A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation.

Science.gov (United States)

Yagita, M; Huang, C L; Umehara, H; Matsuo, Y; Tabata, R; Miyake, M; Konaka, Y; Takatsuki, K

2000-05-01

We present the establishment of a natural killer (NK) leukemia cell line, designated KHYG-1, from the blood of a patient with aggressive NK leukemia, which both possessed the same p53 point mutation. The immunophenotype of the primary leukemia cells was CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16+, CD56+, CD57+ and HLA-DR+. A new cell line (KHYG-1) was established by culturing peripheral leukemia cells with 100 units of recombinant interleukin (IL)-2. The KHYG-1 cells showed LGL morphology with a large nucleus, coarse chromatin, conspicuous nucleoli, and abundant basophilic cytoplasm with many azurophilic granules. The immunophenotype of KHYG-1 cells was CD1-, CD2+, surface CD3-, cytoplasmic CD3epsilon+, CD7+, CD8alphaalpha+, CD16-, CD25-, CD33+, CD34-, CD56+, CD57-, CD122+, CD132+, and TdT-. Southern blot analysis of these cells revealed a normal germline configuration for the beta, delta, and gamma chains of the T cell receptor and the immunoglobulin heavy-chain genes. Moreover, the KHYG-1 cells displayed NK cell activity and IL-2-dependent proliferation in vitro, suggesting that they are of NK cell origin. Epstein-Barr virus (EBV) DNA was not detected in KHYG-1 cells by Southern blot analysis with a terminal repeat probe from an EBV genome. A point mutation in exon 7 of the p53 gene was detected in the KHYG-1 cells by PCR/SSCP analysis, and direct sequencing revealed the conversion of C to T at nucleotide 877 in codon 248. The primary leukemia cells also carried the same point mutation. Although the precise role of the p53 point mutation in leukemogenesis remains to be clarified, the establishment of an NK leukemia cell line with a p53 point mutation could be valuable in the study of leukemogenesis.

Transgenic cassava lines carrying heterologous alternative oxidase ...

African Journals Online (AJOL)

Afuape

2013-07-03

Jul 3, 2013 ... production of flowers, apomixis (Nassar et al., 2000; ... In order to increase the stress tolerance capacity of ... stress-related procedure due to the activities of auxin ... the evaluation of the transgenic lines for rate of OES .... Some transgenic lines carrying the 35S-AOX fragment amplified using 35S303F1 and.

Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

Science.gov (United States)

Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

2013-12-15

The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level. Copyright © 2013 Elsevier GmbH. All rights reserved.

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

Science.gov (United States)

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions.

Science.gov (United States)

Sambhara, S R; Upadhya, A G; Miller, R G

1990-06-12

We describe a microculture system for the generation of CTL and T helper cells against peptides. Tryptic digest and cyanogen bromide fragments of chicken ovalbumin and synthetic peptides of ovalbumin (323-339) and influenza virus (NP 365-380) were used to generate CTL and T helper lines from unprimed T cells. These lines were both peptide-specific and MHC-restricted. The relative ease of generating peptide-specific, MHC-restricted CTL and helper T cell lines with as few as 10(6) unprimed lymphocytes can be an efficient method of detecting potential immunogenic determinants of an antigen.

Progesterone-specific stimulation of triglyceride biosynthesis in a breast cancer cell line (T-47D)

International Nuclear Information System (INIS)

Judge, S.M.; Chatterton, R.T. Jr.

1983-01-01

The purpose of this study was to examine the lactogenic response of human mammary cancer cell lines to hormones in vitro. Progesterone was found to stimulate the incorporation of 14C from [14C]acetate into triglycerides (TG) and to promote accumulation of TG with a fatty acid composition similar to that of human milk fat in T-47D cells. Lipid droplets were observed in larger numbers without concomitant accumulation of casein granules in cells incubated with progesterone, but secretion of lipid into the medium did not occur. An effect of progesterone on TG accumulation was detectable after 12 hr and was maximal at 72 hr. Increasing doses of progesterone (10(-9) to 10(-5) M) caused a progressive increase in TG accumulation. The presence of cortisol and/or prolactin did not alter TG formation nor the dose response of the cells to progesterone. The growth rate of T-47D cells was not altered by the presence of progesterone in the medium. Neither of the human mammary cancer cell lines, MCF-7 and HBL-100, nor the human fibroblast cell lines, 28 and 857, responded to progesterone. The data indicate that, while the normally lactogenic hormones do not stimulate milk product biosynthesis in the cell lines tested, progesterone specifically stimulated synthesis and accumulation of TG in the T-47D cells

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

Science.gov (United States)

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-01-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

Generation of human iPSC line from a patient with laterality defects and associated congenital heart anomalies carrying a DAND5 missense alteration

Directory of Open Access Journals (Sweden)

Fernando Cristo

2017-12-01

Full Text Available A human iPSC line was generated from exfoliated renal epithelial (ERE cells of a patient affected with Congenital Heart Disease (CHD and Laterality Defects carrying tshe variant p.R152H in the DAND5 gene. The transgene-free iPSCs were generated with the human OSKM transcription factor using the Sendai-virus reprogramming system. The established iPSC line had the specific heterozygous alteration, a stable karyotype, expressed pluripotency markers and generated embryoid bodies that can differentiate towards the three germ layers in vitro. This iPSC line offers a useful resource to study the molecular mechanisms of cardiomyocyte proliferation, as well as for drug testing.

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

Science.gov (United States)

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

2017-06-01

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

Transgenic cassava lines carrying heterologous alternative oxidase ...

African Journals Online (AJOL)

Afuape

2013-07-03

Jul 3, 2013 ... Organized embryogenic callus development: In our experiment, somatic embryos were developed from leaf lobes collected from transgenic cassava lines carrying the AtAOX1a gene. Immature leaf lobes measuring about 1 to 6 mm obtained from about six weeks old in vitro derived plants were used.

Using Regularization to Infer Cell Line Specificity in Logical Network Models of Signaling Pathways

Directory of Open Access Journals (Sweden)

Sébastien De Landtsheer

2018-05-01

Full Text Available Understanding the functional properties of cells of different origins is a long-standing challenge of personalized medicine. Especially in cancer, the high heterogeneity observed in patients slows down the development of effective cures. The molecular differences between cell types or between healthy and diseased cellular states are usually determined by the wiring of regulatory networks. Understanding these molecular and cellular differences at the systems level would improve patient stratification and facilitate the design of rational intervention strategies. Models of cellular regulatory networks frequently make weak assumptions about the distribution of model parameters across cell types or patients. These assumptions are usually expressed in the form of regularization of the objective function of the optimization problem. We propose a new method of regularization for network models of signaling pathways based on the local density of the inferred parameter values within the parameter space. Our method reduces the complexity of models by creating groups of cell line-specific parameters which can then be optimized together. We demonstrate the use of our method by recovering the correct topology and inferring accurate values of the parameters of a small synthetic model. To show the value of our method in a realistic setting, we re-analyze a recently published phosphoproteomic dataset from a panel of 14 colon cancer cell lines. We conclude that our method efficiently reduces model complexity and helps recovering context-specific regulatory information.

Tissue-specific signatures in the transcriptional response to Anaplasma phagocytophilum infection of Ixodes scapularis and Ixodes ricinus tick cell lines

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Pilar eAlberdi

2016-02-01

Full Text Available Anaplasma phagocytophilum are transmitted by Ixodes spp. ticks and have become one of the most common and relevant tick-borne pathogens due to their impact on human and animal health. Recent results have increased our understanding of the molecular interactions between Ixodes scapularis and A. phagocytophilum through the demonstration of tissue-specific molecular pathways that ensure pathogen infection, development and transmission by ticks. However, little is known about the Ixodes ricinus genes and proteins involved in the response to A. phagocytophilum infection. The tick species I. scapularis and I. ricinus are evolutionarily closely related and therefore similar responses are expected in A. phagocytophilum-infected cells. However, differences may exist between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cells associated with tissue-specific signatures of these cell lines. To address this hypothesis, the transcriptional response to A. phagocytophilum infection was characterized by RNA sequencing and compared between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cell lines. The transcriptional response to infection of I. scapularis ISE6 cells resembled that of tick hemocytes while the response in I. ricinus IRE/CTVM20 cells was more closely related to that reported previously in infected tick midguts. The inhibition of cell apoptosis by A. phagocytophilum appears to be a key adaptation mechanism to facilitate infection of both vertebrate and tick cells and was used to investigate further the tissue-specific response of tick cell lines to pathogen infection. The results supported a role for the intrinsic pathway in the inhibition of cell apoptosis by A. phagocytophilum infection of I. scapularis ISE6 cells. In contrast, the results in I. ricinus IRE/CTVM20 cells were similar to those obtained in tick midguts and suggested a role for the JAK/STAT pathway in the inhibition of apoptosis in tick cells infected with A. phagocytophilum

Establishment of cell lines with rat spermatogonial stem cell characteristics

NARCIS (Netherlands)

van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

2002-01-01

Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

Susceptibility testing of fish cell lines for virus isolation

DEFF Research Database (Denmark)

Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

2009-01-01

and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

2-DE analysis of breast cancer cell lines 1833 and 4175 with distinct metastatic organ-specific potentials: Comparison with parental cell line MDA-MB-231

Czech Academy of Sciences Publication Activity Database

Selicharová, Irena; Šanda, Miloslav; Mládková, Jana; Ohri, S. S.; Vashishta, A.; Fusek, M.; Jiráček, Jiří; Vetvicka, V.

2008-01-01

Roč. 19, č. 5 (2008), s. 1237-1244 ISSN 1021-335X R&D Projects: GA MZd NR8323 Grant - others:NIH(US) ROI CAA082159-03 Institutional research plan: CEZ:AV0Z40550506 Keywords : breast cancer * cell line * 2-DE * organ-specific metastases Subject RIV: CE - Biochemistry Impact factor: 1.524, year: 2008

Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

Science.gov (United States)

Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

2017-05-01

MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

Science.gov (United States)

Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

2018-07-01

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

Novel strong tissue specific promoter for gene expression in human germ cells

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Kuzmin Denis

2010-08-01

Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

Molecular characterization of breast cancer cell lines through multiple omic approaches.

Science.gov (United States)

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated

Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

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Ana Gracanin

Full Text Available Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, β-catenin, GSK3β, CK1α and Axin1 and have a functional β-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms.

Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.

Science.gov (United States)

Kunzelmann, K; Lei, D C; Eng, K; Escobar, L C; Koslowsky, T; Gruenert, D C

1995-09-01

Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

DEFF Research Database (Denmark)

Momcilovic, Olga; Sivapatham, Renuka; Oron, Tal Ronnen

2016-01-01

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight...... not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis........ We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may...

Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

Directory of Open Access Journals (Sweden)

Ashley R.P. Hinson

2013-07-01

Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

Science.gov (United States)

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

DEFF Research Database (Denmark)

Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

2013-01-01

Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

Establishment and characterization of rat portal myofibroblast cell lines.

Directory of Open Access Journals (Sweden)

Michel Fausther

Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

International Nuclear Information System (INIS)

Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

2007-01-01

Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

DEFF Research Database (Denmark)

Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

2006-01-01

Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

Monitoring cell line identity in collections of human induced pluripotent stem cells

Directory of Open Access Journals (Sweden)

Raquel Sarafian

2018-04-01

Full Text Available The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Keywords: Induced pluripotent stem cells, Genotyping, Cell line identification, Short tandem repeats, Quality control

Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

Science.gov (United States)

Hedberg, Annica; Knutsen, Erik; Løvhaugen, Anne Silje; Jørgensen, Tor Erik; Perander, Maria; Johansen, Steinar D

2018-04-19

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

Science.gov (United States)

Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

LET effects on normal and radiosensitive cell lines

International Nuclear Information System (INIS)

Geard, C.R.; Travisano, M.

1986-01-01

Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

Characterization of a human cell line stably over-expressing the candidate oncogene, dual specificity phosphatase 12.

Directory of Open Access Journals (Sweden)

Erica L Cain

2011-04-01

Full Text Available Analysis of chromosomal rearrangements within primary tumors has been influential in the identification of novel oncogenes. Identification of the "driver" gene(s within cancer-derived amplicons is, however, hampered by the fact that most amplicons contain many gene products. Amplification of 1q21-1q23 is strongly associated with liposarcomas and microarray-based comparative genomic hybridization narrowed down the likely candidate oncogenes to two: the activating transcription factor 6 (atf6 and the dual specificity phosphatase 12 (dusp12. While atf6 is an established transcriptional regulator of the unfolded protein response, the potential role of dusp12 in cancer remains uncharacterized.To evaluate the oncogenic potential of dusp12, we established stable cell lines that ectopically over-express dusp12 in isolation and determined whether this cell line acquired properties frequently associated with transformed cells. Here, we demonstrate that cells over-expressing dusp12 display increased cell motility and resistance to apoptosis. Additionally, over-expression of dusp12 promoted increased expression of the c-met proto-oncogene and the collagen and laminin receptor intergrin alpha 1 (itga1 which is implicated in metastasis.Collectively, these results suggest that dusp12 is oncologically relevant and exposes a potential association between dusp12 and established oncogenes that could be therapeutically targeted.

Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

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Hans eKlingemann

2016-03-01

Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

Science.gov (United States)

Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

(Asteraceae) Fraction against Human Cancer Cell Lines

African Journals Online (AJOL)

Purpose: To investigate the anti-proliferative and apoptotic activity of crude and dichloromethane fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and L929). Methods: A. sieberi was extracted with methanol and further purification was carried out using liquidliquid extraction ...

Folic acid induces cell type-specific changes in the transcriptome of breast cancer cell lines: a proof-of-concept study.

