang, Guo-Qiang; Tan, Guang; Gong, Peng; Luo, Hai-Feng; Li, Hui-Min; Du, Jian; Ning, Zhen; Xin, Yi; Wang, Zhong-Yu

2017-03-01

Hepatocellular carcinoma (HCC) accounts for approximately 90% of all cases of primary liver cancer, and the majority of patients with HCC are deprived of effective curative methods. Osthole is a Chinese herbal medicine which has been reported to possess various pharmacological functions, including hepatocellular protection. In the present study, we investigated the anticancer activity of osthole using HCC cell lines. We found that osthole inhibited HCC cell proliferation, induced cell cycle arrest, triggered DNA damage and suppressed migration in HCC cell lines. Furthermore, we demonstrated that osthole not only contributed to cell cycle G2/M phase arrest via downregulation of Cdc2 and cyclin B1 levels, but also induced DNA damage via an increase in ERCC1 expression. In addition, osthole inhibited the migration of HCC cell lines by significantly downregulating MMP-2 and MMP-9 levels. Finally, we demonstrated that osthole inhibited epithelial-mesenchymal transition (EMT) via increasing the expression of epithelial biomarkers E-cadherin and β-catenin, and significantly decreasing mesenchymal N-cadherin and vimentin protein expression. These results suggest that osthole may have potential chemotherapeutic activity against HCC.

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.

Manipulation of the sodium-potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures.

PubMed

Wang, Samantha B; Lee-Goldman, Alexandria; Ravikrishnan, Janani; Zheng, Lili; Lin, Henry

2018-04-01

Perfusion processes typically require removal of a continuous or semi-continuous volume of cell culture in order to maintain a desired target cell density. For fast growing cell lines, the product loss from this stream can be upwards of 35%, significantly reducing the overall process yield. As volume removed is directly proportional to cell growth, the ability to modulate growth during perfusion cell culture production thus becomes crucial. Leveraging existing media components to achieve such control without introducing additional supplements is most desirable because it decreases process complexity and eliminates safety and clearance concerns. Here, the impact of extracellular concentrations of sodium (Na) and potassium (K) on cell growth and productivity is explored. High throughput small-scale models of perfusion revealed Na:K ratios below 1 can significantly suppress cell growth by inducing cell cycle arrest in the G0/1 phase. A concomitant increase in cell specific productivity was also observed, reaching as high as 115 pg/cell/day for one cell line studied. Multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrated similar responses to lower Na:K media, indicating the universal applicability of such an approach. Product quality attributes were also assessed and revealed that effects were cell line specific, and can be acceptable or manageable depending on the phase of the drug development. Drastically altering Na and K levels in perfusion media as a lever to impact cell growth and productivity is proposed. © 2017 Wiley Periodicals, Inc.

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines.

PubMed

Nagel, David A; Hill, Eric J; O'Neil, John; Mireur, Alexandra; Coleman, Michael D

2014-11-01

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48h of incubation with increasing concentrations of pesticides ranging from 1 to 1000μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure. Copyright © 2014 Elsevier B.V. All rights reserved.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Transformation of primary chick embryo fibroblasts by Marek's disease virus.

PubMed

Buranathai, C; Rodriguez, J; Grose, C

1997-12-08

Marek's disease virus (MDV) is an alphaherpesvirus, which can mediate the malignant transformation of lymphocytes to form lymphomas in chickens. In this study, we demonstrate that MDV can transform primary chick embryo fibroblasts (CEF). The cell line derived from primary CEF infected with the GA strain of MDV was called CEM(MDV). The fibroblast nature of CEM(MDV) was verified by absence of cytokeratin type II. The CEM(MDV) phenotype differed from either primary CEF or MDV-infected CEF. CEM(MDV) were extensively vacuolated, with unusual multilamellar structures in the cytoplasm, The nuclei were considerably larger than those in primary CEF and were uniformly positive for proliferating cell nuclear antigen. The cell line was subcultured for more than 10 generations; however, CEM(MDV) did not support a fully productive MDV infection, because complete nucleocapsids were not detected and infectivity assays showed that cell line produced no infectious virus. PCR analyses demonstrated that this cell line carried both polypeptide 38 (pp38) and Meq DNA, MDV-specific genes associated with transformation. In addition, examination by laser scanning confocal microscopy revealed that CEM(MDV) constitutively produced MDV MEQ protein in nuclei and pp38 as well as glycoprotein B in the cytoplasm and on the plasma membrane. Growth in soft agar assay demonstrated that CEM(MDV) formed colonies, similar to HeLa and human melanoma cells. Retroviral insertion was not detected in DNA from the CEM(MDV) line.

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

PubMed

Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

2015-10-01

Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines.

PubMed

Souza, Raquel P; Bonfim-Mendonça, Patrícia de S; Gimenes, Fabrícia; Ratti, Bianca A; Kaplum, Vanessa; Bruschi, Marcos L; Nakamura, Celso V; Silva, Sueli O; Maria-Engler, Silvya S; Consolaro, Marcia E L

2017-01-01

Recently, the cytotoxic effects of apigenin (4',5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H 2 O 2 , decreased the Δ ψm , and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

PubMed Central

Souza, Raquel P.; Gimenes, Fabrícia; Ratti, Bianca A.; Kaplum, Vanessa; Bruschi, Marcos L.; Nakamura, Celso V.; Maria-Engler, Silvya S.

2017-01-01

Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes. PMID:28191273

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

PubMed Central

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

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

PubMed

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

2012-12-01

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

Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro

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

Rubin, L.A.; Kurman, C.C.; Fritz, M.E.

1985-11-01

With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2Rmore » by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.« less

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

PubMed

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Dosage and Cell Line Dependent Inhibitory Effect of bFGF Supplement in Human Pluripotent Stem Cell Culture on Inactivated Human Mesenchymal Stem Cells

PubMed Central

Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

2014-01-01

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4–10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system. PMID:24465853

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

PubMed Central

Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

2015-01-01

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

Lawrence Transfer Factor: Transference of Specific Immune Memory by Dialyzable Leukocyte Extract from a CD8+ T Cell Line.

PubMed

Wang, Jason F; Park, Andrew J; Rendini, Tina; Levis, William R

2017-12-01

Lawrence transfer factor (TF) is defined as dialyzable leukocyte extract (DLE) that can transfer antigen-specific cell-mediated immunity from a person testing positive for the antigen in a delayed type hypersensitivity skin test manner to a person negative for the same antigen. A recent article by Myles et al1 has identified a DLE isolated from an established CD8+ T cell line capable of transferring antigen-specific immunity. The DLE contains a portion of the beta chain of the T cell receptor and additional nucleotide and protein factors that are being subjected to further modern biochemical analysis. After months of study that included interviews of TF physician-scientists, we conclude that an antigen-specific TF exists for most, if not all, antigens. By working from a CD8+ T cell line with modern biochemical technology, it should be possible to identify and patent products capable of treating infectious diseases, antigen-responsive cancers, and autoimmune disorders.

Platelets Inhibit Migration of Canine Osteosarcoma Cells.

PubMed

Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

2017-01-01

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

PubMed

Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša

2013-03-01

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor 2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.

Selective effects of quercetin on the cell growth and antioxidant defense system in normal versus transformed mouse hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Kook, Sung-Ho; Lee, Jeong-Chae; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2004-10-19

Quercetin is a dietary anticancer chemical that is capable of inducing apoptosis in tumor cells. However, little is known about its biological effect on nonmalignant cells, although the effect is one of the critical criteria to evaluate the clinical efficacy of the anticancer agent. In this study, we investigated the effects of quercetin on cell growth and apoptosis using embryonic normal hepatic cell line (BNL CL.2) and its SV40-transformed cell line (BNL SV A.8). We also evaluated the effects of quercetin on the antioxidant defense system in those cells. BNL SV A.8 cells were more sensitive to quercetin-mediated cytotoxicity than BNL CL.2 cells. In addition, the enzyme assays showed that quercetin actively stimulated the antioxidant defense systems including superoxide dismutase, catalase, glutathione, and glutathione reductase only in the BNL CL.2 cells. In particular, quercetin significantly reduced superoxide dismutase activity and increased the malonaldehyde content in BNL SV A.8 cells. These are thought to be closely related to quercetin-mediated apoptosis. Our findings suggest that quercetin is a dietary flavonoid that is capable of inducing selective growth inhibition and apoptosis in hepatic tumor cells, but not in normal cells.

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition

PubMed Central

Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-01-01

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis. Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants. PMID:28159923

The anti-neoplastic activity of Vandetanib against high-risk medulloblastoma variants is profoundly enhanced by additional PI3K inhibition.

PubMed

Craveiro, Rogerio B; Ehrhardt, Michael; Velz, Julia; Olschewski, Martin; Goetz, Barbara; Pietsch, Torsten; Dilloo, Dagmar

2017-07-18

Medulloblastoma is comprised of at least four molecular subgroups with distinct clinical outcome (WHO classification 2016). SHH-TP53-mutated as well as MYC-amplified Non-WNT/Non-SHH medulloblastoma show the worst prognosis.Here we present evidence that single application of the multi-kinase inhibitor Vandetanib displays anti-neoplastic efficacy against cell lines derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/Non-SHH medulloblastoma. The narrow target spectrum of Vandetanib along with a favourable toxicity profile renders this drug ideal for multimodal treatment approaches. In this context our investigation documents that Vandetanib in combination with the clinically available PI3K inhibitor GDC-0941 leads to enhanced cytotoxicity against MYC-amplified and SHH-TP53-mutated medulloblastoma. In line with these findings we show for MYC-amplified medulloblastoma a profound reduction in activity of the oncogenes STAT3 and AKT. Furthermore, we document that Vandetanib and the standard chemotherapeutic Etoposide display additive anti-neoplastic efficacy in the investigated medulloblastoma cell lines that could be further enhanced by PI3K inhibition. Of note, the combination of Vandetanib, GDC-0941 and Etoposide results in MYC-amplified and SHH-TP53-mutated cell lines in complete loss of cell viability. Our findings therefore provide a rational to further evaluate Vandetanib in combination with PI3K inhibitors as well as standard chemotherapeutics in vivo for the treatment of most aggressive medulloblastoma variants.