Science.gov (United States)

Price, R Jordan; Lillycrop, Karen A; Burdge, Graham C

2016-01-01

The effect of folic acid (FA) on breast cancer (BC) risk is uncertain. We hypothesised that this uncertainty may be due, in part, to differential effects of FA between BC cells with different phenotypes. To test this we investigated the effect of treatment with FA concentrations within the range of unmetabolised FA reported in humans on the expression of the transcriptome of non-transformed (MCF10A) and cancerous (MCF7 and Hs578T) BC cells. The total number of transcripts altered was: MCF10A, seventy-five (seventy up-regulated); MCF7, twenty-four (fourteen up-regulated); and Hs578T, 328 (156 up-regulated). Only the cancer-associated gene TAGLN was altered by FA in all three cell lines. In MCF10A and Hs578T cells, FA treatment decreased pathways associated with apoptosis, cell death and senescence, but increased those associated with cell proliferation. The folate transporters SLC19A1, SLC46A1 and FOLR1 were differentially expressed between cell lines tested. However, the level of expression was not altered by FA treatment. These findings suggest that physiological concentrations of FA can induce cell type-specific changes in gene regulation in a manner that is consistent with proliferative phenotype. This has implications for understanding the role of FA in BC risk. In addition, these findings support the suggestion that differences in gene expression induced by FA may involve differential activities of folate transporters. Together these findings indicate the need for further studies of the effect of FA on BC.

Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

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Alessandra M. Welker

2016-02-01

Full Text Available Glioblastoma (GBM is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

Science.gov (United States)

Meraviglia, Viviana; Benzoni, Patrizia; Landi, Sara; Murano, Carmen; Langione, Marianna; Motta, Benedetta M; Baratto, Serena; Silipigni, Rosamaria; Di Segni, Marina; Pramstaller, Peter P; DiFrancesco, Dario; Gazzerro, Elisabetta; Barbuti, Andrea; Rossini, Alessandra

2018-03-01

Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

DEFF Research Database (Denmark)

Efrat, S; Linde, S; Kofod, Hans

1988-01-01

Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement.

Science.gov (United States)

Basolo, Fulvio; Giannini, Riccardo; Toniolo, Antonio; Casalone, Rosario; Nikiforova, Marina; Pacini, Furio; Elisei, Rossella; Miccoli, Paolo; Berti, Piero; Faviana, Pinuccia; Fiore, Lisa; Monaco, Carmen; Pierantoni, Giovanna Maria; Fedele, Monica; Nikiforov, Yuri E; Santoro, Massimo; Fusco, Alfredo

2002-02-10

A novel human thyroid papillary carcinoma cell line (FB-2) has been established and characterized. FB-2 cells harbor the RET/PTC1 chimeric oncogene in which the RET kinase domain is fused to the H4 gene. FB-2 cells neither formed colonies in semisolid media nor induced tumors after heterotransplant into severe combined immunodeficient mice. However, HMGI(Y), HMGI-C and c-myc genes, which are associated to thyroid cell transformation, were abundantly expressed in FB-2 cells but not in normal thyroid cells. FB-2 cells only partially retained the differentiated thyroid phenotype. In fact, the PAX-8 gene, which codes for a transcriptional factor required for thyroid cell differentiation, was expressed, while thyroglobulin, TSH-receptor and thyroperoxidase genes were not. Moreover, FB-2 cells produced high levels of interleukin (IL)-6 and IL-8. Copyright 2001 Wiley-Liss, Inc.

Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

Directory of Open Access Journals (Sweden)

Biljana Dumevska

2016-03-01

Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

DEFF Research Database (Denmark)

Theilade, M D; Gram, G J; Jensen, P B

1999-01-01

Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

Induction of apoptosis by opium in some tumor cell lines.

Science.gov (United States)

Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

2016-09-30

The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status.

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Simon Memmel

Full Text Available Glioblastoma multiforme (GBM is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM, the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm(2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19 showed the highest C m values of 3.7-4.0 µF/cm(2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the

Generation of genome-modified Drosophila cell lines using SwAP.

Science.gov (United States)

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

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

Killing cancer cells by targeted drug-carrying phage nanomedicines

Directory of Open Access Journals (Sweden)

Yacoby Iftach

2008-04-01

Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

Killing cancer cells by targeted drug-carrying phage nanomedicines

Science.gov (United States)

Bar, Hagit; Yacoby, Iftach; Benhar, Itai

2008-01-01

Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates. PMID:18387177

Development of one control and one tumor-specific induced pluripotent stem cell line from laryngeal carcinoma patient

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Yamin Zhang

2017-12-01

Full Text Available Skin fibroblasts and tumor fibroblasts were extracted from a 64-year old male patient clinically diagnosed with laryngeal carcinoma. Control and tumor specific induced pluripotent stem cells were reprogrammed with 5 reprogramming factors, Klf-4, c-Myc, Oct-4, Sox-2, and Lin-28, using the messenger RNA reprogramming system. The transgene-free iPSC lines showed pluripotency, confirmed by immunofluorescence staining. The iPSC lines also showed normal karyotype, and could form embryoid bodies in vitro and differentiate into the 3 germ layers in vivo. This in vitro cellular model can be used to study the oncogenesis and pathogenesis of laryngeal carcinoma.

Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe

International Nuclear Information System (INIS)

Kalejs, Martins; Ivanov, Andrey; Plakhins, Gregory; Cragg, Mark S; Emzinsh, Dzintars; Illidge, Timothy M; Erenpreisa, Jekaterina

2006-01-01

We have previously reported that p53 mutated radioresistant lymphoma cell lines undergo mitotic catastrophe after irradiation, resulting in metaphase arrest and the generation of endopolyploid cells. A proportion of these endopolyploid cells then undergo a process of de-polyploidisation, stages of which are partially reminiscent of meiotic prophase. Furthermore, expression of meiosis-specific proteins of the cancer/testis antigens group of genes has previously been reported in tumours. We therefore investigated whether expression of meiosis-specific genes was associated with the polyploidy response in our tumour model. Three lymphoma cell lines, Namalwa, WI-L2-NS and TK6, of varying p53 status were exposed to a single 10 Gy dose of gamma radiation and their responses assessed over an extended time course. DNA flow cytometry and mitotic counts were used to assess the kinetics and extent of polyploidisation and mitotic progression. Expression of meiotic genes was analysed using RT-PCR and western blotting. In addition, localisation of the meiotic cohesin REC8 and its relation to centromeres was analysed by immunofluorescence. The principal meiotic regulator MOS was found to be significantly post-transcriptionally up-regulated after irradiation in p53 mutated but not p53 wild-type lymphoma cells. The maximum expression of MOS coincided with the maximal fraction of metaphase arrested cells and was directly proportional to both the extent of the arrest and the number of endopolyploid cells that subsequently emerged. The meiotic cohesin REC8 was also found to be up-regulated after irradiation, linking sister chromatid centromeres in the metaphase-arrested and subsequent giant cells. Finally, RT-PCR revealed expression of the meiosis-prophase genes, DMC1, STAG3, SYCP3 and SYCP1. We conclude that multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation. Furthermore, we suggest that the coordinated expression

Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

Science.gov (United States)

Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

2017-07-05

Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L + HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L + HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L + HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

Generation of induced pluripotent stem cells (iPSCs) from an Alzheimer's disease patient carrying an A79V mutation in PSEN1

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Li, Tong; Pires, Carlota; Nielsen, Troels Tolstrup

2016-01-01

Skin fibroblasts were obtained from a 48-year-old presymptomatic woman carrying a A79V mutation in the presenilin 1 gene (PSEN1), causing Alzheimer's disease (AD). Induced pluripotent stem cell (iPSCs) were derived via transfection with episomal vectors carrying hOCT4, hSOX2, hKLF2, hL-MYC, hLIN28...... and shTP53 genes. A79V-iPSCs were free of genomically integrated reprogramming genes, had the specific mutation but no additional genomic aberrancies, expressed the expected pluripotency markers and displayed in vitro differentiation potential to the three germ layers. The reported A79V-iPSCs line may...

Presence of dopamine D-2 receptors in human tumoral cell lines

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Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. (Centre Paul Broca, Paris (France))

1989-07-31

({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations

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Gentilcore Giusy

2013-01-01

Full Text Available Abstract Background Conventional therapeutic agents are largely unsatisfactory into the treatment of malignant melanoma. Recently, an innovative approach based on inhibitors of the mutated BRAF gene (which represents the most prevalent alteration in melanoma patients appears very promising from the clinical point of view. On this regard, a new compound, dabrafenib (GSK2118436, has been demonstrated to be effective in patients carrying the BRAFV600E/K mutations. We here tested dabrafenib for its capability to inhibit cell growth on primary melanoma cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations. Methods Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D. The MTT assays were performed using standardized approaches. To evaluate the inhibition of MAPK pathway and the consequent inhibition of cellular proliferation, the phosphorylation of ERK was examined by Western Blot analysis performed on total protein extracts from cell lines after treatment with dabrafenib. Results Our experiments demonstrated an effective action of Dabrafenib (GSK2118436 and the inhibition of MAPK pathway in melanoma cell lines carrying BRAFV600D/R mutations. Conclusion These results could be helpful to enlarge the number of melanoma patients who may benefit of a more effective targeted treatment.

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

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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.

Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

International Nuclear Information System (INIS)

López, Jacqueline; Poitevin, Adela; Mendoza-Martínez, Veverly; Pérez-Plasencia, Carlos; García-Carrancá, Alejandro

2012-01-01

Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I). Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 10 3 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 10 5 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR). We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, Si

AEGY-28 Cell Line of Aedes aegypti (Diptera Culicidae is Infection Refractory to Dengue 2 and Yellow Fever Virus

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Nadia Y. Castañeda

2007-07-01

Full Text Available Mosquito cell derived cultures are useful tools for arbovirus isolation, identification or characterization. For studying dengue (DENV and yellow fever viruses (YFV Aedes albopictus C6/36 or Aedes pseudoscutellaris AP-61 cell lines, are normally used. The Aedes aegypti AEGY-28 cell line was obtained from embryonic tissues and characterized previously by one of us. In order to evaluate its susceptibility to two Flavivirus, AEGY- 28 cells were inoculated with different multiplicity of infection (MOI with type 2 DENV (COL-789, MOI: 1 and 5 and YFV clinical isolates (V-341, MOI 0,02 then processed at different times post infection (p.i.. Immunostai ning and fluorometric cell-ELISA were carried out to identify and quantify viral antigens. C6/36 and Vero cells were used as positive controls. Unexpectedly, immunoreactivity was not found in inoculated AEGY-28 cells, even in higher MOI or late times p.i., therefore antigen quantification using fluorometric cell-ELISA were not  plausible. Reverse transcriptase PCR with specific primers did not detect viral RNA in AEGY-28 inoculated cells. We can conclude that Aedes aegypti AEGY-28 cell line is not susceptible to dengue and yellow fever Flavivirus, a finding possibly related with the lacking of specific molecules at the plasma membrane or absence of cell machinery necessary for viral replication.

The transcriptional diversity of 25 Drosophila cell lines

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Cherbas, Lucy [Indiana Univ., Bloomington, IN (United States); Willingham, Aarron [Affymetrix Inc., Santa Clara, CA (United States); Zhang, Dayu [Indiana Univ., Bloomington, IN (United States); Yang, Li [University of Connecticut Health Center, Farmington, Connecticut (United States); Zou, Yi [Indiana Univ., Bloomington, IN (United States); Eads, Brian D. [Indiana Univ., Bloomington, IN (United States); Carlson, Joseph W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Landolin, Jane M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kapranov, Philipp [Affymetrix Inc., Santa Clara, CA (United States); Dumais, Jacqueline [Affymetrix Inc., Santa Clara, CA (United States); Samsonova, Anastasia [Harvard Medical School, Boston, MA (United States); Choi, Jeong-Hyeon [Indiana Univ., Bloomington, IN (United States); Roberts, Johnny [Indiana Univ., Bloomington, IN (United States); Davis, Carrie A. [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Tang, Haixu [Indiana Univ., Bloomington, IN (United States); van Baren, Marijke J. [Washington Univ., St. Louis, MO (United States); Ghosh, Srinka [Affymetrix Inc., Santa Clara, CA (United States); Dobin, Alexander [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Bell, Kim [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Lin, Wei [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Langton, Laura [Washington Univ., St. Louis, MO (United States); Duff, Michael O. [University of Connecticut Health Center, Farmington, Connecticut (United States); Tenney, Aaron E. [Washington Univ., St. Louis, MO (United States); Zaleski, Chris [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Brent, Michael R. [Washington Univ., St. Louis, MO (United States); Hoskins, Roger A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kaufman, Thomas C. [Indiana University, Bloomington, Indiana (United States); Andrews, Justen [Indiana University, Bloomington, Indiana (United States); Graveley, Brenton R. [University of Connecticut Health Center, Farmington, Connecticut (United States); Perrimon, Norbert [Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Institute, Boston, MA (United States); Celniker, Susan E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gingeras, Thomas R. [Affymetrix Inc., Santa Clara, CA (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Cherbas, Peter [Indiana Univ., Bloomington, IN (United States)

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 signaling 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

Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

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Viviana Meraviglia

2018-03-01

Full Text Available Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3, encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-AL1CAV3 (EURACi002-AN1CAV3 (EURACi003-AInstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini (alessandra.rossini@eurac.eduType of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCsMethod of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hULMultiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-AHeterozygous CAV3 c.303 TGG > TGC (EURACi002-AHeterozygous CAV3 c.233 ACG > AAG (EURACi003-AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-ASeptember 2016 (EURACi002-AMay 2016 (EURACi003-ACell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the

Derivation of NEM2 affected human embryonic stem cell line Genea079

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Biljana Dumevska

2016-03-01

Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

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Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

2015-08-01

Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC?1

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Fujita, Mayumi; Imadome, Kaori; Imai, Takashi

2017-01-01

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC?1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC?1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC?1 cells, metabolome analysis of the invaded PANC?1 compared with the whole cultured PANC?1 was performed using CE?TOFMS, and concentrations of 110 met...