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

PubMed

Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

2017-02-01

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

SU-F-T-674: In Vitro Study of 5-Aminolevulinic Acid-Mediated Photo Dynamic Therapy in Human Cancer Cell Lines

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

Cvetkovic, D; Wang, B; Gupta, R

Purpose: Photodynamic therapy (PTD) is a promising cancer treatment modality. 5-sminolevulinic acid (ALA) is a clinically approved photosensitizer. Here we studied the effect of 5-ALA administration with irradiation on several cell lines in vitro. Methods: Human head and neck (FaDu), lung (A549) and prostate (LNCaP) cancer cells (104/well) were seeded overnight in 96-well plates (Figure 1). 5-ALA at a range from 0.1 to 30.0mg/ml was added to confluent cells 3h before irradiation in 100ul of culture medium. 15MV photon beams from a Siemens Artiste linear accelerator were used to deliver 2 Gy dose in one fraction to the cells. Cellmore » viability was evaluated by WST1 assay. The development of orange color was measured 3h after the addition of WST-1 reagent at 450nm on an Envision Multilabel Reader (Figure 2) and directly correlated to cell number. Control, untreated cells were incubated without 5-ALA. The experiment was performed twice for each cell line. Results: The cell viability rates for the head and neck cancer line are shown in Figure 3. FaDu cell viability was reduced significantly to 36.5% (5-ALA) and 18.1% (5-ALA + RT) only at the highest concentration of 5-ALA, 30mg/ml. This effect was observed in neither A549, nor LNCaP cell line. No toxicity was detected at lower 5-ALA concentrations. Conclusion: Application of 5-ALA and subsequent PDT was found to be cytotoxic at the highest dose of the photosensitizer used in the FaDu head and neck cell line, and their effect was synergistic. Further efforts are necessary to study the potential therapeutic effects of 5-ALA PTD in vitro and in vivo. Our results suggest 5-ALA may improve the efficacy of radiotherapy by acting as a radiomediator in head and neck cancer.« less

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

PubMed

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

2017-01-25

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

Chilling-induced physiological, anatomical and biochemical responses in the leaves of Miscanthus × giganteus and maize (Zea mays L.).

PubMed

Bilska-Kos, Anna; Panek, Piotr; Szulc-Głaz, Anna; Ochodzki, Piotr; Cisło, Aneta; Zebrowski, Jacek

2018-06-08

Miscanthus × giganteus and Zea mays, closely-related C 4 grasses, originated from warm climates react differently to low temperature. To investigate the response to cold (12-14 °C) in these species, the photosynthetic and anatomical parameters as well as biochemical properties of the cell wall were studied. The research was performed using M. giganteus (MG) and two Z. mays lines differentiated for chilling-sensitivity: chilling-tolerant (Zm-T) and chilling-sensitive (Zm-S). The chilled plants of Zm-S line demonstrated strong inhibition of net CO 2 assimilation and a clear decrease in F' v /F' m , F v /F m and ɸ PSII, while in MG and Zm-T plants these parameters were almost unchanged. The anatomical studies revealed that MG plants had thinner leaves, epidermis and mesophyll cell layer as well as thicker cell walls in the comparison to both maize lines. Cold led to an increase in leaf thickness and mesophyll cell layer thickness in the Zm-T maize line, while the opposite response was observed in Zm-S. In turn, in chilled plants of MG and Zm-T lines, some anatomical parameters associated with bundle sheath cells were higher. In addition, Zm-S line showed the strong increase in the cell wall thickness at cold for mesophyll and bundle sheath cells. Chilling-treatment induced the changes in the cell wall biochemistry of tested species, mainly in the content of glucuronoarabinoxylan, uronic acid, β-glucan and phenolic compounds. This work presents a new approach in searching of mechanism(s) of tolerance/sensitivity to low temperature in two thermophilic plants: Miscanthus and maize. Copyright © 2018 Elsevier GmbH. All rights reserved.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

PubMed

Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

2011-08-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

PubMed Central

Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

2012-01-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype.

PubMed

Lee, H; Li, D; Prior, T; Casto, B C; Weghorst, C M; Shuler, C F; Milo, G E

1997-10-01

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells' ability to exhibit a malignant potential in nude mice.

Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM.

PubMed

Burgman, Paul; O'Donoghue, Joseph A; Lewis, Jason S; Welch, Michael J; Humm, John L; Ling, C Clifton

2005-08-01

Cu-diacetyl-bis(N(4)-methylthiosemicarbazone) [Cu-ATSM] is a potential marker for tumor hypoxia that has been under evaluation for clinical use. In this study, we examined the mechanisms underlying the uptake of (64)Cu in cells incubated with (64)Cu-ATSM. The in vitro uptake of (64)Cu was determined as a function of oxygenation conditions and incubation time with (64)Cu-ATSM using four and two tumor cell lines of human origin and rodent origin, respectively. Additionally, the rate of (64)Cu efflux and Cu-ATSM metabolism was determined. (64)Cu accumulation is rapid during the first 0.5-1 h of incubation. It is highest in anoxic cells but is also significant in normoxic cells. After this initial period, the level of intracellular (64)Cu varies depending on the cell line and the oxygenation conditions and, in some circumstances, may decrease. During the first 0.5-1 h, the ratio of (64)Cu levels between anoxic and normoxic cells is approximately 2:10 and that between hypoxic (0.5% O(2)) and normoxic cells is approximately 1:2.5, depending on the cell line. These ratios generally decrease at longer times. The (64)Cu-ATSM compound was found to be metabolized during incubation in a manner dependent on oxygenation conditions. Within 2 h under anoxic conditions, (64)Cu-ATSM could no longer be detected, although 60-90% of the amount of (64)Cu added as (64)Cu-ATSM was present in the medium. Non-ATSM (64)Cu was taken up by the cells, albeit at a much slower rate. Efflux rates of (64)Cu were found to be cell line dependent and appeared to be inversely correlated with the final (64)Cu uptake levels under anoxic conditions. The uptake and retention of (64)Cu and their relation to oxygenation conditions were found to be cell line dependent. Given the complexities in the oxygen dependence and cell line-dependent kinetics of uptake and retention of Cu following exposure to Cu-ATSM, the clinical utility of this compound may be disease site specific.

In vitro determination of the efficacy of scorpion venoms as anti-cancer agents against colorectal cancer cells: a nano-liposomal delivery approach.

PubMed

Al-Asmari, Abdulrahman K; Ullah, Zabih; Al Balowi, Ali; Islam, Mozaffarul

2017-01-01

The use of liposomes in biological and medicinal sciences is a relatively new approach. The liposomal strategy greatly depends on the technological advancement in the formation of vesicles of various sizes and properties. In the current study, we encapsulated the venoms obtained from medically important scorpions such as Androctonus bicolor (AB), Androctonus crassicauda (AC), and Leiurus quinquestriatus (LQ). To begin with, our first and foremost aim was to prepare biocompatible and biodegradable nanovesicles. Additionally, we intended to enhance the anti-cancer potential of these encapsulated venoms. The liposomal venoms were prepared by rehydration and dehydration methods. Morphology, particle size, and size distribution of the liposomes were examined by scanning electron microscope (SEM), transmission electron microscope (TEM), and Zetasizer. We found that the prepared liposomes had a smooth surface and a spherical/ovoid shape and existed mainly as single unilamellar vesicles (SUVs). Furthermore, the liposomal formulation of all three venoms exhibited excellent stability and good encapsulation efficiency (EE). Additionally, the anti-cancer potential of the encapsulated venoms was also evaluated on a colorectal cancer cell line (HCT-8). The venom-loaded liposomes showed elevated anti-cancer properties such as low rate of cell survival, higher reactive oxygen species (ROS) generation, and enhancement in the number of apoptotic cells. In addition to this, cell cycle analysis revealed G0/G1 enrichment upon venom treatment. The effect of treatment was more pronounced when venom-liposome was used as compared to free venom on the HCT-8 cell line. Furthermore, we did not observe any interference of liposomal lipids used in these preparations on the progression of cancer cells. Considering these findings, we can conclude that the encapsulated scorpion venoms exhibit better efficacy and act more vigorously as an anti-cancer agent on the colorectal cancer cell line when compared with their free counterpart.

In vitro determination of the efficacy of scorpion venoms as anti-cancer agents against colorectal cancer cells: a nano-liposomal delivery approach

PubMed Central

Al-Asmari, Abdulrahman K; Ullah, Zabih; Al Balowi, Ali; Islam, Mozaffarul

2017-01-01

The use of liposomes in biological and medicinal sciences is a relatively new approach. The liposomal strategy greatly depends on the technological advancement in the formation of vesicles of various sizes and properties. In the current study, we encapsulated the venoms obtained from medically important scorpions such as Androctonus bicolor (AB), Androctonus crassicauda (AC), and Leiurus quinquestriatus (LQ). To begin with, our first and foremost aim was to prepare biocompatible and biodegradable nanovesicles. Additionally, we intended to enhance the anti-cancer potential of these encapsulated venoms. The liposomal venoms were prepared by rehydration and dehydration methods. Morphology, particle size, and size distribution of the liposomes were examined by scanning electron microscope (SEM), transmission electron microscope (TEM), and Zetasizer. We found that the prepared liposomes had a smooth surface and a spherical/ovoid shape and existed mainly as single unilamellar vesicles (SUVs). Furthermore, the liposomal formulation of all three venoms exhibited excellent stability and good encapsulation efficiency (EE). Additionally, the anti-cancer potential of the encapsulated venoms was also evaluated on a colorectal cancer cell line (HCT-8). The venom-loaded liposomes showed elevated anti-cancer properties such as low rate of cell survival, higher reactive oxygen species (ROS) generation, and enhancement in the number of apoptotic cells. In addition to this, cell cycle analysis revealed G0/G1 enrichment upon venom treatment. The effect of treatment was more pronounced when venom–liposome was used as compared to free venom on the HCT-8 cell line. Furthermore, we did not observe any interference of liposomal lipids used in these preparations on the progression of cancer cells. Considering these findings, we can conclude that the encapsulated scorpion venoms exhibit better efficacy and act more vigorously as an anti-cancer agent on the colorectal cancer cell line when compared with their free counterpart. PMID:28144138

Pleiotropic effects of bisphosphonates on osteosarcoma.

PubMed

Ohba, Tetsuro; Cates, Justin M M; Cole, Heather A; Slosky, David A; Haro, Hirotaka; Ichikawa, Jiro; Ando, Takashi; Schwartz, Herbert S; Schoenecker, Jonathan G

2014-06-01

Osteosarcoma is the most common primary malignant tumor of bone and accounts for half of all primary skeletal malignancies in children and teenagers. The prognosis for patients who fail or progress on first-line chemotherapy protocols is poor, therefore, additional adjuvant therapeutic strategies are needed. A recent feasibility study has demonstrated that the nitrogen-containing bisphosphonate zoledronic acid (ZOL) can be combined safely with conventional chemotherapy. However, the pharmacodynamics of bisphosphonate therapy is not well characterized. Osteosarcoma is a highly angiogenic tumor. Recent reports of the anti-angiogenic effects of bisphosphonates prompted us to determine whether nitrogen-containing bisphosphonate (ZOL and alendronate) treatment attenuates osteosarcoma growth by inhibition of osteoclast activity, tumor-mediated angiogenesis, or direct inhibitory effects on osteosarcoma. Here, we demonstrate that bisphosphonates directly inhibit VEGFR2 expression in endothelial cells, as well as endothelial cell proliferation and migration. Additionally, bisphosphonates also decrease VEGF-A expression in osteosarcoma (K7M3) cells, resulting in reduced stimulation of endothelial cell migration in co-culture assays. ZOL also decreases VEGFR1 expression in aggressive osteosarcoma cell lines (K7M3, 143B) and induces apoptosis of these cells, but has negligible effects on less aggressive osteosarcoma cell lines (K12 and TE85). In vivo ZOL treatment results in significant reduction in osteosarcoma-initiated angiogenesis and tumor growth in a murine model of osteosarcoma. In conclusion, bisphosphonates have diverse growth inhibitory effects on osteosarcoma through: (1) activation of apoptosis and inhibition of cell proliferation, (2) inhibition of VEGF-A and VEGFR1 expression by tumor cells, (3) inhibition of tumor-induced angiogenesis, and (4) direct inhibitory actions on endothelial cells. Published by Elsevier Inc.

Paracrine-mediated osteoclastogenesis by the osteosarcoma MG63 cell line: is RANKL/RANK signalling really important?

PubMed

Costa-Rodrigues, J; Teixeira, C A; Fernandes, M H

2011-08-01

Although in the past little attention has been paid to the influence of osteosarcoma cells in osteoclast function, recent studies suggest a close relationship between osteosarcoma aggressiveness and osteoclastic activity. The present study addresses the paracrine effects of MG63 cells, a human osteosarcoma-derived cell line, on the differentiation of peripheral blood osteoclast precursor cells (PBMC). PBMC were cultured for 21 days in the presence of conditioned media from MG63 cell cultures (CM) collected at 48 h (CM_MG1), 7 days (CM_MG2) and 14 days (CM_MG3). MG63 cell cultures displayed the expression of ALP and BMP-2 and, also, the osteoclastogenic genes M-CSF and RANKL, although with a low expression of RANKL. PBMC cultures supplemented with CM presented an evident osteoclastogenic behavior, which was dependent on the culture period of the MG63 cells. The inductive effect appeared to be more relevant for the differentiation and activation genes, c-myc and c-src, and lower for genes associated with osteoclast function. In addition, PBMC cultures displayed increased functional parameters, including calcium phosphate resorbing activity. Assessment of the PBMC cultures in the presence of U0126, PDTC, and indomethacin suggested that in addition to MEK and NFkB pathways, other signaling mechanisms, probably not involving RANKL/RANK interaction, might be activated in the presence of conditioned medium from MG63. In conclusion, MG63 cell line appears to induce a significant paracrine-mediated osteoclastogenic response. Understanding the mechanisms underlying the interaction of osteosarcoma cells and osteoclasts may contribute to the development of new potential approaches in the treatment of such bone metabolic diseases.

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin.

PubMed

Wan, Yong-Jie; Zhang, Yan-Li; Zhou, Zheng-Rong; Jia, Ruo-Xin; Li, Meng; Song, Hui; Wang, Zi-Yu; Wang, Li-Zhong; Zhang, Guo-Min; You, Ji-Hao; Wang, Feng

2012-08-01

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT. Copyright © 2012 Elsevier Inc. All rights reserved.

Secretion of cytokines in breast cancer cells: the molecular mechanism of procathepsin D proliferative effects.

PubMed

Fusek, Martin; Vetvickova, Jana; Vetvicka, Vaclav

2007-03-01

Procathepsin D (pCD) is a major secreted protein in estrogen receptor-positive (ER+) breast cancer cell lines. Several independent studies have documented pronounced mitogenic effect of secreted pCD on cancer tissue-derived cell lines, including those from breast, lung, and prostate cancer. It has also been shown that the proliferative effect of pCD involves both autocrine and paracrine modes of action. Recent studies have suggested that pCD could act as a key paracrine communicator between cancer and stromal cells. We have shown earlier that the proliferative activity of pCD depends on the activation peptide sequence of pCD. The present study casts light on the mechanism by which pCD influences the proliferation of cancer cells expressing the ER. Results described in the current paper clearly show that pCD initiates secretion of cytokines interleukin-4 (IL-4), IL-8, IL-10, IL-13, macrophage inflammatory protein-1beta and (MIP-1beta) from such tumor cells. Secreted cytokines take part in the proliferation of the cancer cells, as proven by selective inhibition using antibodies. In addition, expression of cytokine receptors on tested cell lines corresponded to the effects of individual cytokines. An analogous pattern was also observed for fibroblasts, which, under physiologic conditions, are the cells in closest contact with the tumor tissue and play a role in tumor growth and invasion. Our observations were further supported by coculture experiments that are in agreement. Although very similar in response to addition of pCD, the invasive ER- cells do not secrete cytokines. Together with previous in vivo results, these data point to pCD as one of key molecules for therapeutic attack in breast cancer.

Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells.

PubMed

Ting, Sherwin; Lecina, Marti; Chan, Yau-Chi; Tse, Hung Fat; Reuveny, Shaul; Oh, Steve Kw

2013-07-26

To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells. Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction. hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar. The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.

Anti-cancer Effect of Luminacin, a Marine Microbial Extract, in Head and Neck Squamous Cell Carcinoma Progression via Autophagic Cell Death.

PubMed

Shin, Yoo Seob; Cha, Hyun Young; Lee, Bok-Soon; Kang, Sung Un; Hwang, Hye Sook; Kwon, Hak Cheol; Kim, Chul-Ho; Choi, Eun Chang

2016-04-01

The purpose of this study is to determine whether luminacin, a marine microbial extract from the Streptomyces species, has anti-tumor effects on head and neck squamous cell carcinoma (HNSCC) cell lines via autophagic cell death. Inhibition of cell survival and increased cell death was measured using cell viability, colony forming, and apoptosis assays. Migration and invasion abilities of head and cancer cells were evaluated using wound healing, scattering, and invasion assays. Changes in the signal pathway related to autophagic cell death were investigated. Drug toxicity of luminacin was examined in in vitro HaCaT cells and an in vivo zebrafish model. Luminacin showed potent cytotoxicity in HNSCC cells in cell viability, colony forming, and fluorescence-activated cell sorting analysis. In vitro migration and invasion of HNSCC cells were attenuated by luminacin treatment. Combined with Beclin-1 and LC3B, Luminacin induced autophagic cell death in head and neck cancer cells. In addition, in a zebrafish model and human keratinocyte cell line used for toxicity testing, luminacin treatment with a cytotoxic concentration to HNSCC cells did not cause toxicity. Taken together, these results demonstrate that luminacin induces the inhibition of growth and cancer progression via autophagic cell death in HNSCC cell lines, indicating a possible alternative chemotherapeutic approach for treatment of HNSCC.

ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro.

PubMed

Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Huijie; Yuan, Yuan; Ji, Zhenyu; Qiao, Baoping; Wen, Jian-Guo; Zhang, Mingzhi; Kvalheim, Gunnar; Nesland, Jahn M; Suo, Zhenhe

2015-12-15

Cancer stem cells (CSCs) are associated with cancer recurrence and metastasis. Prostate cancer cells often metastasize to the bone with a complex microenvironment of cytokines favoring cell survival. In this study, the cell stemness influence of a group of interleukins including IL-3, 6, 10, 11 and 24 on human prostate cancer cell lines LNCaP and PC-3 was explored in vitro. Sulforhodamine B(SRB) and 5-ethynyl-2'-deoxyuridine (EdU) assays were applied to examine the effect on cell proliferation, and wound healing and transwell assays were used for migration and invasion studies, in addition to colony formation, Western blotting and flowcytometry for the expression of stemness factors and chemotherapy sensitivity. We observed that ILs-3, 6 and 11 stimulated while ILs-10 and 24 inhibited the growth, invasion and migration of both cell lines. Interestingly, ILs-3, 6 and 11 significantly promoted colony formation and increased the expression of SOX2, CD44 and ABCG2 in both prostate cancer cell lines. However, ILs-10 and 24 showed the opposite effect on the expression of these factors. In line with the above findings, treatment with either IL-3 or IL-6 or IL-11 decreased the chemosensitivity to docetaxel while treatment with either IL-10 or IL-24 increased the sensitivity of docetaxel chemotherapy. In conclusion, our results suggest that ILs-3, 6 and 11 function as tumor promoters while ILs-10 and 24 function as tumor suppressors in the prostate cancer cell lines PC-3 and LNCaP in vitro, and such differences may attribute to their different effect on the stemness of PCa cells.

Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models.

PubMed

Baker, Amanda F; Hanke, Neale T; Sands, Barbara J; Carbajal, Liliana; Anderl, Janet L; Garland, Linda L

2014-12-31

Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models. A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ. CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC50 values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC50 values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP). CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell.

PubMed

Hu, Zhilan; Guo, Donglin; Yip, Shirley S M; Zhan, Dejin; Misaghi, Shahram; Joly, John C; Snedecor, Bradley R; Shen, Amy Y

2013-01-01

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR-deficient DG44, and DUXB11-based DHFR deficient CHO. Current Genentech commercial full-length antibody products have all been produced in the DUXB11-derived DHFR-deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11-derived DHFR-deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11-based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14-day fed batch cultures in shake flasks. In contrast, the DUXB11-based host produced ∼0.1 g/l for both antibodies in the same 14-day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ∼2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers.

ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro

PubMed Central

Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Huijie; Yuan, Yuan; Ji, Zhenyu; Qiao, Baoping; Wen, Jian-Guo; Zhang, Mingzhi; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2015-01-01

Cancer stem cells (CSCs) are associated with cancer recurrence and metastasis. Prostate cancer cells often metastasize to the bone with a complex microenvironment of cytokines favoring cell survival. In this study, the cell stemness influence of a group of interleukins including IL-3, 6, 10, 11 and 24 on human prostate cancer cell lines LNCaP and PC-3 was explored in vitro. Sulforhodamine B(SRB) and 5-ethynyl-2′-deoxyuridine (EdU) assays were applied to examine the effect on cell proliferation, and wound healing and transwell assays were used for migration and invasion studies, in addition to colony formation, Western blotting and flowcytometry for the expression of stemness factors and chemotherapy sensitivity. We observed that ILs-3, 6 and 11 stimulated while ILs-10 and 24 inhibited the growth, invasion and migration of both cell lines. Interestingly, ILs-3, 6 and 11 significantly promoted colony formation and increased the expression of SOX2, CD44 and ABCG2 in both prostate cancer cell lines. However, ILs-10 and 24 showed the opposite effect on the expression of these factors. In line with the above findings, treatment with either IL-3 or IL-6 or IL-11 decreased the chemosensitivity to docetaxel while treatment with either IL-10 or IL-24 increased the sensitivity of docetaxel chemotherapy. In conclusion, our results suggest that ILs-3, 6 and 11 function as tumor promoters while ILs-10 and 24 function as tumor suppressors in the prostate cancer cell lines PC-3 and LNCaP in vitro, and such differences may attribute to their different effect on the stemness of PCa cells. PMID:26528857

Tectonic-1 contributes to the growth and migration of prostate cancer cells in vitro

PubMed Central

WANG, ZHIJUN; GAO, YI; LIU, YUSHAN; CHEN, JIE; WANG, JUNKAI; GAN, SISHUN; XU, DANFENG; CUI, XINGANG

2015-01-01

Tectonic-1 (TCTN1) is an upstream gene involved in embryonic development. The aim of the present study was to investigate the effect of the TCTN1 gene on the viability and migration of prostate cancer cells. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of TCTN1 in PC-3 and DU145 prostate cancer cells. Cell viability and proliferation were measured using MTT and colony formation assays, and the distribution of cells in phases of the cell cycle was determined using flow cytometry. Cell migration was detected using a Transwell assay. The results demonstrated that TCTN1 was widely expressed in several human prostate cancer cell lines. Knockdown of the TCTN1 gene by RNA interference markedly suppressed cell viability and colony formation in the PC-3 and DU145 cell lines. Cell cycle progression was also arrested by TCTN1 silencing. In addition, knockdown of the TCTN1 gene led to the inhibition of cell migration in the two cell lines. These findings confirmed the direct association between the TCTN1 gene and prostate cancer growth in vitro. With further understanding and clinical investigation, this indicates the potential for future development of a novel marker for early detection and gene therapy for prostate cancer. PMID:26310786

Dacarbazine and the Agonistic TRAIL Receptor-2 Antibody Lexatumumab Induce Synergistic Anticancer Effects in Melanoma

PubMed Central

Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

2012-01-01

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients. PMID:23029050

Dacarbazine and the agonistic TRAIL receptor-2 antibody lexatumumab induce synergistic anticancer effects in melanoma.

PubMed

Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

2012-01-01

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients.

Establishment and genetic characterization of ANGM-CSS, a novel, immortal cell line derived from a human glioblastoma multiforme.

PubMed

Notarangelo, Angelantonio; Trombetta, Domenico; D'Angelo, Vincenzo; Parrella, Paola; Palumbo, Orazio; Storlazzi, Clelia Tiziana; Impera, Luciana; Muscarella, Lucia Anna; La Torre, Antonella; Affuso, Andrea; Fazio, Vito Michele; Carella, Massimo; Zelante, Leopoldo

2014-03-01

Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells

PubMed Central

Gu, Ha Ra; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-01-01

Background/Aims Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Methods Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Results Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Conclusions Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future. PMID:25834802

Combined treatment with silibinin and either sorafenib or gefitinib enhances their growth-inhibiting effects in hepatocellular carcinoma cells.

PubMed

Gu, Ha Ra; Park, Su Cheol; Choi, Su Jin; Lee, Jae Cheol; Kim, You Cheoul; Han, Chul Ju; Kim, Jin; Yang, Ki Young; Kim, Yeon Joo; Noh, Geum Youb; No, So Hyeon; Jeong, Jae-Hoon

2015-03-01

Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.

Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts

PubMed Central

He, Yingzi; Cai, Chengfu; Tang, Dongmei; Sun, Shan; Li, Huawei

2014-01-01

In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21Cip1 and p27Kip1 expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line. PMID:25431550

IGF-1R/MDM2 relationship confers enhanced sensitivity to RITA in Ewing sarcoma cells.

PubMed

Di Conza, Giusy; Buttarelli, Marianna; Monti, Olimpia; Pellegrino, Marsha; Mancini, Francesca; Pontecorvi, Alfredo; Scotlandi, Katia; Moretti, Fabiola

2012-06-01

Ewing sarcoma is one of the most frequent bone cancers in adolescence. Although multidisciplinary therapy has improved the survival rate for localized tumors, a critical step is the development of new drugs to improve the long-term outcome of recurrent and metastatic disease and to reduce side effects of conventional therapy. Here, we show that the small molecule reactivation of p53 and induction of tumor cell apoptosis (RITA, NSC652287) is highly effective in reducing growth and tumorigenic potential of Ewing sarcoma cell lines. These effects occur both in the presence of wt-p53 as well as of mutant or truncated forms of p53, or in its absence, suggesting the presence of additional targets in this tumor histotype. Further experiments provided evidence that RITA modulates an important oncogenic mark of these cell lines, insulin-like growth factor receptor 1 (IGF-1R). Particularly, RITA causes downregulation of IGF-1R protein levels. MDM2 degradative activity is involved in this phenomenon. Indeed, inhibition of MDM2 function by genetic or pharmacologic approaches reduces RITA sensitivity of Ewing sarcoma cell lines. Overall, these data suggest that in the cell context of Ewing sarcoma, RITA may adopt additional mechanism of action besides targeting p53, expanding its field of application. Noteworthy, these results envisage the promising utilization of RITA or its derivative as a potential treatment for Ewing sarcomas. ©2012 AACR

Tight Junction–Associated Signaling Pathways Modulate Cell Proliferation in Uveal Melanoma

PubMed Central

Jayagopal, Ashwath; Yang, Jin-Long; Haselton, Frederick R.; Chang, Min S.

2011-01-01

Purpose. To investigate the role of tight junction (TJ)–associated signaling pathways in the proliferation of uveal melanoma. Methods. Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1–associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways. Results. Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity. Conclusions. TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma. PMID:20861479

Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

NASA Astrophysics Data System (ADS)

Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

2016-11-01

In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Phenformin inhibits cell proliferation and induces cell apoptosis and autophagy in cholangiocarcinoma.

PubMed

Hu, Shuyang; Ouyang, Qing; Cheng, Qingbao; Wang, Jinghan; Feng, Feiling; Qiao, Liang; Gan, Wei; Shi, Yang; Wu, Demin; Jiang, Xiaoqing

2018-04-01

Cholangiocarcinoma (CCA) is an aggressive malignant tumor and the prognosis of patients with advanced stage disease remains poor. Therefore, the identification of novel treatment agents for CCA is required. In the present study, the biological effects of the diabetes therapeutic agent, phenformin, in CCA cell lines was investigated. Cell Counting Kit‑8 cell viability, cellular clone formation and subcutaneous tumor formation assays were performed, which revealed that phenformin inhibited CCA cell proliferation and growth both in vitro and in vivo. In addition, phenformin induced CCA cell apoptosis and autophagy. Phenformin partly activated the liver kinase B1 (LKB1)/5' AMP‑activated protein kinase signaling pathway to exert its biological effects on CCA cell lines, as demonstrated by knockdown of LKB1, which reversed these effects. In conclusion, the present study demonstrated the biological effects of phenformin in CCA and suggested that phenformin may be a potential novel agent for CCA treatment.

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

Beyond the Mouse Monopoly: Studying the Male Germ Line in Domestic Animal Models

PubMed Central

González, Raquel; Dobrinski, Ina

2015-01-01

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals. PMID:25991701

ONYX-411, a conditionally replicative oncolytic adenovirus, induces cell death in anaplastic thyroid carcinoma cell lines and suppresses the growth of xenograft tumors in nude mice

PubMed Central

Reddi, HV; Madde, P; Reichert-Eberhardt, AJ; Galanis, EC; Copland, JA; McIver, B; Grebe, SKG; Eberhardt, NL

2011-01-01

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer variant, accounting for 1–2% of all cases, but 33% of deaths, and exhibiting an average life expectancy of 5 months. ATC is largely unresponsive to radioactive iodine, chemotherapy, external beam radiation or surgery, underscoring the need for new and effective therapies. We evaluated the therapeutic potential of an oncolytic adenovirus, ONYX-411, that replicates selectively in and kills cells with dysfunction of the retinoblastoma (RB) pathway. In the present study, we report that ONYX-411 is able to induce cell death in eight human anaplastic carcinoma cell lines in vitro. The cytopathic effect of the virus is specific to cells with RB dysfunction, which appears to be frequent in ATC. We confirmed the expression of the coxsackie adenovirus receptor, CAR, in all ATC cell lines, demonstrating the potentially universal application of this oncolytic viral therapy to ATC. In addition, the growth of xenograft tumors induced in athymic mice with the ARO and DRO cell lines was significantly reduced by ONYX-411 treatment. These results indicate that ONYX-411 can be a potential therapeutic agent for the treatment of ATC, rendering this class of conditionally replicating adenoviruses an attractive candidate for clinical trials. PMID:18583996

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell population and proteomic modifications.

PubMed

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB.

Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

PubMed Central

Zanini, Cristina; Ercole, Elisabetta; Mandili, Giorgia; Salaroli, Roberta; Poli, Alice; Renna, Cristiano; Papa, Valentina; Cenacchi, Giovanna; Forni, Marco

2013-01-01

Background Medulloblastoma (MB) is an aggressive pediatric tumor of the Central Nervous System (CNS) usually treated according to a refined risk stratification. The study of cancer stem cells (CSC) in MB is a promising approach aimed at finding new treatment strategies. Methodology/Principal Findings The CSC compartment was studied in three characterized MB cell lines (DAOY, UW228 and ONS-76) grown in standard adhesion as well as being grown as spheres, which enables expansion of the CSC population. MB cell lines, grown in adherence and as spheres, were subjected to morphologic analysis at the light and electron microscopic level, as well as cytofluorimetric determinations. Medullospheres (MBS) were shown to express increasingly immature features, along with the stem cells markers: CD133, Nestin and β-catenin. Proteomic analysis highlighted the differences between MB cell lines, demonstrating a unique protein profile for each cell line, and minor differences when grown as spheres. In MBS, MALDI-TOF also identified some proteins, that have been linked to tumor progression and resistance, such as Nucleophosmin (NPM). In addition, immunocytochemistry detected Sox-2 as a stemness marker of MBS, as well as confirming high NPM expression. Conclusions/Significance Culture conditioning based on low attachment flasks and specialized medium may provide new data on the staminal compartment of CNS tumors, although a proteomic profile of CSC is still elusive for MB. PMID:23717474

The myotoxic effect of bupivacaine and ropivacaine on myotubes in primary mouse cell culture and an immortalized cell line.

PubMed

Hofmann, Petra; Metterlein, Thomas; Bollwein, Gabriele; Gruber, Michael; Plank, Christoph; Graf, Bernhard M; Zink, Wolfgang

2013-09-01

The 2 local anesthetics (LAs) bupivacaine and ropivacaine have acute cytotoxic effects on different tissues. In this respect, LA-induced myotoxicity has been subject to various studies; however, the exact mechanisms are still not fully understood. Most in vitro studies use immortalized cell lines because of feasibility. Thus, establishing a primary cell line might result in more accurate results. In this study, we examined the effects of immortalization on bupivacaine- and ropivacaine-induced myotoxicity in vitro. An immortalized (N = 6) and a primary cell line (N = 8) of the same tissue and species were established, and differentiation in myotubes was induced. Cells were exposed to increasing concentrations of bupivacaine and ropivacaine for 1 or 2 hours, respectively. Twenty-four and 48 hours after treatment, the fractions of dead and vital cells were measured using flow cytometry. Significance was tested through 1-way analysis of variance with post hoc Dunnett T3 test. Medians of dataset pairs were compared by T test. In both cell lines, increasing concentrations of both LAs resulted in decreased cell survival (e.g., P < 0.001 for 5000 ppm bupivacaine, 1 or 2 hours of incubation, and 24 hours recovery in both cell lines). For the same LA concentrations, survival was significantly higher in the immortalized cell culture (e.g., P < 0.001 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). In addition, equal concentrations of bupivacaine resulted in significantly fewer vital cells compared with ropivacaine (e.g., P = 0.032 for 2500 ppm ropivacaine, 1 hour of incubation, and 24 hours recovery). Two hours of incubation resulted in a significantly higher rate of dead cells compared with 1 hour of incubation (e.g., P = 0.004 for C2C12 cells, 2500 ppm bupivacaine, and 24 hours recovery). Primary skeletal muscle cells are more vulnerable to LAs than immortalized cells. The higher myotoxic potential of bupivacaine compared with ropivacaine in vivo can be reproduced in vitro. Incubation time has an influence on cell survival.

Genistein enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nuclear factor kappa B in nonsmall cell lung cancer cell lines.

PubMed

Gadgeel, Shirish M; Ali, Shadan; Philip, Philip A; Wozniak, Antoinette; Sarkar, Fazlul H

2009-05-15

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown modest clinical benefit in patients with relapsed nonsmall cell lung cancer (NSCLC). Down-regulation of Akt appears to correlate with the antitumor activity of EGFR-TKIs. Akt activates nuclear factor kappa B (NF-kappaB), which transcribes genes important for cell survival, invasion, and metastasis. The authors hypothesized that genistein, through the inhibition of NF-kappaB, could enhance the activity of EGFR-TKIs in NSCLCs. Three NSCLC cell lines with various EGFR mutation status and sensitivities to EGFR-TKIs were selected: H3255 (L858R), H1650 (del E746-A750), and H1781 (wild-type EGFR). Cells were treated with erlotinib, gefitinib, genistein, or the combination of each of the EGFR-TKIs with genistein. Cell survival and apoptosis were assessed, and expression levels of EGFR, pAkt, cyclooxygenase-2 (COX-2), E-cadherin, prostaglandin E(2) (PGE(2)), and NF-kappaB were measured. Both EGFR-TKIs demonstrated growth inhibition and apoptosis in each of the cell lines, but H1650 and H1781 were much less sensitive. Genistein demonstrated some antitumor activity in all cell lines, but enhanced growth inhibition and apoptosis when combined with the EGFR-TKIs in each of the cell lines. Both combinations down-regulated NF-kappaB significantly more than either agent alone in H3255. In addition, the combinations reduced the expression of EGFR, pAkt, COX-2, and PGE(2,) consistent with inactivation of NF-kappaB. The authors concluded that genistein enhances the antitumor effects of EGFR-TKIs in 3 separate NSCLC cell lines. This enhanced activity is in part because of greater reduction in the DNA-binding activity of NF-kappaB when EGFR-TKIs were combined with genistein.

Inactivated Tianjin strain, a novel genotype of Sendai virus, induces apoptosis in HeLa, NCI-H446 and Hep3B cells.

PubMed

Chen, Jun; Han, Han; Wang, Bin; Shi, Liying

2016-07-01

The Sendai virus strain Tianjin is a novel genotype of the Sendai virus. In previous studies, ultraviolet-inactivated Sendai virus strain Tianjin (UV-Tianjin) demonstrated antitumor effects on human breast cancer cells. The aim of the present study was to investigate the in vitro antitumor effects of UV-Tianjin on the human cervical carcinoma HeLa, human small cell lung cancer NCI-H446 and human hepatocellular carcinoma Hep 3B cell lines, and the possible underlying mechanisms of these antitumor effects. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay revealed that UV-Tianjin treatment inhibited the proliferation of HeLa, NCI-H446 and Hep 3B cells in a dose- and time-dependent manner. Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide double staining indicated that UV-Tianjin induced dose-dependent apoptosis in all three cell lines with the most significant effect observed in the HeLa cell line. In the HeLa cell line, UV-Tianjin-induced apoptosis was further confirmed by the disruption of the mitochondria membrane potential and the activation of caspases, as demonstrated by fluorescent cationic dye and colorimetric assays, respectively. In addition, western blot analysis revealed that UV-Tianjin treatment resulted in significant upregulation of cytochrome c , apoptosis protease activating factor-1, Fas, Fas ligand and Fas-associated protein with death domain, and activated caspase-9, -8 and -3 in HeLa cells. Based on these results, it is hypothesized that UV-Tianjin exhibits anticancer activity in HeLa, NCI-H446 and Hep 3B cell lines via the induction of apoptosis. In conclusion, the results of the present study indicate that in the HeLa cell line, intrinsic and extrinsic apoptotic pathways may be involved in UV-Tianjin-induced apoptosis.

Chronophin regulates active vitamin B6 levels and transcriptomic features of glioblastoma cell lines cultured under non-adherent, serum-free conditions.

PubMed

Schulze, Markus; Hutterer, Maria; Sabo, Anja; Hoja, Sabine; Lorenz, Julia; Rothhammer-Hampl, Tanja; Herold-Mende, Christel; Floßbach, Lucia; Monoranu, Camelia; Riemenschneider, Markus J

2018-05-03

The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

PubMed Central

1984-01-01

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts. PMID:6538202

Artepillin C induces selective oxidative stress and inhibits migration and invasion in a comprehensive panel of human cervical cancer cell lines.

PubMed

Souza, Raquel Pantarotto; de Souza Bonfim-Mendonca, Patricia; Damke, Gabrielle Marconi Zago Ferreira; De Assis Carvalho, Analine Rosa Barquez; Ratti, Bianca Altrao; de Oliveira Dembogurski, Djaceli Sampaio; da Silva, Vania Ramos Sela; Silva, Sueli Oliveira; da Silva, Denise Brentan; Bruschi, Marcos Luciano; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

2018-06-03

Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is the main bioactive component of Brazilian green propolis, and possesses, among other things, anticancer properties. However, to the best of our knowledge, there are no studies of artepillin C in cervical cancer. To explore a new therapeutic candidate for cervical cancer, we have evaluated the effects of artepillin C on cellular viability in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16- and 18-positive) and C33A (HPV-negative) cells compared to a spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that artepillin C had a selective effect on cellular viability and could induce apoptosis possibly by intrinsic pathway, likely a result of oxidative stress, in all cancer-derived cell lines but not in HaCaT. Additionally, artepillin C was able to inhibit the migration and invasion of cancer cells. Thus, artepillin C appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV types. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Chondracanthus tenellus (Harvey) hommersand extract protects the human keratinocyte cell line by blocking free radicals and UVB radiation-induced cell damage.

PubMed

Piao, Mei Jing; Hyun, Yu Jae; Oh, Tae-Heon; Kang, Hee Kyoung; Yoo, Eun Sook; Koh, Young Sang; Lee, Nam Ho; Suh, In Soo; Hyun, Jin Won

2012-12-01

The aim of this study was to investigate the protective effects of the ethanol extract of the red algae Chondracanthus tenellus (Harvey) Hommersand (CTE) on cultured human keratinocyte cell line. The cellular protection conferred by CTE was evidenced by the ability of the extract to absorb ultraviolet B (UVB; 280-320 nm) and to scavenge the radical 1,1-diphenyl-2-picrylhydrazyl, as well as intracellular reactive oxygen species (ROS), induced by either hydrogen peroxide (H(2)O(2)) or UVB radiation. In addition, both superoxide anion generated by the xanthine/xanthine oxidase system and hydroxyl radical generated by the Fenton reaction (FeSO(4) + H(2)O(2)) were scavenged by CTE, as confirmed using electron spin resonance spectrometry. In the human keratinocyte cell line, CTE decreased the degree of injury resulting from UVB-induced oxidative stress to lipids, proteins, and DNA. CTE-treated cells also showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies and less DNA fragmentation. Taken together, these results suggest that CTE confers protection on the human keratinocyte cell line against UVB-induced oxidative stress by absorbing UVB ray and scavenging ROS, thereby reducing injury to cellular constituents.