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

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Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

Science.gov (United States)

Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

2013-08-01

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Ag85A-specific CD4+ T cell lines derived after boosting BCG-vaccinated cattle with Ad5-85A possess both mycobacterial growth inhibition and anti-inflammatory properties.

Science.gov (United States)

Metcalfe, Hannah J; Biffar, Lucia; Steinbach, Sabine; Guzman, Efrain; Connelley, Tim; Morrison, Ivan; Vordermeier, H Martin; Villarreal-Ramos, Bernardo

2018-05-11

There is a need to improve the efficacy of the BCG vaccine against human and bovine tuberculosis. Previous data showed that boosting bacilli Calmette-Guerin (BCG)-vaccinated cattle with a recombinant attenuated human type 5 adenovirally vectored subunit vaccine (Ad5-85A) increased BCG protection and was associated with increased frequency of Ag85A-specific CD4 + T cells post-boosting. Here, the capacity of Ag85A-specific CD4 + T cell lines - derived before and after viral boosting - to interact with BCG-infected macrophages was evaluated. No difference before and after boosting was found in the capacity of these Ag85A-specific CD4 + T cell lines to restrict mycobacterial growth, but the secretion of IL-10 in vitro post-boost increased significantly. Furthermore, cell lines derived post-boost had no statistically significant difference in the secretion of pro-inflammatory cytokines (IL-1β, IL-12, IFNγ or TNFα) compared to pre-boost lines. In conclusion, the protection associated with the increased number of Ag85A-specific CD4 + T cells restricting mycobacterial growth may be associated with anti-inflammatory properties to limit immune-pathology. Copyright © 2018 Department for Environment Food and Rural Affairs. Published by Elsevier Ltd.. All rights reserved.

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

Science.gov (United States)

Touat-Hamici, Zahia; Bulteau, Anne-Laure; Bianga, Juliusz; Jean-Jacques, Hélène; Szpunar, Joanna; Lobinski, Ryszard; Chavatte, Laurent

2018-04-13

Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76 Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

1992-08-01

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

GENETIC TRANSFORMATION AND ANALYSIS OF WHEAT TRANSGENIC CELL LINES BY IRAP-PCR

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Bavol A. V.

2013-12-01

Full Text Available The transgenic wheat cell lines were obtained via biolistic transformation of the callus cultures initiated from the 3-day-old sterile seedling shoot apexes. The pAHC25 vector construction used for 14- and 28-day-old callus cultures transformation carried the selective phosphinothricin-N-acetyltransferase (bar gene and reporter ?-glucuronidase gene. The cell line selection was carried out on the media with phosphinothricin by means of graduated cell selection. The transgenic status of the obtained forms was proved by PCR-analysis. The presence of new relatively high molecular (more than 1 000 bp amplicons were found out for three transformed lines by means of IRAP PCRanalysis with the primers coding for long termainal repeats sequences of SIRE 1 retrotransposon. This fact may prove transposition of this mobile genetic element. The new DNA fragments were detected for three of the seven analyzed lines but for the control callus. It is possible to assume at induction of SIRE 1 transposition bis probably caused by the genomic stress of foreign DNA inserting or associated with the transformation process (mechanical wounding, cultivation on selective media.

CD4+ T-cell lines used to evaluate a Mycobacterium avium subsp. paratuberculosis (MAP) peptide vaccine

DEFF Research Database (Denmark)

Lybeck, Kari; Sjurseth, Siri K.; Al-Touama, Zainab

The aim of the study was to establish a protocol for generation of MAP-specific T-cell lines and to use these lines for evaluation of a peptide vaccine. A protocol for culturing T-cell lines from peripheral blood of goats naturally infected with MAP was established. CD4+ T cells were positively...... selected using an anti CD4 mAb and Dynabeads. Sorted CD4+ cells were cultivated with purified protein derivative from MAP (PPDj) or E. coli sonicate, IL-2, and IL-15. After two cultivation cycles, T cells were tested for recall responses in a proliferative T-cell assay. T-cell line responses were...... in average 92 % for PPDj, and -3 % for E. coli sonicate. CD4+ T-cell lines stimulated with PPDj showed a 6 fold increase in IFN- γ production compared to controls. These results indicated that the T-cell lines were MAP-specific. The protocol was subsequently used to evaluate MAP-specific peptides as vaccine...

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

International Nuclear Information System (INIS)

Chromik, Ansgar M; Weyhe, Dirk; Mittelkötter, Ulrich; Uhl, Waldemar; Hahn, Stephan A; Daigeler, Adrien; Flier, Annegret; Bulut, Daniel; May, Christina; Harati, Kamran; Roschinsky, Jan; Sülberg, Dominique

2010-01-01

The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis

Recombinant protein production from stable mammalian cell lines and pools.

Science.gov (United States)

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

Science.gov (United States)

Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-05-31

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

Radiation sensitivity of Merkel cell carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines

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Yongwen LI

2015-05-01

Full Text Available Background and objective It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. Methods Real-time quantitative PCR and methylation-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D. DNA sequencing was used to confirm the methylation results. Results The level of miR-182 expression significantly differs among these lung cancer cell lines. The highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines significantly increases when treated with 10 μM 5’-Aza-dC. Conclusion DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occurrence and development.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

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Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

Science.gov (United States)

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

International Nuclear Information System (INIS)

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

International Nuclear Information System (INIS)

Playle, L.C.

2001-03-01

The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

Science.gov (United States)

Dastjerdi, M Nikbakht; Babazadeh, Z; Rabbani, M; Gharagozloo, M; Esmaeili, A; Narimani, M

2014-01-01

Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 μM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (pPANC-1 through p21 and Bax pathway but not through RASSF1A.

CLO : The cell line ontology

NARCIS (Netherlands)

Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

2014-01-01

Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

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Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

2016-05-20

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Angiocrine functions of organ-specific endothelial cells

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Rafii, Shahin; Butler, Jason M; Ding, Bi-Sen

2016-01-01

Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

Epstein-Barr virus (EBV) provides survival factors to EBV+ diffuse large B-cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+  DLBCL.

Science.gov (United States)

Wu, Liang; Ehlin-Henriksson, Barbro; Zhou, Xiaoying; Zhu, Hong; Ernberg, Ingemar; Kis, Lorand L; Klein, George

2017-12-01

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin-21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications. © 2017 John Wiley & Sons Ltd.

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

Science.gov (United States)

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

2017-01-01

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

Establishment and culture optimization of a new type of pituitary immortalized cell line

Energy Technology Data Exchange (ETDEWEB)

Kokubu, Yuko [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Asashima, Makoto [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577 (Japan); Kurisaki, Akira, E-mail: akikuri@hotmail.com [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan)

2015-08-07

The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.

Mitochondrial Alterations by PARKIN in Dopaminergic Neurons Using PARK2 Patient-Specific and PARK2 Knockout Isogenic iPSC Lines

Directory of Open Access Journals (Sweden)

Atossa Shaltouki

2015-05-01

Full Text Available In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction. The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism.

Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

Science.gov (United States)

Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J.; Levy-Lahad, Ephrat; Renbaum, Paul

2012-01-01

Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

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Ryo Kurita

Full Text Available Transfusion of red blood cells (RBCs is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

Investigation of therapeutic efficiency of phenytoin (PHT) labeled with radioactive 131I in the cancer cell lines

International Nuclear Information System (INIS)

Cansu Uzaras; Ugur Avcibasi; Hasan Demiroglu; Emin Ilker Medine; Ayfer Yurt KiIcar; Fazilet Zuemruet Biber Mueftueler; Perihan Uenak

2016-01-01

The aim of this study is to determine the incorporations of PHT radiolabeled with 131 I ( 131 I-PHT) on U-87 MG, Daoy and A549 cancerous cell lines. For this, cold and radio-labeling studies were carried out. The radiolabeling yield of 131 I-PHT was obtained about 95 %. Subsequently, cell culture studies were carried out and radio-labeling yields of 131 I, 131 I-PHT on U-87 MG, Daoy and A549 cancerous cells were investigated. Cell culture studies demonstrated that the incorporation values of 131 IPHT on the three cell lines decreased with increasing radioactivity. Consequently, 131 I-PHT may be a good radiopharmaceutical for targeting radionuclide therapy of Central Nervous System Tumors. (author)

Abnormal A-type lamin organization in a human lung carcinoma cell line

NARCIS (Netherlands)

Machiels, BM; Broers, JL; Raymond, Y; de Leij, Louis; Kuijpers, HJH; Caberg, NEH; Ramaekers, Frans C. S.

We have studied the expression of lamins A and C (A-type lamins) in a lung carcinoma cell line using type-specific monoclonal antibodies, Using immunofluorescence and immunoblotting studies it was noted that several irregularities in lamin expression exist in the cell line GLC-A1, derived from an

Accessory signals in T-T cell interactions between antigen- and alloantigen-specific, human memory T cells generated in vitro

DEFF Research Database (Denmark)

Odum, N; Ryder, L P; Georgsen, J

1990-01-01

The potential of activated HLA class II-positive T cells as antigen-/alloantigen-presenting cells remains controversial. In our model system we use in vitro-primed, HLA class II-specific T cells of the memory T-cell phenotype, CD4+, CD29+ (4B4+), and CD45RO+ (UCHL-1). We have previously shown......), or a calcium ionophore (A23187) enabled Ta to elicit alloantigen-specific memory T-cell responses and to present purified protein derivative (PPD) to PPD-specific T-cell lines. The addition of irradiated, Epstein-Barr virus-transformed B-cell lines (EBV-LCL) (but not their supernatants) had a similar but less...

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

Science.gov (United States)

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

2016-06-01

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation of a primate embryonic stem cell line.

OpenAIRE

Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

1995-01-01

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Mouse DRG Cell Line with Properties of Nociceptors.

Science.gov (United States)

Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

2015-01-01

In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

International Nuclear Information System (INIS)

Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.; Wilson, George D.; Marples, Brian

2010-01-01

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity was assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing γH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2μg/mL], Trinovin [10μg/mL], and Prostate Rx [50 μg/mL]). However, both Trinovin (10μg/mL) and Prostate Rx (6μg/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.

Global Conservation of Protein Status between Cell Lines and Xenografts

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Julian Biau

2016-08-01

Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

International Nuclear Information System (INIS)

Fellig, Yakov; Almogy, Gidon; Galun, Eithan; Ketzinel-Gilad, Mali

2004-01-01

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10 II , exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10 II cell genome. The presence of HBV in the FLC4A10 II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10 II , can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents

Effect of the coffee ingredient cafestol on head and neck squamous cell carcinoma cell lines

International Nuclear Information System (INIS)

Kotowski, Ulana; Heiduschka, Gregor; Eckl-Dorna, Julia; Kranebitter, Veronika; Stanisz, Isabella; Brunner, Markus; Lill, Claudia; Thurnher, Dietmar; Seemann, Rudolf; Schmid, Rainer

2015-01-01

Cafestol is a diterpene molecule found in coffee beans and has anticarcinogenic properties. The aim of the study was to examine the effects of cafestol in head and neck squamous cell carcinoma (HNSCC) cells. Three HNSCC cell lines (SCC25, CAL27 and FaDu) were treated with increasing doses of cafestol. Then combination experiments with cisplatin and irradiation were carried out. Drug interactions and possible synergy were calculated using the combination index analysis. Clonogenic assays were performed after irradiation with 2, 4, 6 and 8 Gy, respectively, and the rate of apoptosis was measured with flow cytometry. Treatment of HNSCC cells with cafestol leads to a dose-dependent reduction of cell viability and to induction of apoptosis. Combination with irradiation shows a reduction of clonogenic survival compared to each treatment method alone. In two of the cell lines a significant additive effect was observed. Cafestol is a naturally occurring effective compound with growth-inhibiting properties in head and neck cancer cells. Moreover, it leads to a significant inhibition of colony formation. (orig.) [de

Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

International Nuclear Information System (INIS)

Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

2005-01-01

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

1α-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines

Science.gov (United States)

Lagishetty, Venu; Chun, Rene F.; Liu, Nancy Q.; Lisse, Thomas S.; Adams, John S.; Hewison, Martin

2010-01-01

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase). In cells such as macrophages, expression of 1α-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and β-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1α-hydroxylase. Alternative activators of 1α-hydroxylase such as transforming growth factor β were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. PMID:20152900

Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

DEFF Research Database (Denmark)

Odum, Niels; Ledbetter, J A; Martin, P

1991-01-01

Major histocompatibility complex (MHC) class II molecules have been implicated in cell adhesion in two ways. In addition to the well-established role of class II antigens in low-affinity adhesion provided by interactions between class II and CD4, recent data indicated that class II may also induce...... adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human...... class II-positive monocytic (I937) and T leukemic (HUT78) tumor cell lines and Epstein-Barr virus (EBV) transformed B-lymphoid cell lines (EBV-LCL). Class II-negative cell lines (U-937 and the EBV-LCL mutant line 616) were not induced to aggregate. An HLA-G-transfected EBV-LCL, 221-AGN...

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

Directory of Open Access Journals (Sweden)

Kazuya Takahashi

Full Text Available Prognosis of childhood acute lymphoblastic leukemia (ALL has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ, a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+ ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ, a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19 ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19 ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19 ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

Science.gov (United States)

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid

In vitro cytotoxicity of alpha conjugates for human pancreatic cancer cell lines

International Nuclear Information System (INIS)

Qu, C.; Li, Y.; Rizvi, M.A.; Allen, B.; Samra, J.; Smith, R.

2003-01-01

Targeted Alpha therapy (TAT) can inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The aim of this study is to demonstrate the cytotoxicity of different alpha conjugates in vitro to human metastatic pancreatic cancer cell lines (CAPAN-1, CFPAN-1 and PANC-1). We are labeling the C595 and J591 (non-specific controls) monoclonal antibodies (Mabs) with 213 Bi were performed according to the standard methods in our laboratory. 213 Bi-C595 is specifically cytotoxic to CAPAN-1, CFPAN-1 and PANC-1cell lines in a concentration-dependent fashion. While non-specific alpha conjugates only killed very small fractions of pancreatic cancer cells. These alpha conjugates might be useful agents for the treatment of micro-metastases in pancreatic cancer patients with over-expression of the targeted receptors

Anticancer activity of Cynodon dactylon and Oxalis corniculata on Hep2 cell line.

Science.gov (United States)

Salahuddin, H; Mansoor, Q; Batool, R; Farooqi, A A; Mahmood, T; Ismail, M

2016-04-30

Bioactive chemicals isolated from plants have attracted considerable attention over the years and overwhelmingly increasing laboratory findings are emphasizing on tumor suppressing properties of these natural agents in genetically and chemically induced animal carcinogenesis models. We studied in vitro anticancer activity of organic extracts of Cynodon dactylon and Oxalis corniculata on Hep2 cell line and it was compared with normal human corneal epithelial cells (HCEC) by using MTT assay. Real Time PCR was conducted for p53 and PTEN genes in treated cancer cell line. DNA fragmentation assay was also carried out to note DNA damaging effects of the extracts. The minimally effective concentration of ethanolic extract of Cynodon dactylon and methanolic extract of Oxalis corniculata that was nontoxic to HCEC but toxic to Hep2 was recorded (IC50) at a concentration of 0.042mg/ml (49.48 % cell death) and 0.048mg/ml (47.93% cell death) respectively, which was comparable to the positive control. Our results indicated dose dependent increase in cell death. P53 and PTEN did not show significant increase in treated cell line. Moreover, DNA damaging effects were also not detected in treated cancer cell line. Anticancer activity of these plants on the cancer cell line showed the presence of anticancer components which should be characterized to be used as anticancer therapy.

Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

Directory of Open Access Journals (Sweden)

Tobias Hannes

2015-01-01

Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

DNA fingerprinting of glioma cell lines and considerations on similarity measurements.

Science.gov (United States)

Bady, Pierre; Diserens, Annie-Claire; Castella, Vincent; Kalt, Stefanie; Heinimann, Karl; Hamou, Marie-France; Delorenzi, Mauro; Hegi, Monika E

2012-06-01

Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.

Virus specific antigens in mammalian cells infected with herpes simplex virus

Science.gov (United States)

Watson, D. H.; Shedden, W. I. H.; Elliot, A.; Tetsuka, T.; Wildy, P.; Bourgaux-Ramoisy, D.; Gold, E.

1966-01-01

Antisera to specific proteins in herpes simplex infected cells were produced by immunization of rabbits with infected rabbit kidney cells. These antisera were highly virus specific and produced up to twelve lines in immunodiffusion tests against infected cell extracts. Acrylamide electrophoresis and immunoelectrophoresis revealed up to ten virus specific proteins of varying size. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5 PMID:4288648

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

Science.gov (United States)

Ma, Hailun; Galvin, Teresa A; Glasner, Dustin R; Shaheduzzaman, Syed; Khan, Arifa S

2014-06-01

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell

A novel RNA sequencing data analysis method for cell line authentication.

Directory of Open Access Journals (Sweden)

Erik Fasterius

Full Text Available We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories.

Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

DEFF Research Database (Denmark)

Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna

2015-01-01

Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...... based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted...... for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies....

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Science.gov (United States)

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Characteristics of replication and radiation response of Aedes Albopictus cell line in vitro

International Nuclear Information System (INIS)

Lee, C.K.

1974-01-01

The radiosensitivity of the line of Aedes albopictus cells was investigated by scoring x-ray-induced chromosome aberrations as a function of dose and of time after irradiation as well as the modification by dose fractionation. In order to obtain these data, a series of studies, e.g., karyotype, cell life cycle, radiation- induced mitotic delay, and frequency and type of spontaneous aberrations, were carried out. Cells from this line had three pairs of chromosomes as a stem line chromosome number. The morphology of the chromosomes is metacentric. Somatic pairing between homologous chromosomes was observed as a common event and there was a high frequency of achromatic gaps on the chromosomes. These observations are in good agreement with those made in other laboratories. The generation time of A. albopictus cell line was approximately 32 hours, in which G 1 , S, and G 2 phases were 2 1 / 2 , 24 and 5 1 / 2 hours respectively. This generation time is in agreement with the population doubling time observed in our cell growth studies. By contrast, in most mammalian cells, G 1 usually is the longest phase and S is comparatively short. Dose fractionation studies indicated that A. albopictus cells in culture may have a faster chromosome repair system than mammalian and other cell systems. This may, at least in part; explain the difference in radiosensitivity between A. albopictus cell line and other cell systems. (Diss. Abstr.)

Interaction between x-irradiated plateau-phase bone marrow stromal cell lines and co-cultivated factor-dependent cell lines leading to leukemogenesis in vitro

International Nuclear Information System (INIS)

Naparstek, E.; Anklesaria, P.; FitzGerald, T.J.; Sakakeeny, M.A.; Greenberger, J.S.

1987-01-01

Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia

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

Science.gov (United States)

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

2016-08-01

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

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

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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

Application of the inter-line PCR for the analyse of genomic rearrangements in radiation-transformed mammalian cell lines

International Nuclear Information System (INIS)

Leibhard, S.; Smida, J.

1996-01-01

Repetitive DNA sequences of the LINE-family (long interspersed elements) that are widely distributed among the mammalian genome can be activated or altered by the exposure to ionizing radiation [1]. By the integration at new sites in the genome alterations in the expression of genes that are involved in cell transformation and/or carcinogenesis may occur [2, 3]. A new technique -the inter-LINE PCR - has been developed in order to detect and analyse such genomic rearrangements in radiation-transformed cell lines. From the sites of transformation- or tumour-specific changes in the genome it might be possible to develop new tumour markers for diagnostic purpose. (orig.) [de

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

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Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

OpenAIRE

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

Science.gov (United States)

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

Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin

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Rothuizen Jan

2006-09-01

Full Text Available Abstract Background Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE, mammary carcinoma (P114, and osteosarcoma (D17. These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay. Results All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively. Conclusion XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors.

Differential biological effects of iodoacetate in mammalian cell lines; radio sensitization and radio protection

International Nuclear Information System (INIS)

Yadav, Usha; Anjaria, K.B.; Desai, Utkarsha N.; Chaurasia, Rajesh K.; Shirsath, K.B.; Bhat, Nagesh N.; Balakrishnan, Sreedevi; Sapra, B.K.; Nairy, Rajesha

2014-01-01

There are several studies where it has been shown that Iodoacetate (IA) possesses in vivo anti-tumor activity. The fact that it is a model glycolytic inhibitor makes it more interesting. As seen in recent trends, glycolytic inhibitors are emerging as new strategy for cancer therapeutic research taking advantage of glycolytic phenotype of cancerous tissues. IA has been reported to have radioprotective effects in yeast cells and human lymphocytes. Biological effects of IA in response to radiation in mammalian cell lines are not well documented. We screened IA for cytotoxicity using clonogenic assay at different concentrations ranging from 0.1 to 2.5 μg/ml using three different mammalian cell lines; A-549 (human lung carcinoma cell line), MCF-7 (human mammary cancer cell line) and a noncancerous CHO (Chinese hamster ovary cell line). For studying radioprotective/radio sensitizing efficacy, cells were exposed to 4 Gy of 60 Co-γ radiation using a teletherapy source at a dose rate of 1 Gy/min, following which IA post-treatment was carried out. Clonogenic and micronucleus assay were performed to assess radioprotection/sensitization. The results indicated that IA was highly cytotoxic in cancerous cell lines A-549 (IC 50 =1.25 μg/ml) and MCF-7 (IC 50 = 1.9 μg/ml). In contrast, it was totally non-toxic in non-cancerous cell line, viz. CHO, in the same concentration range. In addition, IA exhibited radio protective effect in CHO cell line, whereas in other two cancer cell lines, viz. A-549 and MCF-7, radio sensitizing effect was seen as judged by induction of cell killing and micronuclei. In conclusion, lA, a model glycolytic inhibitor, was found to be selectively cytotoxic in cancer cells as compared to normal cells. Further, it reduced radiation induced damage (micronuclei and cell killing) in normal cells but increased it in cancer cells indicating its potential use in cancer therapy. (author)

1alpha-hydroxylase and innate immune responses to 25-hydroxyvitamin D in colonic cell lines.

Science.gov (United States)

Lagishetty, Venu; Chun, Rene F; Liu, Nancy Q; Lisse, Thomas S; Adams, John S; Hewison, Martin

2010-07-01

Vitamin D-insufficiency is a prevalent condition in populations throughout the world, with low serum levels of 25-hydroxyvitamin D (25OHD) linked to a variety of human health concerns including cancer, autoimmune disease and infection. Current data suggest that 25OHD action involves localized extra-renal conversion to 1,25-dihydroxyvitamin D (1,25(OH)2D) via tissue-specific expression of the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase). In cells such as macrophages, expression of 1alpha-hydroxylase is intimately associated with toll-like receptor (TLR) recognition of pathogens. However, this mechanism may not be exclusive to extra-renal generation of 1,25(OH)2D. To investigate the relationship between TLR-mediated pathogen recognition and vitamin D-induced antibacterial activity, intracrine responses to 25OHD metabolism were explored in vitro using the established colonic cell lines Caco-2 and Caco-2 clone BBe. Analysis of antibacterial factors such as cathelicidin (LL37) and beta-defensin-4 (DEFB4) was carried out following co-treatment with TLR ligands. Data indicate that, unlike macrophages, Caco-2 and BBe colonic cell lines are unresponsive to TLR-induced 1alpha-hydroxylase. Alternative activators of 1alpha-hydroxylase such as transforming growth factor beta were also ineffective at priming intracrine responses to 25OHD. Thus, in common with other barrier sites such as the skin or placenta, colonic epithelial cells may require specific factors to initiate intracrine responses to vitamin D. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Generation of an induced pluripotent stem cell line, IBMS-iPSC-014-05, from a female autosomal dominant polycystic kidney disease patient carrying a common mutation of R803X in PKD2

Directory of Open Access Journals (Sweden)

Ming-Ching Ho

2017-12-01

Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is one of the most commonly inherited forms of polycystic kidney disease, and is characterized by the growth of numerous cysts in both kidneys. Here we generated an induced pluripotent stem cell (iPSC line from the peripheral blood mononuclear cells (PBMCs of a 63-year-old female ADPKD patient carrying an R803X mutation in the PKD2 gene using the Sendai-virus delivery system. Downstream characterization of these iPSCs showed that they possessed normal karyotyping, were free of genomic integration, retained the disease-causing PKD2 mutation, expressed pluripotency markers and could differentiate into three germ layers.

Generation of human induced pluripotent stem cell line from a patient with a long QT syndrome type 2

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Azra Fatima

2016-03-01

Full Text Available We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55 in KCNH2 gene leading to long QT syndrome type 2 (LQT2. We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC.

Adaptive response to ionizing radiation induced by low doses of gamma rays in human cell lines

International Nuclear Information System (INIS)

Seong, Jinsil; Chang, Ok Suh; Gwi, Eon Kim

1995-01-01

Purpose: The aim of this study was to investigate whether the adaptive response could be induced in human lymphoblastoid cell lines and human tumor cell lines. The time necessary for the expression of the adaptive response was also investigated. Materials and Methods: Three lymphoblastoid cell lines from ataxia telangiectasia (AT) homozygote (GM 1526), AT heterozygote (GM 3382), and normal individual (3402p) and two hepatoma cell lines, Hep G2 and Hep 3B, were used in this study. Experiments were carried out by delivering 0.01 Gy followed by 0.5 Gy of gamma radiation to the exponentially growing cells. The time necessary for the expression of the adaptive response was determined by varying the time interval between the two doses from 1 to 72 h. In some experiments, 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, was added immediately after the 0.5 Gy exposure. The cultures were fixed 30 min (for the G 2 chromatid) and 6 h (for the S chromatid) after the 0.5 Gy exposure. Metaphase chromosome assay was carried out to score chromatid breaks as an end point. Results: A prior exposure to 0.01 Gy of gamma rays significantly reduced the number of chromatid breaks induced by subsequent higher doses (0.5 Gy) in all the tested cell lines. The magnitude of the adaptive response was similar among the cell lines despite their different radiosensitivities. In the G 2 chromatids, the adaptive response was observed both at short-time intervals, as early as 1 h, and at long-time intervals. In the S chromatids, however, the adaptive response was shown only at long-time intervals. When 3-aminobenzamide was added after the 0.5 Gy, the adaptive responses were abolished in all the experimental groups. Conclusion: The adaptive response was observed in human lymphoblastoid cell lines and hepatoma cell lines. The magnitude of the adaptive response did not seem to be related to the radiosensitivity of the cells. The elimination of the adaptive response with 3

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

International Nuclear Information System (INIS)

Fujii, Hodaka

2007-01-01

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Science.gov (United States)

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

The Cellosaurus, a Cell-Line Knowledge Resource

Science.gov (United States)

Bairoch, Amos

2018-01-01

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

Establishment of induced pluripotent stem cell line (ZZUi010-A from an Alzheimer's disease patient carrying an APP gene mutation

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Zhilei Wang

2017-12-01

Full Text Available Alzheimer's disease (AD is one of the most common neurodegenerative disorders. Previous studies have identified mutations in several genes, such as amyloid precursor protein (APP, presenilin-1 (PSEN1, and presenilin-2 (PSEN2, in patients with early-onset (<65 years familial AD. Recently, a patient with an APP gene mutation was identified; the dermal fibroblasts of the patient were obtained and a line of induced pluripotent stem cells (iPSCs was successfully generated using the Sendai-virus (SeV delivery system. The iPSC line will be useful for further study of the pathomechanism and drug screening for AD.

Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

International Nuclear Information System (INIS)

Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara

2011-01-01

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

Science.gov (United States)

Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

2017-10-01

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

Science.gov (United States)

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

In vitro radiosensitivity of six human cell lines. A comparative study with different statistical models

International Nuclear Information System (INIS)

Fertil, B.; Deschavanne, P.J.; Lachet, B.; Malaise, E.P.

1980-01-01

The intrinsic radiosensitivity of human cell lines (five tumor and one nontransformed fibroblastic) was studied in vitro. The survival curves were fitted by the single-hit multitarget, the two-hit multitarget, the single-hit multitarget with initial slope, and the quadratic models. The accuracy of the experimental results permitted evaluation of the various fittings. Both a statistical test (comparison of variances left unexplained by the four models) and a biological consideration (check for independence of the fitted parameters vis-a-vis the portion of the survival curve in question) were carried out. The quadratic model came out best with each of them. It described the low-dose effects satisfactorily, revealing a single-hit lethal component. This finding and the fact that the six survival curves displayed a continuous curvature ruled out the adoption of the target models as well as the widely used linear regression. As calculated by the quadratic model, the parameters of the six cell lines lead to the following conclusions: (a) the intrinsic radiosensitivity varies greatly among the different cell lines; (b) the interpretation of the fibroblast survival curve is not basically different from that of the tumor cell lines; and (c) the radiosensitivity of these human cell lines is comparable to that of other mammalian cell lines

Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration

International Nuclear Information System (INIS)

Guelden, Michael; Moerchel, Sabine; Seibert, Hasso

2005-01-01

Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC 50 values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC 50 values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC 50 values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

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Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transcriptome variations among human embryonic stem cell lines are associated with their differentiation propensity.

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Changbin Sun

Full Text Available Human embryonic stem cells (hESCs have the potential to form any cell type in the body, making them attractive cell sources in drug screening, regenerative medicine, disease and developmental processes modeling. However, not all hESC lines have the equal potency to generate desired cell types in vitro. Significant variations have been observed for the differentiation efficiency of various human ESC lines. The precise underpinning molecular mechanisms are still unclear. In this work, we compared transcriptome variations of four hESC lines H7, HUES1, HUES8 and HUES9. We found that hESC lines have different gene expression profiles, and these differentially expressed genes (DEGs are significantly enriched in developmental processes, such as ectodermal, mesodermal and endodermal development. The enrichment difference between hESC lines was consistent with its lineage bias. Among these DEGs, some pluripotency factors and genes involved in signaling transduction showed great variations as well. The pleiotropic functions of these genes in controlling hESC identity and early lineage specification, implicated that different hESC lines may utilize distinct balance mechanisms to maintain pluripotent state. When the balance is broken in a certain environment, gene expression variation between them could impact on their different lineage specification behavior.

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

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Zaborski Margarete

2009-01-01

Full Text Available Abstract Background SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9(q34.11q34.13 has recently been described in T-cell acute lymphoblastic leukemia (T-ALL and in one case of acute myeloid leukemia (AML. The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene. Results Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH and array-based copy number analysis were both consistent with del(9(q34.11q34.13 as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein. Conclusion Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

A universal mammalian vaccine cell line substrate.

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Jackelyn Murray

Full Text Available Using genome-wide small interfering RNA (siRNA screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205 showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

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Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Investigation of Cytocidal Activity of Bacillus Thuringiensis Parasporal Toxin on CCRF-CEM Cell Line

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Elham Moazamian

2013-03-01

Full Text Available Background & Objective: Parasporin is a parasporal protein of Bacillus thuringiensis and exhibits special cytocidal activity against human cancer cells. Similar to other insecticidal Bacillus thuringiensis crystal toxins, parasporin shows target specificity and damages the cellular membrane. In this study, different strains of Bacillus thuringiensis isolated from various regions of Iran and their cytocidal activity against CCRF-CEM cell line and human erythrocyte were investigated.   Materials & Methods: Fifty soil samples were collected from different Iranian provinces, and characterization was performed based on protein crystal morphology by phase-contrast microscope and variations of Cry protein toxin using SDS-PAGE. After parasporin was processed with proteinase K, the active form was produced and protein activity on the cell line was evaluated. Results: Parasporal inclusion proteins showed different cytotoxicity against acute lymphoblastic leukemia cells (ALL, but not against normal lymphocyte. Isolated parasporin demonstrated no hemolytic activity against human erythrocyte. It appears that these proteins have the ability to differentiate between normal lymphocytes and leukemia cells and have specific receptors on specific cancer cell lines. Conclusion: Our results provide evidence that the parasporin-producing organism is a common member in Bacillus thuringiensis populations occurring in the natural environments of Iran.

C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.

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Michael W Holliday

Full Text Available We previously reported that fenretinide (4-HPR was cytotoxic to acute lymphoblastic leukemia (ALL cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04 and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004, but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001 and cytotoxicity (P ≤ 0.001. These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides

Gene expression-signature of belinostat in cell lines is specific for histone deacetylase inhibitor treatment, with a corresponding signature in xenografts

DEFF Research Database (Denmark)

Monks, A.; Hose, C.D.; Pezzoli, P.

2009-01-01

gene modulation were significantly correlated. A belinostat-gene profile was specific for HDACi in three cell lines when compared with equipotent concentrations of four mechanistically different chemotherapeutic agents: 5-fluorouracil, cisplatin, paclitaxel, and thiotepa. Belinostat- and trichostatin...... in a drug-sensitive tumor than a more resistant model. We have demonstrated a gene signature that is selectively regulated by HDACi when compared with other clinical agents allowing us to distinguish HDACi responses from those related to other mechanisms Udgivelsesdato: 2009/9...

Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines

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Imrich Amy

2008-05-01

Full Text Available Abstract Background Alveolar macrophages (AM avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/- mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice. Results We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF, we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2, fluorescent latex beads and bacteria (Staphylococcus aureus compared with the primary AMs from wild type (WT C57BL/6 mice. Conclusion Our results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6 were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define

Derivation and characterization of monkey embryonic stem cells

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Wolf Don P

2004-06-01

Full Text Available Abstract Embryonic stem (ES cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

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Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

Science.gov (United States)

Norris, J S; Kohler, P O

1978-01-01

Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

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Stringer Saundra L

2006-10-01

Full Text Available Abstract Background Loss of heterozygosity (LOH contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. Results As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. Conclusion Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors

Quantitative analysis of genes regulating sensitivity to heavy ion irradiation in cultured cell lines of malignant choroid melanoma

International Nuclear Information System (INIS)

Kumagai, Ken; Adachi, Nanao; Nimura, Yoshinori

2004-01-01

As a treatment strategy for malignant melanoma, heavy ion irradiation has been planned in National Institute of Radiological Sciences (NIRS). However, the molecular biology of the malignant melanoma cell after irradiation of heavy ion is still unknown. In this study, we used resistant and sensitive cell lines of malignant melanoma to study the effects of heavy ion irradiation. Furthermore, gene expression profiling of early response genes for heavy ion irradiation was carried out on these cell lines using microarray technology. (author)

Quantitative analysis of genes regulating sensitivity to heavy ion irradiation in cultured cell lines of malignant choroid melanoma

International Nuclear Information System (INIS)

Kumagai, Ken; Nimura, Yoshinori; Kato, Masaki; Seki, Naohiko; Miyahara, Nobuyuki; Aoki, Mizuho; Shino, Yayoi; Furusawa, Yoshiya; Mizota, Atsushi

2005-01-01

As a treatment strategy for malignant melanoma, heavy ion irradiation has been planned in National Institute of Radiological Sciences (NIRS). However, the molecular biology of the malignant melanoma cell after irradiation of heavy ion is still unknown. In this study, we used resistant and sensitive cell lines of malignant melanoma to study the effects of heavy ion irradiation. Furthermore, gene expression profiling of early response genes for heavy ion irradiation was carried out on these cell lines using microarray technology. (author)

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Science.gov (United States)

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Construction of a novel lentiviral vector carrying human B-domain ...

African Journals Online (AJOL)

... integration were detected in all cell lines after transfection. A novel lentiviral vector carrying human FVIII³BD was constructed, which was able to transfect different mammalian cell types accompanied by high-level activity. This lentiviral vector may provide a theoretical basis for the gene therapy of patients with hemophilia ...

Characterization of stem-like cells in a new astroblastoma cell line

Energy Technology Data Exchange (ETDEWEB)

Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

2017-03-15

Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

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Silver, Kristopher; Jiang, Hongbo; Fu, Jinping; Phillips, Thomas W; Beeman, Richard W; Park, Yoonseong

2014-10-30

The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

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

Science.gov (United States)

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

DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

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Sahar Houshdaran

2010-02-01

Full Text Available Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies.We obtained DNA methylation profiles of 1,505 CpG sites (808 genes in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes that exhibited 'subtype-specific' DNA methylation patterns (FDR<1% among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene.The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of

The effect of the immunophilin ligands rapamycin and FK506 on proliferation of mast cells and other hematopoietic cell lines.

Science.gov (United States)

Hultsch, T; Martin, R; Hohman, R J

1992-01-01

The immunosuppressive drugs FK506 and cyclosporin A have an identical spectrum of activities with respect to IgE receptor (Fc epsilon RI)-mediated exocytosis from mast cells and T cell receptor-mediated transcription of IL-2. These findings suggest a common step in receptor-mediated signal transduction leading to exocytosis and transcription and imply that immunosuppressive drugs target specific signal transduction pathways, rather than specific cell types. This hypothesis is supported by studies on the effect of rapamycin on IL-3 dependent proliferation of the rodent mast cell line PT18. Rapamycin inhibits proliferation of PT18 cells, achieving a plateau of 80% inhibition at 1 nM. This inhibition is prevented in a competitive manner by FK506, a structural analogue of rapamycin. Proliferation of rat basophilic leukemia cells and WEHI-3 cells was also inhibited, at doses comparable to those shown previously to inhibit IL-2-dependent proliferation of cytotoxic T lymphocyte line (CTLL) cells. In contrast, proliferation of A-431 cells, a epidermoid cell line, was not affected by rapamycin. DNA histograms indicate that complexes formed between the rapamycin-FK506-binding protein (FKBP) and rapamycin arrest-proliferating PT18 cells in the G0/G1-phase. It is concluded that FKBP-rapamycin complexes may inhibit proliferative signals emanating from IL-3 receptors, resulting in growth arrest of cytokine-dependent, hematopoietic cells. PMID:1384815

Cytotoxicity of Portuguese Propolis: The Proximity of the In Vitro Doses for Tumor and Normal Cell Lines

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Ricardo C. Calhelha

2014-01-01

Full Text Available With a complex chemical composition rich in phenolic compounds, propolis (resinous substance collected by Apis mellifera from various tree buds exhibits a broad spectrum of biological activities. Recently, in vitro and in vivo data suggest that propolis has anticancer properties, but is the cytoxicity of propolis specific for tumor cells? To answer this question, the cytotoxicity of phenolic extracts from Portuguese propolis of different origins was evaluated using human tumor cell lines (MCF7—breast adenocarcinoma, NCI-H460—non-small cell lung carcinoma, HCT15—colon carcinoma, HeLa—cervical carcinoma, and HepG2—hepatocellular carcinoma, and non-tumor primary cells (PLP2. The studied propolis presented high cytotoxic potential for human tumor cell lines, mostly for HCT15. Nevertheless, excluding HCT15 cell line, the extracts at the GI50 obtained for tumor cell lines showed, in general, cytotoxicity for normal cells (PLP2. Propolis phenolic extracts comprise phytochemicals that should be further studied for their bioactive properties against human colon carcinoma. In the other cases, the proximity of the in vitro cytotoxic doses for tumor and normal cell lines should be confirmed by in vivo tests and may highlight the need for selection of specific compounds within the propolis extract.

Differentiation of Induced Pluripotent Stem Cells to Lentoid Bodies Expressing a Lens Cell-Specific Fluorescent Reporter.

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Taruna Anand

Full Text Available Curative approaches for eye cataracts and other eye abnormalities, such as myopia and hyperopia currently suffer from a lack of appropriate models. Here, we present a new approach for in vitro growth of lentoid bodies from induced pluripotent stem (iPS cells as a tool for ophthalmological research. We generated a transgenic mouse line with lens-specific expression of a fluorescent reporter driven by the alphaA crystallin promoter. Fetal fibroblasts were isolated from transgenic fetuses, reprogrammed to iPS cells, and differentiated to lentoid bodies exploiting the specific fluorescence of the lens cell-specific reporter. The employment of cell type-specific reporters for establishing and optimizing differentiation in vitro seems to be an efficient and generally applicable approach for developing differentiation protocols for desired cell populations.