Biomarkers in Tumorigenesis Using Cancer Cell Lines: A Systematic Review

PubMed Central

K, Lizbeth Raju; Augustine, Dominic; Rao, Roopa S; SV, Sowmya; Haragannavar, Vanishri C; Nambiar, Shwetha; Prasad, Kavitha; Awan, Kamran Habib; Patil, Shankargouda

2017-01-01

Cancer is a leading cause of death worldwide. Despite many research advancements in the field, the genetic changes regulating the transformation of normal oral cells into malignant cells have not been fully elucidated. Several studies have evaluated carcinogenesis at the molecular level. Cancer cell lines are commonly used in biomedical research because they provide an unlimited source of cells and represent various stages of initiation and progression of carcinogenesis in vitro. Aims: The objective of the study was to review original research articles using cancer cell lines as a tool to understand carcinogenesis and to identify the genes involved in tumor development. Additionally, we also examined the application of the genes as predictive biomarkers. Methods and Materials: Several databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 1985 to December 2016 using various combinations of the following key words: “mouth neoplasm”, “cell lines”, and “tumorigenesis”. Original experimental studies published in English were included. We excluded letters to the editor, historic reviews, and unpublished data from the analysis. Results: There were 17 studies (in vitro) included in the analysis. There were 14 genes and 4 miRNAs involved in malignant transformation of oral keratinocytes into cancer cells. The most commonly studied genes were p53, cyclin D1, and hTERT. Conclusion: Additional reviews and studies are needed to identify a panel of genes specific to various potentially malignant disorders and to aid in the early detection of oral squamous cell carcinoma (OSCC) because tumorigenesis involves the mutation of multiple genes. Furthermore, improving advanced cost-effective diagnostic methods may benefit the public health sector. PMID:28950674

The BET bromodomain inhibitor CPI203 improves lenalidomide and dexamethasone activity in in vitro and in vivo models of multiple myeloma by blockade of Ikaros and MYC signaling

PubMed Central

Díaz, Tania; Rodríguez, Vanina; Lozano, Ester; Mena, Mari-Pau; Calderón, Marcos; Rosiñol, Laura; Martínez, Antonio; Tovar, Natalia; Pérez-Galán, Patricia; Bladé, Joan; Roué, Gaël; de Larrea, Carlos Fernández

2017-01-01

Most patients with multiple myeloma treated with current therapies, including immunomodulatory drugs, eventually develop relapsed/refractory disease. Clinical activity of lenalidomide relies on degradation of Ikaros and the consequent reduction in IRF4 expression, both required for myeloma cell survival and involved in the regulation of MYC transcription. Thus, we sought to determine the combinational effect of an MYC-interfering therapy with lenalidomide/dexamethasone. We analyzed the potential therapeutic effect of the combination of the BET bromodomain inhibitor CPI203 with the lenalidomide/dexamethasone regimen in myeloma cell lines. CPI203 exerted a dose-dependent cell growth inhibition in cell lines, indeed in lenalidomide/dexamethasone-resistant cells (median response at 0.5 μM: 65.4%), characterized by G1 cell cycle blockade and a concomitant inhibition of MYC and Ikaros signaling. These effects were potentiated by the addition of lenalidomide/dexamethasone. Results were validated in primary plasma cells from patients with multiple myeloma co-cultured with the mesenchymal stromal cell line stromaNKtert. Consistently, the drug combination evoked a 50% reduction in cell proliferation and correlated with basal Ikaros mRNA expression levels (P=0.04). Finally, in a SCID mouse xenotransplant model of myeloma, addition of CPI203 to lenalidomide/dexamethasone decreased tumor burden, evidenced by a lower glucose uptake and increase in the growth arrest marker GADD45B, with simultaneous downregulation of key transcription factors such as MYC, Ikaros and IRF4. Taken together, our data show that the combination of a BET bromodomain inhibitor with a lenalidomide-based regimen may represent a therapeutic approach to improve the response in relapsed/refractory patients with multiple myeloma, even in cases with suboptimal prior response to immunomodulatory drugs. PMID:28751557

Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

PubMed

Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

2015-10-15

Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling. Copyright © 2015 the American Physiological Society.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

PubMed

Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

2016-06-01

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

PubMed

Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

2016-01-01

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

PI3K and Inhibitor of Apoptosis Proteins Modulate Gentamicin- Induced Hair Cell Death in the Zebrafish Lateral Line

PubMed Central

Wiedenhoft, Heather; Hayashi, Lauren; Coffin, Allison B.

2017-01-01

Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure. PMID:29093665

Untangling the mechanism of 3-methyladenine (3-MA) in enhancing the specific productivity: Transcriptome analysis of recombinant CHO cells treated with 3-MA.

PubMed

Baek, Eric; Lee, Jae Seong; Lee, Gyun Min

2018-06-25

3-Methyladenine (3-MA) is a chemical additive that enhances the specific productivity (q p ) in recombinant Chinese hamster ovary (rCHO) cell lines. Different from its widely known function of inhibiting autophagy, 3-MA has instead shown to increase autophagic flux in various rCHO cell lines. Thus, the mechanism by which 3-MA enhances the q p requires investigation. To evaluate the effect of 3-MA on transcriptome dynamics in rCHO cells, RNA-seq was performed with Fc-fusion protein-producing rCHO cells treated with 3-MA. By analyzing genes that were differentially expressed following the addition of 3-MA during culture, the role of 3-MA in the biological processes of rCHO cells was identified. One pathway markedly influenced by the addition of 3-MA was the unfolded protein response (UPR). Having a close relationship with autophagy, the UPR reestablishes protein folding homeostasis under endoplasmic reticulum (ER) stress. The addition of 3-MA increased the expression of key regulators of the UPR, such as Atf4, Ddit3, and Creb3l3, further supporting the idea that the enhancement of ER capacity acts as a key in increasing the q p . Consequently, the downstream effectors of UPR, which include autophagy-promoting genes, were upregulated as well. Hence, the role of 3-MA in increasing UPR pathway could have made a salient contribution to the increased autophagic flux in rCHO cells. Taken together, transcriptome analysis improved the understanding of the role of 3-MA in gene expression dynamics in rCHO cells and its mechanism in enhancing the q p . This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

PubMed

Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

2014-03-30

Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

Antitumor Effects and Mechanism of Novel Emodin Rhamnoside Derivatives against Human Cancer Cells In Vitro

PubMed Central

Deng, Jun-peng; Jiang, Ling-zhi; Xiong, Ping; Yang, Bin-jie; Liu, Shan-shan

2015-01-01

A series of novel anthracene L-rhamnopyranosides compounds were designed and synthesized and their anti-proliferative activities on cancer cell lines were investigated. We found that one derivative S-8 (EM-d-Rha) strongly inhibited cell proliferation of a panel of different human cancer cell lines including A549, HepG2, OVCAR-3, HeLa and K562 and SGC-790 cell lines, and displayed IC50 values in low micro-molar ranges, which are ten folds more effective than emodin. In addition, we found EM-d-Rha (3-(2”,3”-Di-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2’,3’-di-O-acetyl-α-L-rhamnopyranosyl)-emodin) substantially induced cellular apoptosis of HepG2 and OVCAR-3 cells in the early growth stage. Furthermore, EM-d-Rha led to the decrease of mitochondrial transmembrane potential, and up-regulated the express of cells apoptosis factors in a concentration- and time-dependent manner. The results indicated the EM-d-Rha may inhibit the growth and proliferation of HepG2 cells through the pathway of apoptosis induction, and the possible molecular mechanism may due to the activation of intrinsic apoptotic signal pathway. PMID:26682731

Differential Expression of Ccn4 and Other Genes Between Metastatic and Non-metastatic EL4 Mouse Lymphoma Cells

PubMed Central

S. CHAHAL, MANPREET; TERESA KU, H.; ZHANG, ZHIHONG; M. LEGASPI, CHRISTIAN; LUO, ANGELA; M. HOPKINS, MANDI; E. MEIER, KATHRYN

2016-01-01

Background: Previous work characterized variants of the EL4 murine lymphoma cell line. Some are non-metastatic, and others metastatic, in syngenic mice. In addition, metastatic EL4 cells were stably transfected with phospholipase D2 (PLD2), which further enhanced metastasis. Materials and Methods: Microarray analyses of mRNA expression was performed for non-metastatic, metastatic, and PLD2-expressing metastatic EL4 cells. Results: Many differences were observed between non-metastatic and metastatic cell lines. One of the most striking new findings was up-regulation of mRNA for the matricellular protein WNT1-inducible signaling pathway protein 1 (CCN4) in metastatic cells; increased protein expression was verified by immunoblotting and immunocytochemistry. Other differentially expressed genes included those for reproductive homeobox 5 (Rhox5; increased in metastatic) and cystatin 7 (Cst7; decreased in metastatic). Differences between PLD2-expressing and parental cell lines were limited but included the signaling proteins Ras guanyl releasing protein 1 (RGS18; increased with PLD2) and suppressor of cytokine signaling 2 (SOCS2; decreased with PLD2). Conclusion: The results provide insights into signaling pathways potentially involved in conferring metastatic ability on lymphoma cells. PMID:27807066

Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents.

PubMed

Tokala, Ramya; Bale, Swarna; Janrao, Ingle Pavan; Vennela, Aluri; Kumar, Niggula Praveen; Senwar, Kishna Ram; Godugu, Chandraiah; Shankaraiah, Nagula

2018-06-01

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC 50 value of 7.22 ± 0.47 µM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile. Copyright © 2018 Elsevier Ltd. All rights reserved.

THz spectroscopy of whole blood, plasma and cells in mice of SHR line with various pathology

NASA Astrophysics Data System (ADS)

Panchenko, A.; Tyndyk, M.; Smolyanskaya, O.; Sulatskiy, M.; Kravtsenyuk, O.; Balbekin, N.; Khodzitsky, M.

2016-08-01

This paper is devoted to studying of optical properties of whole blood and blood plasma in SHR mice grafted Ehrlich's carcinoma and mice with chronic inflammation at the terahertz frequency range. Additionally physiological saline solution suspension of ascites Ehrlich's carcinoma cells was explored.

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

PubMed

Portella, G; Vitagliano, D; Li, Z; Sferratore, F; Santoro, M; Vecchio, G; Fusco, A

1998-01-01

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Inhibiting DNA-PK{sub CS} radiosensitizes human osteosarcoma cells

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

Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK{sub CS}), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK{sub CS} in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK{sub CS} inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK{submore » CS} was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK{sub CS} inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.« less

Construction and analysis of a modular model of caspase activation in apoptosis

PubMed Central

Harrington, Heather A; Ho, Kenneth L; Ghosh, Samik; Tung, KC

2008-01-01

Background A key physiological mechanism employed by multicellular organisms is apoptosis, or programmed cell death. Apoptosis is triggered by the activation of caspases in response to both extracellular (extrinsic) and intracellular (intrinsic) signals. The extrinsic and intrinsic pathways are characterized by the formation of the death-inducing signaling complex (DISC) and the apoptosome, respectively; both the DISC and the apoptosome are oligomers with complex formation dynamics. Additionally, the extrinsic and intrinsic pathways are coupled through the mitochondrial apoptosis-induced channel via the Bcl-2 family of proteins. Results A model of caspase activation is constructed and analyzed. The apoptosis signaling network is simplified through modularization methodologies and equilibrium abstractions for three functional modules. The mathematical model is composed of a system of ordinary differential equations which is numerically solved. Multiple linear regression analysis investigates the role of each module and reduced models are constructed to identify key contributions of the extrinsic and intrinsic pathways in triggering apoptosis for different cell lines. Conclusion Through linear regression techniques, we identified the feedbacks, dissociation of complexes, and negative regulators as the key components in apoptosis. The analysis and reduced models for our model formulation reveal that the chosen cell lines predominately exhibit strong extrinsic caspase, typical of type I cell, behavior. Furthermore, under the simplified model framework, the selected cells lines exhibit different modes by which caspase activation may occur. Finally the proposed modularized model of apoptosis may generalize behavior for additional cells and tissues, specifically identifying and predicting components responsible for the transition from type I to type II cell behavior. PMID:19077196

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood–brain barrier model for drug permeability studies

PubMed Central

2013-01-01

Background Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Methods Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. Results The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Conclusions Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products. PMID:24262108

Comparative study of four immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies.

PubMed

Eigenmann, Daniela E; Xue, Gongda; Kim, Kwang S; Moses, Ashlee V; Hamburger, Matthias; Oufir, Mouhssin

2013-11-22

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.

Usefulness of sural nerve biopsy in the genomic era.

PubMed

Kanda, Takashi

2009-08-01

The value of peripheral nerve biopsy is now sometimes questioned due to the high complication rate and the recent development of noninvasive molecular techniques for diagnosis of hereditary neuropathy. However, the disorders that can be diagnosed by genetic analysis are limited and sural nerve biopsy is still a powerful tool for making a correct diagnosis of peripheral neuropathy. Histological evaluation of the sural nerve has long focused on changes of the two major components of peripheral nerves, axons and myelin, as well as on the detection of diagnostic changes such as amyloid deposits, sarcoid tubercles, and vasculitis. In addition to these components, the sural nerve biopsy specimen contains various important cells, including perineurial cells, mast cells, endothelial cells, pericytes, and lymphocytes. Among these cells, the endothelial cells and pericytes form the blood-nerve barrier (BNB) and investigation of these cells can reveal important information, especially in inflammatory neuropathies. To better understand the biological basis of BNB, we established rat and human immortal cell lines from the endothelial cells and pericytes of endoneurial microvessels. Characterization of these cell lines is now underway at our laboratory. These BNB cell lines should provide useful information concerning the pathophysiology of peripheral neuropathy, and we should obtain a new perspective for the investigation of nerve biopsy specimens after understanding the molecular background of the BNB.

Antitumorigenic effect of atmospheric-pressure dielectric barrier discharge on human colorectal cancer cells via regulation of Sp1 transcription factor

NASA Astrophysics Data System (ADS)

Han, Duksun; Cho, Jin Hyoung; Lee, Ra Ham; Bang, Woong; Park, Kyungho; Kim, Minseok S.; Shim, Jung-Hyun; Chae, Jung-Il; Moon, Se Youn

2017-02-01

Human colorectal cancer cell lines (HT29 and HCT116) were exposed to dielectric barrier discharge (DBD) plasma at atmospheric pressure to investigate the anticancer capacity of the plasma. The dose- and time-dependent effects of DBDP on cell viability, regulation of transcription factor Sp1, cell-cycle analysis, and colony formation were investigated by means of MTS assay, DAPI staining, propidium iodide staining, annexin V-FITC staining, Western blot analysis, RT-PCR analysis, fluorescence microscopy, and anchorage-independent cell transformation assay. By increasing the duration of plasma dose times, significant reductions in the levels of both Sp1 protein and Sp1 mRNA were observed in both cell lines. Also, expression of negative regulators related to the cell cycle (such as p53, p21, and p27) was increased and of the positive regulator cyclin D1 was decreased, indicating that the plasma treatment led to apoptosis and cell-cycle arrest. In addition, the sizes and quantities of colony formation were significantly suppressed even though two cancer promoters, such as TPA and epidermal growth factor, accompanied the plasma treatment. Thus, plasma treatment inhibited cell viability and colony formation by suppressing Sp1, which induced apoptosis and cell-cycle arrest in these two human colorectal cancer cell lines.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra-Soheila; Essmann, Frank; Deezagi, Abdolkhaleg; Engers, Rainer; Goering, Wolfgang; Schulz, Wolfgang A

2010-08-01

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

PubMed

Cavnar, Peter; Inman, Kristina

2015-01-01

HS1-associated protein X-1 (Hax1) is a 35 kDa protein that is ubiquitously expressed. Hax1 is an anti-apoptotic protein with additional roles in cell motility, and autosomal recessive loss of Hax1 results in Kostmann syndrome, a form of severe congenital neutropenia. Because of the important role of Hax1 in neutrophils we demonstrate here validation of two commercially available research antibodies directed against human Hax1 in the human myeloid leukemia cell line PLB-985 cells. We show that both the mouse anti-Hax1 monoclonal IgG directed against amino acids 10-148 of Hax1 and a rabbit anti-Hax1 polyclonal IgG antibody directed against full-length Hax1 reliably and consistently detect Hax1 during immunoblotting of three different PLB-985 cell densities. Using shRNA mediated Hax1 knockdown, we demonstrate the specificity of both Hax1 antibodies. In addition, our results suggest that the rabbit anti-Hax1 polyclonal antibody provides a stronger intensity in detecting Hax1 protein, with detection in as few as 0.1 x 10 (6) cells in 6 total replicates we have performed.

Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray.

PubMed

Sharif, Suhela; Shi, Jie; Bourakba, Mostafa; Ruijtenbeek, Rob; Pieters, Roland J

2017-09-01

O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development. Copyright © 2017 Elsevier Inc. All rights reserved.

Pleiotrophin promotes functional recovery after neural transplantation in rats.

PubMed

Hida, Hideki; Masuda, Tadashi; Sato, Toyohiro; Kim, Tae-Sun; Misumi, Sachiyo; Nishino, Hitoo

2007-01-22

Pleiotrophin promotes survival of dopaminergic neurons in vitro. To investigate whether pleiotrophin promotes survival of grafted dopaminergic neurons in vivo, donor cells from ventral mesencephalon were treated with pleiotrophin (100 ng/ml) during cell preparation and grafted into striatum of hemi-Parkinson model rats. Functional recovery in methamphetamine-induced rotations was improved, and more tyrosine hydroxylase-positive cells survived in the striatum in the pleiotrophin-treated group. Pleiotrophin addition to cells just before transplantation also resulted in better functional recovery; however, no caspase-3 activation was seen during cell preparation. Interestingly, the effect of pleiotrophin on the survival was additive to that of glial-cell line-derived neutropic factor. These results revealed that pleiotrophin had effects on donor cells in neural transplantation in vivo.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

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

Thanan, Raynoo; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002; Techasen, Anchalee

Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H{sub 2}O{sub 2}) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H{sub 2}O{sub 2}-resistant cell line from an immortal cholangiocytemore » cell line (MMNK1) by chronic treatment with low-concentration H{sub 2}O{sub 2} (25 μM). After 72 days of induction, H{sub 2}O{sub 2}-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H{sub 2}O{sub 2}-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H{sub 2}O{sub 2}). These findings suggest that H{sub 2}O{sub 2}-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes. - Highlights: • An H{sub 2}O{sub 2}-resistant ox-MMNK1-L cells was established from immortalized cholangiocytes. • The resistance was acquired by daily treatment of low H{sub 2}O{sub 2} (25 μM) for 15 passages. • The cells highly expressed catalase, SODs and DNMT1 with rapid cell proliferation. • Pseudopodia and the loss of cell-to-cell adhesion appeared by 100 μM H{sub 2}O{sub 2} treatment. • The resistant cells can be used as a model of oxidative stress-related carcinogenesis.« less

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

PubMed Central

Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

2017-01-01

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium. PMID:23288986

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that these cell lines form tight junctions but do not form E-cadherin-based adherence junctions. These data further indicate that the regulatory role of NHS in actin remodeling, suggested in another study, is cell type dependent. In conclusion, the SRA 01/04 and αTN4 lens epithelial cell lines model some characteristics of an epithelium.

Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line

PubMed Central

Zainal Ariffin, Shahrul Hisham; Wan Omar, Wan Haifa Haryani; Zainal Ariffin, Zaidah; Safian, Muhd Fauzi; Senafi, Sahidan; Megat Abdul Wahab, Rohaya

2009-01-01

Background Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity. Results The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 μg mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 μg mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 μg mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 μg mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines. Conclusion Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro. PMID:19257877

Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

PubMed

Pushparajan, Charlotte; Claus, Juan Daniel; Marshall, Sean D G; Visnovsky, Gabriel

2017-12-01

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 10 5 cells ml -1 ) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 10 7 TCID 50 ml -1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm 2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

PubMed

Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

2009-04-01

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Biological effects of induced MYCN hyper-expression in MYCN-amplified neuroblastomas.