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

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Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

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Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2016-01-01

Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

Adaptive Response to ionizing Radiation Induced by Low Doses of Gamma Rays in Human Lymphoblastoid Cell Lines

International Nuclear Information System (INIS)

Seong, Jin Sil; Suh, Chang Ok; Kim, Gwi Eon

1994-01-01

When cells are exposed to low doses of a mutagenic or clastogenic agents, they often become less sensitive to the effects of a higher does administered subsequently. Such adaptive responses were first described in Escherichia coli and mammalian cells to low doses of an alkylating agent. Since most of the studies have been carried out with human lymphocytes, it is urgently necessary to study this effect in different cellular systems. Its relation with inherent cellular radiosensitivity and underlying mechanism also remain to be answered. In this study, adaptive response by 1 cGy of gamma rays was investigated in three human lymphoblastoid cell lines which were derived from ataxia telangiectasia homozygote, ataxia telangiectasia heterozygote, and normal individual. Experiments were carried out by delivering 1 cGy followed by 50 cGy of gamma radiation and chromatid breaks were scored as an endpoint. The results indicate that prior exposure to 1 cGy of gamma rays reduces the number of chromatid breaks induced by subsequent higher does (50 cGy). The expression of this adaptive response was similar among three cell lines despite of their different radiosensitivity. When 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, was added after 50 cGy, adaptive responses were abolished in all the tested cell lines. Therefore it is suggested that the adaptive response can be observed in human lymphoblastoid cell lines. Which was first documented through this study. The expression of adaptive response was similar among the cell lines regardless of their radiosensitivity. The elimination of the adaptive response by 3-aminobenzamide is consistent with the proposal that this adaptive response is the result of the induction of a certain chromosomal repair mechanism

An avian cell line designed for production of highly attenuated viruses.

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Jordan, Ingo; Vos, Ad; Beilfuss, Stefanie; Neubert, Andreas; Breul, Sabine; Sandig, Volker

2009-01-29

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

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Andrew R Dalby

Full Text Available Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Cell fusion induced by ionizing radiation in various cell lines

International Nuclear Information System (INIS)

Khair, M.B.

1994-07-01

Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

Cytokeratin characterization of human prostatic carcinoma and its derived cell lines.

Science.gov (United States)

Nagle, R B; Ahmann, F R; McDaniel, K M; Paquin, M L; Clark, V A; Celniker, A

1987-01-01

Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.

Doxorubicin-conjugated bacteriophages carrying anti-MHC class I chain-related A for targeted cancer therapy in vitro

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Phumyen A

2014-11-01

Full Text Available Achara Phumyen,1–3 Siriporn Jantasorn,1 Amonrat Jumnainsong,1 Chanvit Leelayuwat1–4 1The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL, Faculty of Associated Medical Sciences, 2The Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, 3Research Cluster: Specific Health Problem of Grater Maekong Subregion (SHeP-GMS, 4Department of Clinical Immunology and Transfusion Sciences, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand Background: Cancer therapy by systemic administration of anticancer drugs, besides the effectiveness shown on cancer cells, demonstrated the side effects and cytotoxicity on normal cells. The targeted drug-carrying nanoparticles may decrease the required drug concentration at the site and the distribution of drugs to normal tissues. Overexpression of major histocompatibility complex class I chain–related A (MICA in cancer is useful as a targeted molecule for the delivery of doxorubicin to MICA-expressing cell lines. Methods: The application of 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC chemistry was employed to conjugate the major coat protein of bacteriophages carrying anti-MICA and doxorubicin in a mildly acid condition. Doxorubicin (Dox on phages was determined by double fluorescence of phage particles stained by M13-fluorescein isothiocyanate (FITC and drug autofluorescence by flow cytometry. The ability of anti-MICA on phages to bind MICA after doxorubicin conjugation was evaluated by indirect enzyme-linked immunosorbent assay. One cervical cancer and four cholangiocarcinoma cell lines expressing MICA were used as models to evaluate targeting activity by cell cytotoxicity test. Results: Flow cytometry and indirect enzyme-linked immunosorbent assay demonstrated that most of the phages (82% could be conjugated with doxorubicin, and the Dox-carrying phage-displaying anti-MICA (Dox-phage remained the binding activity against MICA

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

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

2009-09-01

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

Analysis of cell line variation in biochemical production of protoporphyrin IX

Science.gov (United States)

Gibbs, Summer L.; Chen, Bin; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

2006-02-01

Protoporphyrin IX (PpIX) is produced via the heme synthesis pathway by the cell following administration of aminolevulinic acid (ALA). ALA synthase, the enzyme that produces ALA in the cell from glycine and succinyl-coenzyme A, is inhibited in a feedback mechanism by heme and thus is the rate limiting enzyme in the heme synthesis pathway. Since ALA is administered systemically, the rate limiting step that naturally exists in the cells is bypassed, however it is currently unclear why cells have different rate limiting steps in the ALA-PpIX synthesis pathway, and more specifically which types of cancer cells are most productive. It has been determined that when the same amount of ALA is administered to a wide panel of cancer cells in vitro that vastly differing amounts of PpIX are produced. The steps for the ALA-PpIX pathway occur in and around the mitochondria of the cell, but interestingly no correlation is seen between PpIX production and mitochondrial content of the cell, following ALA administration. However, total cell area shows positive correlation with PpIX production. Administration of the iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed and thus increases the detectable amount of PpIX in each cell line. The cell lines that have the lowest PpIX production following administration of ALA alone show the largest increase in production following the combined administration of ALA and L1. PpIX fluorescence is thought to be a measure of cellular activity and the goal of the current study was to determine which cell lines would be the most promising targets for fluorescence detection or monitoring response to therapy. The results indicate that the cells with larger size and larger numbers of mitochondria may be good potential targets for this therapy. While this conclusion may appear obvious, it is not universally true, and cellular specific

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

Science.gov (United States)

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Fibronectin synthesized by a human hepatoma cell line

International Nuclear Information System (INIS)

Glasgow, J.E.; Colman, R.W.

1984-01-01

Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-[ 35 S]methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

Science.gov (United States)

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.

Application of DNA fingerprints for cell-line individualization.

Science.gov (United States)

Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

1990-09-01

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

Application of fish cell lines for evaluating the chromium induced cytotoxicity, genotoxicity and oxidative stress.

Science.gov (United States)

Taju, G; Abdul Majeed, S; Nambi, K S N; Sahul Hameed, A S

2017-10-01

In the present study, we hypothesize that cytotoxicity, genotoxicity and oxidative stress play a key role in chromium induced toxicity in SISS, SISK, IEE, IEK, IEG, SICH and ICG cell lines after 24 h exposure. Three fish species namely Lates calcarifer, Etroplus suratensis and Catla catla were exposed to the concentrations of 0, 10, 20, 30, 40 and 50 mg/L of chromium for 96 h under static conditions for conducting acute toxicity tests. LC 50 was then calculated. The percentage cell survival was assessed by multiple endpoints such as MTT, NR, AB and CB assays in the seven fish cell lines exposed to different concentrations of chromium and EC 50 values of all the four endpoints were calculated. High significances were noted in the correlations between each in vitro cytotoxicity assays and in vivo mortality data. Cell shrinkage, cell detachment, vacuolations and cell swelling at the highest concentration of chromium (50 mg/L) were seen on microscopic examination of cell morphology. Comet assay and Hoechst staining were carried out to assess DNA damage and nuclear fragmentation in the seven fish lines exposed to chromium. The results of antioxidant parameters obtained indicate a significant reduction in the level of catalase, superoxide dismutase, glutathione S-transferase and Glutathione peroxidase, and increased level of lipid peroxidation in all the cell lines exposed to chromium. These results confirm that fish cell lines could be used as an alternative to whole fish for cytotoxicity, genotoxicity and oxidative stress assessment in chromium toxicity studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines

DEFF Research Database (Denmark)

Sørensen, Rikke Baek; Junker, Niels; Kirkin, Alexei

2009-01-01

Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most-if not the most-frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large...... panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma...... cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing...

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

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Chen Lei

2011-06-01

Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

DEFF Research Database (Denmark)

Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte

2013-01-01

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface...... a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA...

Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis.

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Godfrey, Charlotte L; Mead, Emma J; Daramola, Olalekan; Dunn, Sarah; Hatton, Diane; Field, Ray; Pettman, Gary; Smales, C Mark

2017-08-01

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5' untranslated regions that might be utilised to enhance recombinant transcript translation. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

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Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

Science.gov (United States)

Qin, J-Z; Xin, H; Nickoloff, B J

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

International Nuclear Information System (INIS)

Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

2010-01-01

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

Science.gov (United States)

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

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Kendall K

2013-05-01

Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

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Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

Science.gov (United States)

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for

Functional activity of human sodium/iodide symporter in tumor cell lines

International Nuclear Information System (INIS)

Petrich, T.; Knapp, W.H.; Poetter, E.

2003-01-01

Aim: The sodium/iodide symporter (NIS) actively transports iodide into thyrocytes. Thus, NIS represents a key protein for diagnosis and radioiodine therapy of differentiated thyroid cancer. Additionally, in the future the NIS gene may be used for cancer gene therapy of non-thyroid-derived malignancies. In this study we evaluated the functionality of NIS with respect to iodide uptake in a panel of tumor cell lines and compared this to gene transfer efficiency. Methods: A human NIS-containing expression vector and reporter-gene vectors encoding and beta;-Galactosidase- or EGFP were used for transient transfection of 13 tumor cell lines. Following transfection measurements of NIS-mediated radioiodide uptake using Na 125 I and of transfection efficiency were performed. The latter included β;-Galactosidase activity measurements using a commercial kit and observation by fluorescence microscopy for EGFP expression. Results: In contrast to respective parental cells, most NIS-transfected cell lines displayed high, perchlorate-sensitive radioiodide uptake. Differences in radioiodide uptake between cell lines apparently corresponded to transfection efficiencies, as judged from reporter-gene assays. Conclusion: With respect to iodide uptake we provide evidence that NIS is functional in different cellular context. As iodide uptake capacity appears to be well correlated to gene transfer efficiency, cell type-specific actions on NIS (e. g. post-translational modification such as glycosylation) are not inhibitory to NIS function. Our data support the promising role of NIS in cancer gene therapy strategies. (orig.)

Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

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Lotta E Andersson

Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

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

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Artur Brandt

Full Text Available 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

Science.gov (United States)

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

Synergistic Effects of Cabozantinib and EGFR-Specific CAR-NK-92 Cells in Renal Cell Carcinoma

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Qing Zhang

2017-01-01

Full Text Available The chimeric antigen receptor-modified immune effector cell (CAR-T and CAR-NK therapies are newly developed adoptive treatments of cancers. However, their therapeutic efficacy against solid tumors is limited. Combining CAR-T or CAR-NK cells with chemotherapeutic drugs to treat solid tumor may be a promising strategy. We developed an epidermal growth factor- (EGFR- specific third-generation CAR. NK-92 cells were modified with the CAR by lentivirus infection. The specific killing ability of the CAR-modified NK-92 cells (CAR-NK-92 against renal cell carcinoma (RCC cell lines was confirmed in vitro. The synergistic effects of cabozantinib and EGFR-specific CAR-NK-92 cells were investigated in vitro and in vivo. Our results showed that the CAR-NK-92 cells lyse RCC cells in an EGFR-specific manner. Treatment with cabozantinib could increase EGFR and decrease PD-L1 membrane surface expression in RCC cells and enhance the killing ability of CAR-NK-92 cells against the RCC cells in vitro. Furthermore, the CAR-NK-92 cells show synergistic therapeutic efficacy with cabozantinib against human RCC xenograft models. Our results provided the basis for combination with chemotherapy as a novel strategy for enhancing the therapeutic efficacy of CAR-modified immune effector cells for solid tumors.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

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Wu Xuhong

2006-08-01

Full Text Available Abstract Background INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN. It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. Results We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Conclusion Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

Science.gov (United States)

Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and

Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

Energy Technology Data Exchange (ETDEWEB)

Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo [Nano-optoelectronics Research and Technology Laboratory (NOR.), School of Physics, Universiti Sains Malaysia, 11800, USM, Pulau Pinang (Malaysia); Mohamed, Azman Seeni; Saifuddin, Siti Nazmin [Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Pulau Pinang (Malaysia); Masudi, Sam’an Malik; Mohamad, Dasmawati [Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

2015-04-24

ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

International Nuclear Information System (INIS)

Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo; Mohamed, Azman Seeni; Saifuddin, Siti Nazmin; Masudi, Sam’an Malik; Mohamad, Dasmawati

2015-01-01

ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line

A novel dual-color reporter for identifying insulin-producing beta-cells and classifying heterogeneity of insulinoma cell lines.

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Nan Sook Lee

Full Text Available Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+ beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+ phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+ and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.

Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models

International Nuclear Information System (INIS)

Hara, H.; Seon, B.K.

1987-01-01

In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undersirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. The authors have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Ascitic and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppression the in vivo growth of the ascitic tumor, they divided 40 nude mice that were injected with Ichikawa cells into four groups. None of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment

Establishment and characterization of a novel Hodgkin lymphoma cell line, AM-HLH, carrying the Epstein-Barr virus genome integrated into the host chromosome.

Science.gov (United States)

Hayashida, Masahiko; Daibata, Masanori; Tagami, Erika; Taguchi, Takahiro; Maekawa, Fumiyo; Takeoka, Kayo; Fukutsuka, Katsuhiro; Shimomura, Daiki; Hayashi, Takamasa; Iwatani, Yoshinori; Ohno, Hitoshi

2017-12-01

We describe the establishment and characterization of a cell line, AM-HLH, obtained from a patient with Epstein-Barr virus-positive (EBV + ) nodular sclerosis-type Hodgkin lymphoma (HL). The cells were positive for CD2 and CD30 and negative for CD15. The immunoglobulin heavy- and κ light-chain genes were rearranged. The karyotype was of the triploid range. Southern blotting using the EBV terminal repeat probe detected 3 hybridizing bands that were identical to those of the parental HL material. The cells expressed EBV-encoded RNAs as well as latent genes (EBNA1, EBNA2, LMP1, and LMP2A) and lytic genes (BZLF1 and BALF2). Fluorescence in situ hybridization (FISH) with the cosmid pJB8 clone containing a fragment of EBV DNA as a probe revealed multiple hybridization signals at a marker chromosome. Additional FISH using whole chromosome painting and centromere probes in combination with multicolor FISH determined that multiple EBV copies were clustered within the chromosome 20 materials of the marker chromosome. Culture supernatants of AM-HLH contained IL-10 as measured by the bead-based immunoassay. It is possible that an integrated EBV genome and cellular genes on chromosome 20 were coamplified, leading to the enhanced expression of genes involved in cell growth control. The AM-HLH cell line will be useful to clarify the role of cytokines in the development of EBV + HL. Copyright © 2016 John Wiley & Sons, Ltd.

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

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Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

2015-06-01

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

Establishment of immortalized B lymphoblastoid cell lines of old residents in high background radiation area in Guangdong, China

International Nuclear Information System (INIS)

Lu Xue; Feng Jiangbing; Chen Deqing; Liu Qingjie; Cha Yongru; Zou Jianming

2008-01-01

Objective: To establish the immortalized cell lines of peripheral blood lymphocytes for old male residents in high background radiation area (HBRA) in Guangdong, China, in order to preserve the specific genomic resources of residents in HBRA for the further genetic and molecular biological study on HBRA. Methods: The peripheral blood samples of 20 old male residents in HBRA were collected after informed consent. The immortalized B lymphoblastoid cell lines, 2 fox each resident, were established with Epstein-Barr virus. After being frozen and recovered, the cell viability, the contamination of bacterium and mycoplasma were analyzed. The stabilization of cell lines was decided by comparing the karyotypes of the peripheral blood lymphocytes and the cell lines. Results: 40 cell lines for 20 residents in HBRA were successfully established.. The recovery rate of cell lines after being frozen was 100% . All the cell viablity after recovery was higher than 90%, and no contamination of bacteria and mycoplasma occurred. The karyotypes of the 20th generation cell lines were not change. Conclusion: The immortalized cell lines established in this study could provide biological resources for further study on genetics and molecular biology in HBRA. (authors)

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

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Yoichiro Fukao

2016-01-01

Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Dosimetry of irradiation models. The 96-well clonogenic assay for testing radiosensitivity of cell lines

International Nuclear Information System (INIS)

Kulmala, J.; Rantanen, V.; Turku Univ.; Pekkola-Heino, K.; Turku Univ.; Tuominen, J.; Grenman, R.; Turku Univ.

1995-01-01

Radiation experiments with cells in single cell suspension in test tubes and on 96-well plates were carried out and compared. The cells originated from cell lines established from carcinomas of the floor of the mouth and from endometrical carcinoma. Two irradiation models were constructed. Both models allowed the absorbed doses to the cells to be administered with a high accuracy in both experimental settings (better than 5.0%). These irradiation models were compared on cancer cell lines with dissimilar inherent radiation sensitivity and histologic type (UM-SCC-1 resistant, UM-SCC-14A sensitive, and UT-EC-2B highly sensitive); various radiation doses were used. The fractions of surviving cells as a function of radiation dose were compared: there was no significant difference between cells irradiated in test tubes and cells irradiated in 96-well plates. Thus, if the absorbed doses in cells suspended in a tube and in a plate were the same, the survival was similar regardless of the type of irradiation model. (orig.)

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

Science.gov (United States)

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

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Henderson, R.F.; Waide, J.J.; Lechner, J.F.

1995-12-01

A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

International Nuclear Information System (INIS)

Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

2003-01-01

Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

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Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

2013-06-01

Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

Diversity in host clone performance within a Chinese hamster ovary cell line.

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O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

2015-01-01

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers.

Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

Energy Technology Data Exchange (ETDEWEB)

Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

1986-01-01

Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

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Valeria Feinshtein

2013-09-01

Full Text Available Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD, a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp and Breast Cancer Resistance Protein (BCRP expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp for comparison. Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3 and rhodamine123(rh123. Cyclosporine A (CsA served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3 and rh123 was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

Science.gov (United States)

Combs, Rodney G; Yu, Erwin; Roe, Susanna; Piatchek, Michele Bailey; Jones, Heather L; Mott, John; Kennard, Malcolm L; Goosney, Danika L; Monteith, Diane

2011-01-01

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

Science.gov (United States)

Wun, Kwok S; Reijneveld, Josephine F; Cheng, Tan-Yun; Ladell, Kristin; Uldrich, Adam P; Le Nours, Jérôme; Miners, Kelly L; McLaren, James E; Grant, Emma J; Haigh, Oscar L; Watkins, Thomas S; Suliman, Sara; Iwany, Sarah; Jimenez, Judith; Calderon, Roger; Tamara, Kattya L; Leon, Segundo R; Murray, Megan B; Mayfield, Jacob A; Altman, John D; Purcell, Anthony W; Miles, John J; Godfrey, Dale I; Gras, Stephanie; Price, David A; Van Rhijn, Ildiko; Moody, D Branch; Rossjohn, Jamie

2018-04-01

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

Effects of cholera toxin on human colon carcinoma cell lines.

Science.gov (United States)

Barkla, D H; Whitehead, R H; Hayward, I P

1992-10-01

This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Specification of primordial germ cells in medaka (Oryzias latipes

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Raz Erez

2007-01-01

Full Text Available Abstract Background Primordial germ cells (PGCs give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression – the vasa homologue in another teleost, medaka (Oryzias latipes. Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species. Results In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm. Conclusion Taken together, these results are consistent with the idea that in medaka the inheritance of

Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines

DEFF Research Database (Denmark)

Liu, Ying; Bodmer, Walter F

2006-01-01

A comprehensive analysis of the TP53 gene and its protein status was carried out on a panel of 56 colorectal cancer cell lines. This analysis was based on a combination of denaturing HPLC mutation screening of all exons of the p53 gene, sequencing the cDNA, and assessing the function of the p53 p...

Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

Science.gov (United States)

Sumiya, Yu; Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2015-08-01

Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Selective cytotoxicity and modification activity of picornaviruses on transformed cell lines

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Avagyan H. R.

2011-10-01

Full Text Available Aim. We do analyze of the dynamics of morphometabolic changes in transformed cells (of susceptoible lines demonstrating resistance to picrnaviral infection. Methods. The study was performed by application of cell culture technology and a complex of cytochemical and cytophotometric assays. Were used picornaviruses from various genu. Results. According to the results obtained, resistant to picornavirus infection cells of different susceptible lines have similar changes in the phenotype. They have decreased number of nucleoli and increased percentage of euploidy (and near euploid. In resistant cells of all cultures the reduction in amount of DNA and RNA both in nucleus and in cytoplasm was found. All these data correlated with the increased euploidy (and near euploid of the resistant population. All picornavirus resistant cells had a less transformed phenotype, and decreased proliferative activity. Decreased nucleolar status becomes apparent by reduction of all nucleolar indices. Conclusions. Picornaviruses on the susceptible cells produce 2 types of changes – selection and modification. Whatever the mechanism, it is specific for an individual virus, since no restrictions occur in case of infection caused by another picornavirus

Generation of an induced pluripotent stem cell (iPSC line from a 40-year-old patient with the A8344G mutation of mitochondrial DNA and MERRF (myoclonic epilepsy with ragged red fibers syndrome

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Yu-Ting Wu

2018-03-01

Full Text Available Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

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

NARCIS (Netherlands)

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

2009-01-01

Individual differences in nutrient sensitivity have been suggested to be related with differences in stress sensitivity. Here we used layer hens divergently selected for high and low specific antibody responses to SRBC (i.e., low line hens and high line hens), reflecting a genetically based

CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

Science.gov (United States)

Chen, Peter; Mesci, Aruz; Carlyle, James R

2011-01-01

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Science.gov (United States)

Kotloff, D B; Cebra, J J

1988-02-01

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

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The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Lithium-Acetate-Mediated Biginelli One-Pot Multicomponent Synthesis under Solvent-Free Conditions and Cytotoxic Activity against the Human Lung Cancer Cell Line A549 and Breast Cancer Cell Line MCF7

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Harshita Sachdeva

2012-01-01

Full Text Available Various Biginelli compounds (dihydropyrimidinones have been synthesized efficiently and in high yields under mild, solvent-free, and eco-friendly conditions in a one-pot reaction of 1,3-dicarbonyl compounds, aldehydes, and urea/thiourea/acetyl thiourea using lithium-acetate as a novel catalyst without the addition of any proton source. Comparative catalytic efficiency of lithium-acetate and polyphosphoric acid to catalyze Biginelli condensation is also studied under neat conditions. The reaction is carried out in the absence of any solvent and represents an improvement of the classical Biginelli protocol and an advantage in comparison with FeCl3·6H2O, NiCl2·6H2O and CoCl2·6H2O that were used with HCl as a cocatalyst. Compared to classical Biginelli reaction conditions, the present method has advantages of good yields, short reaction times, and experimental simplicity. The obtained products have been identified by spectral (1H NMR and IR data and their melting points. The prepared compounds are evaluated for anticancer activity against two human cancer cell lines (lung cancer cell line A549 and breast cancer cell line MCF7.

Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development.

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Ville Härmä

Full Text Available The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D growth conditions using non-transformed prostate epithelial cells (EP156T, an androgen-sensitive prostate cancer cell line (LNCaP, and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.

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

Science.gov (United States)

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.

Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

Institute of Scientific and Technical Information of China (English)

Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

2007-01-01

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Loss of prostasin (PRSS8) in human bladder transitional cell carcinoma cell lines is associated with epithelial-mesenchymal transition (EMT)

International Nuclear Information System (INIS)

Chen, Li-Mei; Verity, Nicole J; Chai, Karl X

2009-01-01

The glycosylphosphatidylinositol (GPI)-anchored epithelial extracellular membrane serine protease prostasin (PRSS8) is expressed abundantly in normal epithelia and essential for terminal epithelial differentiation, but down-regulated in human prostate, breast, and gastric cancers and invasive cancer cell lines. Prostasin is involved in the extracellular proteolytic modulation of the epidermal growth factor receptor (EGFR) and is an invasion suppressor. The aim of this study was to evaluate prostasin expression states in the transitional cell carcinomas (TCC) of the human bladder and in human TCC cell lines. Normal human bladder tissues and TCC on a bladder cancer tissue microarray (TMA) were evaluated for prostasin expression by means of immunohistochemistry. A panel of 16 urothelial and TCC cell lines were evaluated for prostasin and E-cadherin expression by western blot and quantitative PCR, and for prostasin gene promoter region CpG methylation by methylation-specific PCR (MSP). Prostasin is expressed in the normal human urothelium and in a normal human urothelial cell line, but is significantly down-regulated in high-grade TCC and lost in 9 (of 15) TCC cell lines. Loss of prostasin expression in the TCC cell lines correlated with loss of or reduced E-cadherin expression, loss of epithelial morphology, and promoter DNA hypermethylation. Prostasin expression could be reactivated by demethylation or inhibition of histone deacetylase. Re-expression of prostasin or a serine protease-inactive variant resulted in transcriptional up-regulation of E-cadherin. Loss of prostasin expression in bladder transitional cell carcinomas is associated with epithelial-mesenchymal transition (EMT), and may have functional implications in tumor invasion and resistance to chemotherapy

Properties of Cells Carrying the Herpes Simplex Virus Type 2 Thymidine Kinase Gene: Mechanisms of Reversion to a Thymidine Kinase-Negative Phenotype

Science.gov (United States)

Bastow, K. F.; Darby, G.; Wildy, P.; Minson, A. C.

1980-01-01

We have isolated cells with a thymidine kinase-negative (tk−) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10−3 to 10−4, and resistance to either compound conferred simultaneous resistance to the other. tk− revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences. Images PMID:16789205

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

Science.gov (United States)

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.

The role of sex chromosomes in mammalian germ cell differentiation: can the germ cells carrying X and Y chromosomes differentiate into fertile oocytes?

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Teruko Taketo

2015-06-01

Full Text Available The sexual differentiation of germ cells into spermatozoa or oocytes is strictly regulated by their gonadal environment, testis or ovary, which is determined by the presence or absence of the Y chromosome, respectively. Hence, in normal mammalian development, male germ cells differentiate in the presence of X and Y chromosomes, and female germ cells do so in the presence of two X chromosomes. However, gonadal sex reversal occurs in humans as well as in other mammalian species, and the resultant XX males and XY females can lead healthy lives, except for a complete or partial loss of fertility. Germ cells carrying an abnormal set of sex chromosomes are efficiently eliminated by multilayered surveillance mechanisms in the testis, and also, though more variably, in the ovary. Studying the molecular basis for sex-specific responses to a set of sex chromosomes during gametogenesis will promote our understanding of meiotic processes contributing to the evolution of sex determining mechanisms. This review discusses the fate of germ cells carrying various sex chromosomal compositions in mouse models, the limitation of which may be overcome by recent successes in the differentiation of functional germ cells from embryonic stem cells under experimental conditions.

THP-1 cell line: an in vitro cell model for immune modulation approach.

Science.gov (United States)

Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

Establishing clonal cell lines with endothelial-like potential from CD9(hi, SSEA-1(- cells in embryonic stem cell-derived embryoid bodies.