PubMed

Torres, Jaime; Regan, Paul L; Edo, Robby; Leonhardt, Payton; Jeng, Eric I; Rappaport, Eric F; Ikegaki, Naohiko; Tang, Xao X

2010-10-01

Neuroblastoma is a childhood malignancy of the sympathetic nervous system. The tumor exhibits two different phenotypes: favorable and unfavorable. MYCN amplification is associated with rapid tumor progression and the worst neuroblastoma disease outcome. We have previously reported that inhibitors of histone deacetylase (HDAC) and proteasome enhance favorable neuroblastoma gene expression in neuroblastoma cell lines and inhibit growth of these cells. In this study, we investigated the effect of trichostatin A or TSA (an HDAC inhibitor), and epoxomycin (a proteasome inhibitor) on MYCN and p53 expression in MYCN-amplified neuroblastoma cells. It was found that TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression in MYCN-amplified neuroblastoma cell lines. Despite their contrasting effects on MYCN expression, TSA and Epoxomycin caused growth suppression and cell death of the MYCN-amplified cell lines examined. Consistent with these data, forced hyper-expression of MYCN in MYCN-amplified IMR5 cells via transfection resulted in growth suppression and the increased expression of several genes known to suppress growth or induce cell death. Furthermore, Epoxomycin as a single agent and its combination with TSA enhance p53 expression in the MYCN-amplified neuroblastoma cell lines. Unexpectedly, co-transfection of TP53 and MYCN in IMR5 cells resulted in high p53 expression but a reduction of MYCN expression. Together our data suggest that either down regulation or hyper-expression of MYCN results in growth inhibition and/or apoptosis of MYCN-amplified neuroblastoma cells. In addition, elevated p53 expression has a suppressive effect on MYCN expression in these cells.

Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

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

Kolokoltsova, Olga A.; Domina, Aaron M.; Kolokoltsov, Andrey A.

2008-07-20

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expressionmore » in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.« less

Induction of DNA-strand breaks after X-irradiation in murine bone cells of various differentiation capacities

NASA Astrophysics Data System (ADS)

Lau, Patrick; Hellweg, Christine E.; Kirchner, Simone; Baumstark-Khan, Christa

During longterm space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. In addition to weightlessness, exposure to cosmic ionization radiation is another space related factor endangering health and productivity of astronauts. In order to elucidate changes in bone cell metabolism induced by ionizing radiation, ground-based bone cell models have been developed. The differentiation level of the bone cells may influence their radiation sensitivity. Therefore, our cell model comprises a collection of immortalized murine pre-osteoblast, osteoblast and osteocyte cell lines representing discrete stages of differentiation: the subclones 4 and 24 of the osteoblast cell line MC3T3-E1, the osteoblast cell line OCT-1 and the osteocyte cell line MLO-Y4 display varying potential to produce mineralized bone matrix upon incubation with ascorbic acid and β-glycerophosphate (osteogenic medium). The MLO-Y4 cells showed the highest and subclone 24 the lowest proliferation rate. The most intense von Kossa reaction after culture in osteogenic medium was observed in subclone 4, indicating mineralized bone matrix. The bone cell markers alkaline phosphatase and osteocalcin were determined to further characterize the differentiation stage. All cell lines expressed osteocalcin, as determined by reverse transcriptase polymerase chain reaction. The activity of alkaline phosphatase was highest in the cell line OCT-1 and very low in MLO-Y4 and S4. The peculiarity of the markers suggests a characterization of OCT-1 and S24 as preosteoblast, S4 as (mature) osteoblast, and MLO-Y4 as osteocyte. Survival after exposure to X-rays was determined using the colony forming ability test. The resulting dose-effect relationships revealed normal radiation sensitivity (compared to human fibroblasts). Cell clone specific variations (subclones 4 and 24) in the radiation sensitivity may be due to the differentiation level. The survival curve of MLO-Y4 shows a broad shoulder, suggesting a high repair capacity or a high DNA damage or misrepair tolerance. The quantitative acquisition of DNA-strand breaks was performed by fluorescent analysis of DNA unwinding and revealed a high level of DNA damage immediately after X-irradiation, which increases dose dependently. In conclusion, the cell line with the highest differentiation level (MLO-Y4) displays lower radiation sensitivity, regarding the shoulder width of the dose-effect curve, compared to the less differentiated osteoblast cell lines.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

PubMed

Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-05-01

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

Anticancer effects of oligomeric proanthocyanidins on human colorectal cancer cell line, SNU-C4

PubMed Central

Kim, Youn-Jung; Park, Hae-Jeong; Yoon, Seo-Hyun; Kim, Mi-Ja; Leem, Kang-Hyun; Chung, Joo-Ho; Kim, Hye-Kyung

2005-01-01

AIM: Oligomeric proanthocyanidins (OPC), natural polyphenolic compounds found in plants, are known to have antioxidant and anti-cancer effects. We investigated whether the anti-cancer effects of the OPC are induced by apoptosis on human colorectal cancer cell line, SNU-C4. METHODS: Colorectal cancer cell line, SNU-C4 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cytotoxic effect of OPC was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt-etrazolium bromide (MTT) assay. To find out the apoptotic cell death, 4, 6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR), and caspase-3 enzyme assay were performed. RESULTS: In this study, cytotoxic effect of OPC on SNU-C4 cells appeared in a dose-dependent manner. OPC treatment (100 µg/mL) revealed typical morphological apoptotic features. Additionally OPC treatment (100 µg/mL) increased level of BAX and CASPASE-3, and decreased level of BCL-2 mRNA expression. Caspase-3 enzyme activity was also significantly increased by treatment of OPC (100 µg/mL) compared with control. CONCLUSION: These data indicate that OPC caused cell death by apoptosis through caspase pathways on human colorectal cancer cell line, SNU-C4. PMID:16094708

IFN-gamma priming up-regulates IFN-stimulated gene factor 3 (ISGF3) components, augmenting responsiveness of IFN-resistant melanoma cells to type I IFNs.

PubMed

Wong, L H; Hatzinisiriou, I; Devenish, R J; Ralph, S J

1998-06-01

IFN-stimulated gene factor 3 (ISGF3) mediates transcriptional activation of IFN-sensitive genes (ISGs). The component subunits of ISGF3, STAT1alphabeta, STAT2, and p48-ISGF3gamma, are tyrosine phosphorylated before their assembly into a complex. Subsequently, the ISGF3 complex is translocated to the nucleus. We have recently established that the responsiveness of human melanoma cell lines to type I IFNs correlates directly with their intracellular levels of ISGF3 components, particularly STAT1. In the present study, we show that pretreating IFN-resistant melanoma cell lines with IFN-gamma (IFN-gamma priming) before stimulation with type I IFN also results in increased levels of ISGF3 components and enhanced DNA-binding activation of ISGF3. In addition, IFN-gamma priming of IFN-resistant melanoma cell lines increased expression of type I IFN-induced ISG products, including ISG54, 2'-5'-oligoadenylate synthase, HLA class I, B7-1, and ICAM-1 Ags. Furthermore, IFN-gamma priming enhanced the antiviral effect of IFN-beta on the IFN-resistant melanoma cell line, MM96. These results support a role for IFN-gamma priming in up-regulating ISGF3, thereby augmenting the responsiveness of IFN-resistant melanoma cell lines to type I IFN and providing a molecular basis and justification for using sequential IFN therapy, as proposed by others, to enhance the use of IFNs in the treatment of melanoma.

Apigenin inhibited hypoxia induced stem cell marker expression in a head and neck squamous cell carcinoma cell line.

PubMed

Ketkaew, Yuwaporn; Osathanon, Thanaphum; Pavasant, Prasit; Sooampon, Sireerat

2017-02-01

Cancer stem cells contribute to tumor recurrence, and a hypoxic environment is critical for maintaining cancer stem cells. Apigenin is a natural product with anticancer activity. However, the effect of apigenin on cancer stem cells remains unclear. Our aim was to investigate the effect of apigenin on cancer stem cell marker expression in head and neck squamous cell carcinoma cells under hypoxia. We used three head and neck squamous cell carcinoma cell lines; HN-8, HN-30, and HSC-3. The mRNA expression of cancer stem cell markers was determined by semiquantitative RT-PCR and Real-time PCR. The cytotoxic effect of apigenin was determined by MTT colorimetric assay. Flow cytometry was used to reveal the number of cells expressing cancer stem cell surface markers. HN-30 cells, a cancer cell line from the pharynx, showed the greatest response to hypoxia by increasing their expression of CD44, CD105, NANOG, OCT-4, REX-1, and VEGF. Apigenin significantly decreased HN-30 cell viability in dose- and time-dependent manners. In addition, 40μM apigenin significantly down-regulated the mRNA expression of CD44, NANOG, and CD105. Consistent with these results, the hypoxia-induced increase in CD44 + cells, CD105 + cells, and STRO-1 + cells was significantly abolished by apigenin. Apigenin suppresses cancer stem cell marker expression and the number of cells expressing cell surface markers under hypoxia. Copyright © 2016 Elsevier Ltd. All rights reserved.

High EMT Signature Score of Invasive Non-Small Cell Lung Cancer (NSCLC) Cells Correlates with NFκB Driven Colony-Stimulating Factor 2 (CSF2/GM-CSF) Secretion by Neighboring Stromal Fibroblasts

PubMed Central

Rudisch, Albin; Dewhurst, Matthew Richard; Horga, Luminita Gabriela; Kramer, Nina; Harrer, Nathalie; Dong, Meng; van der Kuip, Heiko; Wernitznig, Andreas; Bernthaler, Andreas; Dolznig, Helmut; Sommergruber, Wolfgang

2015-01-01

We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the “cytokine fingerprints” identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay. PMID:25919140

Characterization of newly established bovine intestinal epithelial cell line.

PubMed

Miyazawa, Kohtaro; Hondo, Tetsuya; Kanaya, Takashi; Tanaka, Sachi; Takakura, Ikuro; Itani, Wataru; Rose, Michael T; Kitazawa, Haruki; Yamaguchi, Takahiro; Aso, Hisashi

2010-01-01

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.