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Qizhou Lian

Full Text Available BACKGROUND: Differentiation of embryonic stem cells (ESCs into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs. Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2 = 0.93 while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi, SSEA-1(- while ESCs are CD9(lo, SSEA-1(+. Isolation of CD9(hi, SSEA-1(- cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2 = 0.95 and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells

Spontaneous transformation of human granulosa cell tumours into an aggressive phenotype: a metastasis model cell line

International Nuclear Information System (INIS)

Imai, Misa; Muraki, Miho; Takamatsu, Kiyoshi; Saito, Hidekazu; Seiki, Motoharu; Takahashi, Yuji

2008-01-01

Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. Population doubling was carried out to examine the proliferation capacity of KGN cells. Moreover, the invasive capacity of these cells was determined using the in vitro invasion assay. The expression level of tumour markers in KGN cells at different passages was then determined by Western blot analysis. Finally, the growth and metastasis of KGN cells injected subcutaneously (s.c.) into nude mice was observed 3 months after injection. During in vitro culture, the advanced passage KGN cells grew 2-fold faster than the early passage cells, as determined by the population doubling assay. Moreover, we found that the advanced passage cells were 2-fold more invasive than the early passage cells. The expression pattern of tumour markers, such as p53, osteopontin, BAX and BAG-1, supported the notion that with passage, KGN cells became more aggressive. Strikingly, KGN cells at both early and advanced passages metastasized to the bowel when injected s.c. into nude mice. In addition, more tumour nodules were formed when the advanced passage cells were implanted. KGN cells cultured in vitro acquire an aggressive phenotype, which was confirmed by the analysis of cellular activities and the expression of biomarkers. Interestingly, KGN cells injected s.c. are metastatic with nodule formation occurring mostly in the bowel. Thus, this cell line is a good model for analysing GCT progression and the mechanism of metastasis in vivo

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

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Krishna Das

Full Text Available 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 (sgRNA 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.

Cost-effective master cell bank validation of multiple clinical-grade human pluripotent stem cell lines from a single donor.

Science.gov (United States)

Devito, Liani; Petrova, Anastasia; Miere, Cristian; Codognotto, Stefano; Blakely, Nicola; Lovatt, Archie; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

2014-10-01

Standardization guidelines for human pluripotent stem cells are still very broadly defined, despite ongoing clinical trials in the U.S., U.K., and Japan. The requirements for validation of human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) in general follow the regulations for other clinically compliant biologics already in place but without addressing key differences between cell types or final products. In order to realize the full potential of stem cell therapy, validation criteria, methodology, and, most importantly, strategy, should address the shortfalls and efficiency of current approaches; without this, hESC- and, especially, iPSC-based therapy will not be able to compete with other technologies in a cost-efficient way. We addressed the protocols for testing cell lines for human viral pathogens and propose a novel strategy that would significantly reduce costs. It is highly unlikely that the multiple cell lines derived in parallel from a tissue sample taken from one donor would have different profiles of endogenous viral pathogens; we therefore argue that samples from the Master Cell Banks of sibling lines could be safely pooled for validation. We illustrate this approach with tiered validation of two sibling clinical-grade hESC lines, KCL033 and KCL034 (stage 1, sterility; stage 2, specific human pathogens; and stage 3, nonspecific human pathogens). The results of all tests were negative. This cost-effective strategy could also be applied for validation of Master Cell Banks of multiple clinical-grade iPSC lines derived from a single donor. ©AlphaMed Press.

Differential heat shock response of primary human cell cultures and established cell lines

DEFF Research Database (Denmark)

Richter, W W; Issinger, O G

1986-01-01

degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

Radiation response of haematopoietic cell lines of human origin

International Nuclear Information System (INIS)

Lehnert, S.; Rybka, W.B.; Suissa, S.; Giambattisto, D.

1986-01-01

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radio-sensitivity of the cells varied widely, the D 0 s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivities. (author)

Radiation sensitivity of human lung cancer cell lines

International Nuclear Information System (INIS)

Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

1989-01-01

X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

Directory of Open Access Journals (Sweden)

Sylvie Lachmann

Full Text Available The mammalian protein kinase N (PKN family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

Regulatory domain selectivity in the cell-type specific PKN-dependence of cell migration.

Science.gov (United States)

Lachmann, Sylvie; Jevons, Amy; De Rycker, Manu; Casamassima, Adele; Radtke, Simone; Collazos, Alejandra; Parker, Peter J

2011-01-01

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

Peptidomic analysis of human cell lines

Science.gov (United States)

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

2011-01-01

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

Science.gov (United States)

Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

2018-01-12

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

OpenAIRE

Feng, Yi; Tian, Zhong-Min; Wan, Ming-Xi; Zheng, Zhao-Bin

2007-01-01

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.

Tissue- and Cell-Specific Cytokinin Activity in Populus × canescens Monitored by ARR5::GUS Reporter Lines in Summer and Winter.

Science.gov (United States)

Paul, Shanty; Wildhagen, Henning; Janz, Dennis; Teichmann, Thomas; Hänsch, Robert; Polle, Andrea

2016-01-01

Cytokinins play an important role in vascular development. But knowledge on the cellular localization of this growth hormone in the stem and other organs of woody plants is lacking. The main focus of this study was to investigate the occurrence and cellular localization of active cytokinins in leaves, roots, and along the stem of Populus × canescens and to find out how the pattern is changed between summer and winter. An ARR5::GUS reporter construct was used to monitor distribution of active cytokinins in different tissues of transgenic poplar lines. Three transgenic lines tested under outdoor conditions showed no influence of ARR5::GUS reporter construct on the growth performance compared with the wild-type, but one line lost the reporter activity. ARR5::GUS activity indicated changes in the tissue- and cell type-specific pattern of cytokinin activity during dormancy compared with the growth phase. ARR5::GUS activity, which was present in the root tips in the growing season, disappeared in winter. In the stem apex ground tissue, ARR5::GUS activity was higher in winter than in summer. Immature leaves from tissue-culture grown plants showed inducible ARR5::GUS activity. Leaf primordia in summer showed ARR5::GUS activity, but not the expanded leaves of outdoor plants or leaf primordia in winter. In stem cross sections, the most prominent ARR5::GUS activity was detected in the cortex region and in the rays of bark in summer and in winter. In the cambial zone the ARR5::GUS activity was more pronounced in the dormant than in growth phase. The pith and the ray cells adjacent to the vessels also displayed ARR5::GUS activity. In silico analyses of the tissue-specific expression patterns of the whole PtRR type-A family of poplar showed that PtRR10, the closest ortholog to the Arabidopsis ARR5 gene, was usually the most highly expressed gene in all tissues. In conclusion, gene expression and tissue-localization indicate high activity of cytokinins not only in summer, but

The Expression and Biological Significance of PD-L1 on Lung Cancer Cell Lines

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Cheng CHEN

2009-08-01

Full Text Available Background and objective Tumor-associated PD-L1 expression was recently shown to promote T-cell apoptosis and proposed as a potential mechanism of immune evasion by tumors. On the basis of the ability of tumor-associated PD-L1 to mediate activated T-cell death, it is likely that manipulation of the PD-L1 pathway at defined time points during the development of the T-cell antitumor immune response can enhance the efficacy of T-cell-based immunotherapy. Here, the levels of expression of PD-L1 on lung cancer cell lines and its role in interaction of CTL and target cells was investigated. Methods Human PBMC derived DCs were loaded with apoptotic tumor cells and stimulated by CD40 mAb (5C11. Tumor specific CTL was generated in vitro by autologous T cells co-cultured with mature DCs. Expression of PD-L1 on lung cancer cell lines H1299 and A549 were analyzed by FCM. JAM assay was used to detect the cytolytic activity of CTL with or without blocking PD-L1 by PD-L1 mAb respectively. The concentrations of IFN-γ in supernatants from distinct groups were analyzed by ELISA. Results Tumor cells-loaded mature DCs could induce the generation of the tumor specific CTL. Expression of PD-L1 was low on A549 cell, but high on H1299 cell. Blockade of PD-L1 on A549 could not improve cytolytic effect of CTL on target cells and IFN-γ production, but fragmentation of H1299 cells and IFN-γ production were significantly enhanced by the combination of PD-L1 mAb and CTL. Conclusion Expression of PD-L1 on lung cancer cell line can decrease the cytolytic effect of CTL on target cells.

Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC-1.

Science.gov (United States)

Fujita, Mayumi; Imadome, Kaori; Imai, Takashi

2017-05-01

We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC-1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC-1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC-1 cells, metabolome analysis of the invaded PANC-1 compared with the whole cultured PANC-1 was performed using CE-TOFMS, and concentrations of 110 metabolites were measured. In contrast to the whole cultured cells, the invaded PANC-1 was characterized as a population with reduced levels of amino acids and TCA cycle intermediates, and decreased and increased intermediates in glycolysis and nucleic acid metabolism. In particular, the ratio of both adenosine and guanosine energy charge was reduced in the invaded cells, revealing that the consumption of ATP and GTP was high in the invaded cells, and thus suggesting that ATP- or GTP-generating pathways are stimulated. In addition, the GSH/GSSG ratio was low in the invaded cells, but these cells had a higher surviving fraction after exposure to hydrogen peroxide. Thus, the invaded cells were the population resistant to oxidative stress. Furthermore, reduction in intracellular GSH content inhibited PANC-1 invasiveness, indicated that GSH has an important role in PANC-1 invasiveness. Overall, we propose the invaded cells have several unique metabolic profiles. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

International Nuclear Information System (INIS)

Ascierto, Maria Libera; Bedognetti, Davide; Uccellini, Lorenzo; Rossano, Fabio; Ascierto, Paolo A; Stroncek, David F; Restifo, Nicholas P; Wang, Ena; Szalay, Aladar A; Marincola, Francesco M; Worschech, Andrea; Yu, Zhiya; Adams, Sharon; Reinboth, Jennifer; Chen, Nanhai G; Pos, Zoltan; Roychoudhuri, Rahul; Di Pasquale, Giovanni

2011-01-01

Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo. In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection. We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection. Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection

Surface-modified gold nanorods for specific cell targeting

Science.gov (United States)

Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

2012-05-01

Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

Integrated analysis of breast cancer cell lines reveals unique signaling pathways

Energy Technology Data Exchange (ETDEWEB)

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

2009-03-31

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

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

Science.gov (United States)

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

2009-01-01

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

Investigation of the selenium metabolism in cancer cell lines

DEFF Research Database (Denmark)

Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

2011-01-01

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

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Barr, Martin P.; Gray, Steven G.; Hoffmann, Andreas C.; Hilger, Ralf A.; Thomale, Juergen; O’Flaherty, John D.; Fennell, Dean A.; Richard, Derek; O’Leary, John J.; O’Byrne, Kenneth J.

2013-01-01

Introduction Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing

Generation and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature.

Directory of Open Access Journals (Sweden)

Martin P Barr

Full Text Available Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC. Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460. Over a period of twelve months, cisplatin resistant (CisR cell lines were derived from original, age-matched parent cells (PT and subsequently characterized. Proliferation (MTT and clonogenic survival assays (crystal violet were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX and cellular platinum uptake (ICP-MS was also assessed.Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Directory of Open Access Journals (Sweden)

M.A. Sukoyan

2002-05-01

Full Text Available Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

Science.gov (United States)

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.

SV-BR-1-GM, a Clinically Effective GM-CSF-Secreting Breast Cancer Cell Line, Expresses an Immune Signature and Directly Activates CD4+ T Lymphocytes

Directory of Open Access Journals (Sweden)

Markus D. Lacher

2018-05-01

Full Text Available Targeted cancer immunotherapy with irradiated, granulocyte–macrophage colony-stimulating factor (GM-CSF-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862, a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB, in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B, ADA (encoding adenosine deaminase, ADGRE5 (CD97, CD58 (LFA3, CD74 (encoding invariant chain and CLIP, CD83, CXCL8 (IL8, CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele treated with

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

Science.gov (United States)

Drexler, H G; Matsuo, Y

2000-05-01

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

Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

International Nuclear Information System (INIS)

Hidalgo, Alfredo; Monroy, Alberto; Arana, Rosa Ma; Taja, Lucía; Vázquez, Guelaguetza; Salcedo, Mauricio

2003-01-01

Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma

Menadione inhibits MIBG uptake in two neuroendocrine cell lines

NARCIS (Netherlands)

Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

1997-01-01

In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

Science.gov (United States)

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Science.gov (United States)

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell

Cell line name recognition in support of the identification of synthetic lethality in cancer from text

Science.gov (United States)

Kaewphan, Suwisa; Van Landeghem, Sofie; Ohta, Tomoko; Van de Peer, Yves; Ginter, Filip; Pyysalo, Sampo

2016-01-01

Motivation: The recognition and normalization of cell line names in text is an important task in biomedical text mining research, facilitating for instance the identification of synthetically lethal genes from the literature. While several tools have previously been developed to address cell line recognition, it is unclear whether available systems can perform sufficiently well in realistic and broad-coverage applications such as extracting synthetically lethal genes from the cancer literature. In this study, we revisit the cell line name recognition task, evaluating both available systems and newly introduced methods on various resources to obtain a reliable tagger not tied to any specific subdomain. In support of this task, we introduce two text collections manually annotated for cell line names: the broad-coverage corpus Gellus and CLL, a focused target domain corpus. Results: We find that the best performance is achieved using NERsuite, a machine learning system based on Conditional Random Fields, trained on the Gellus corpus and supported with a dictionary of cell line names. The system achieves an F-score of 88.46% on the test set of Gellus and 85.98% on the independently annotated CLL corpus. It was further applied at large scale to 24 302 102 unannotated articles, resulting in the identification of 5 181 342 cell line mentions, normalized to 11 755 unique cell line database identifiers. Availability and implementation: The manually annotated datasets, the cell line dictionary, derived corpora, NERsuite models and the results of the large-scale run on unannotated texts are available under open licenses at http://turkunlp.github.io/Cell-line-recognition/. Contact: sukaew@utu.fi PMID:26428294

Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines