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

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

Science.gov (United States)

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

2017-09-23

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

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.

Establishment and characterization of a new cell line derived from human colorectal laterally spreading tumor

Science.gov (United States)

Wang, Xin-Ying; Lai, Zhou-Sheng; Yeung, Chung-Man; Wang, Ji-De; Deng, Wen; Li, Hoi Yee; Han, Yu-Jing; Kung, Hsiang-Fu; Jiang, Bo; Lin, Marie Chia-mi

2008-01-01

AIM: To study the molecular mechanism of laterally spreading tumor (LST), a cell line [Laterally Spreading Tumor-Rectum 1 (LST-R1)] was derived and the characteristics of this cell line were investigated. METHODS: A new cell line (LST-R1) originated from laterally spreading tumor was established. Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments. In vitro invasion assay, cDNA microarray and Western blotting were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer. RESULTS: Our study demonstrated that both epithelial special antigen (ESA) and cytokeratin-20 (CK20) were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability. cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells. CONCLUSION: We established and characterized a colorectal cancer cell line, LST-R1 and LST-R1 has an obvious malignant tendency, which maybe partially attributed to the changes of the expression of some adhesion molecules, such as E-cadherin. It is also a versatile tool for exploring the original and progressive mechanisms of laterally spreading tumor and the early colon cancer genesis. PMID:18300345

Derivation of the human embryonic stem cell line RCe006-A (RC-2

Directory of Open Access Journals (Sweden)

P.A. De Sousa

2016-03-01

Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

Establishment and long-term culture of the cell lines derived from gonad tissues of Siberian sturgeon (Acipenser baerii

Directory of Open Access Journals (Sweden)

Jun Hyung Ryu

2016-06-01

Full Text Available Abstract To culture germline stem cells in vitro, establishment of the cell lines that can be used as the feeder cells is a prerequisite. In this study, we tried to establish gonad-derived cell lines in Siberian sturgeon (Acipenser baerii. Five 1-year-old A. baerii were used as a donor of gonad tissues, and gonad-dissociated cells were cultured in vitro. Subsequently, determination of growth conditions, long-term culture, characterization, and cryopreservation of the cell lines were also conducted. Five gonad-derived cell lines were stably established and cultured continuously over at least the 73th passage and 402 culture days under the media containing 20 % fetal bovine serum at 28 °C. All cell lines consisted of two main cell types based on morphology even if the ratio of the two cell types was different depending on cell lines. Despite long-term culture, all cell lines maintained diploid DNA contents and expression of several genes that are known to express in the A. baerii gonad. After freezing and thawing of the cell lines, post-thaw cell viabilities between 57.6 and 92.9 % depending on cell lines were indentified, suggesting that stable cryopreservation is possible. The results and the cell lines established in this study will contribute to the development of an in vitro system for A. baerii germline stem cell culture.

Characterisation of the cell line HC-AFW1 derived from a pediatric hepatocellular carcinoma.

Directory of Open Access Journals (Sweden)

Sorin Armeanu-Ebinger

Full Text Available Current treatment of paediatric hepatocellular carcinoma (HCC is often inefficient due to advanced disease at diagnosis and resistance to common drugs. The aim of this study was to generate a cell line derived from a paediatric HCC in order to expand research in this field. We established the HC-AFW1 cell line from a liver neoplasm of a 4-year-old boy through culturing of primary tumor specimens. The cell line has been stable for over one year of culturing and has a doubling time of 40 h. The tumour cells have an epithelial histology and express HCC-associated proteins such as Alpha-fetoprotein (AFP, Glypican 3, E-cadherin, CD10, CD326, HepPar1 and Vimentin. Forty-nine amino acids in exon 3 of β-Catenin that involve the phosphorylation sites of GSK3 were absent and β-Catenin is detectable in the cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. Several alterations of gene copy numbers were detected by genome-wide SNP array. Among the different drugs tested, cisplatin and irinotecan showed effective inhibition of tumour cell growth in a proliferation assay at concentrations below 5 µg/ml. Subcutaneous xenotransplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumours with high levels of serum AFP. Histological analyses of the primary tumour and xenografts included national and international expert pathological review. Consensus reading characterised the primary tumour and the HC-AFW1-derived tumours as HCC. HC-AFW1 is the first cell line derived from a paediatric HCC without a background of viral hepatitis or cirrhosis and represents a valuable tool for investigating the biology of and therapeutic strategies for childhood HCC.

Chemo-radioresistance of small cell lung cancer cell lines derived from untreated primary tumors obtained by diagnostic bronchofiberscopy

International Nuclear Information System (INIS)

Tanio, Yoshiro; Watanabe, Masatoshi; Inoue, Tamotsu

1990-01-01

New cell lines of small cell lung cancer (SCLC) were established from specimens of untreated primary tumors biopsied by diagnostic bronchofiberscopy. The advantage of this method was ease of obtaining specimens from lung tumors. Establishment of cell lines was successful with 4 of 13 specimens (30%). Clinical responses of the tumors showed considerable variation, but were well correlated with the in vitro sensitivity of the respective cell lines to chemotherapeutic drugs and irradiation. One of the cell lines was resistant to all drugs tested and irradiation, while another was sensitive to all of them. Although the acquired resistance of SCLC is the biggest problem in treatment, the natural resistance to therapy is another significant problem. Either acquired or natural, resistance mechanisms of SCLC may be elucidated by the use of such cell lines derived from untreated tumors. This method and these SCLC cell lines are expected to be useful for the serial study of biologic and genetic changes of untreated and pre-treated tumors, or primary and secondary tumors. (author)

In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines

Directory of Open Access Journals (Sweden)

Das Bhaskar C

2011-09-01

Full Text Available Abstract Background Rhabdoid tumors (RTs are aggressive pediatric malignancies with poor prognosis. N-(4-hydroxy phenyl retinamide (4-HPR or fenretinide is a potential chemotherapeutic for RTs with activity correlated to its ability to down-modulate Cyclin D1. Previously, we synthesized novel halogen-substituted and peptidomimetic-derivatives of 4-HPR that retained activity in MON RT cells. Here we analyzed the effect of 4-HPR in inhibiting the growth of several RT, glioma, and breast cancer cell lines and tested their effect on cell cycle, apoptosis and Cyclin D1 expression. Methods Effect of compounds on RT cell cycle profiles, and cell death were assessed by MTS cell survival assays and FACS analysis. The effects of treatment on Cyclin D1 expression were determined by immunoblotting. The efficacy of these compounds on glioma and breast cancer cell lines was also determined using MTS assays. Results Low micromolar concentrations of 4-HPR derivatives inhibited cell survival of all RT cells tested. The 4-HPR derivatives altered RT cell cycle profiles and induced high levels of cell death that was correlated with their potency. ATRA exhibited high IC50 values in all cell lines tested and did not cause cell death. In MON RT cells, the iodo-substituted compounds were more active than 4-HPR in inducing cell cycle arrest and apoptosis. Additionally, the activity of the compounds correlated with their ability to down-modulate Cyclin D1: while active compounds reduced Cyclin D1 levels, inactive ATRA did not. In glioma and breast cancer cell lines, 4-HPR and 4-HPR derivatives showed variable efficacy. Conclusions Here we demonstrate, for the first time, that the inhibitory activities of novel halogen-substituted and peptidomimetic derivatives of 4-HPR are correlated to their ability to induce cell death and down-modulate Cyclin D1. These 4-HPR derivatives showed varied potencies in breast cancer and glioma cell lines. These data indicate that further

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

Directory of Open Access Journals (Sweden)

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

Derivation of the human embryonic stem cell line RCe014-A (RC-10

Directory of Open Access Journals (Sweden)

P.A. De Sousa

2016-03-01

Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Derivation of the human embryonic stem cell line RCe010-A (RC-6

Directory of Open Access Journals (Sweden)

P.A. De Sousa

2016-03-01

Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

Establishment and characterization of a unique 1 microm diameter liver-derived progenitor cell line.

Science.gov (United States)

Aravalli, Rajagopal N; Behnan Sahin, M; Cressman, Erik N K; Steer, Clifford J

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 microm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers. Copyright 2009 Elsevier Inc. All rights reserved.

Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

DEFF Research Database (Denmark)

Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss

2012-01-01

The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

Derivation and characterization of the NYSCFe003-A human embryonic stem cell line

Directory of Open Access Journals (Sweden)

Ana Sevilla

2017-12-01

Full Text Available The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free.

Derivation of the human embryonic stem cell line RCe012-A (RC-8

Directory of Open Access Journals (Sweden)

P.A. De Sousa

2016-03-01

Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri.

Science.gov (United States)

Lin, Jiaying; Liu, Xishi; Ding, Ding

2015-01-01

The cancer stem cell (CSC) paradigm is one possible way to understand the genesis of cancer, and cervical cancer in particular. We quantified and enriched ALDH1(+) cells within cervical cancer cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). ALDH1 expression in spheroid-derived cells (SDC) and the parental monolayer-derived cell (MDC) line was compared by flow-cytometry. Invasion capability was evaluated by Matrigel assay and expression of EMT-related genes Twist 1, Twist 2, Snail 1, Snail 2, Vimentin and E-cadherin by real-time PCR. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. SDC expressed lower levels of E-cadherin and elevated levels of Twist 1, Twist 2, Snail 1, Snail 2 and Vimentin compared to MDC. Cervical cancer cell lines harbor potential CSC, characterized by ALDH1 expression as well as properties like invasiveness, colony-forming ability, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

Directory of Open Access Journals (Sweden)

Hong Xin

Full Text Available Hepatocellular carcinoma (HCC is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903 by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3 was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

Genetic and Genomic Characterization of 462 Melanoma Patient-Derived Xenografts, Tumor Biopsies, and Cell Lines

Directory of Open Access Journals (Sweden)

Bradley Garman

2017-11-01

Full Text Available Summary: Tumor-sequencing studies have revealed the widespread genetic diversity of melanoma. Sequencing of 108 genes previously implicated in melanomagenesis was performed on 462 patient-derived xenografts (PDXs, cell lines, and tumors to identify mutational and copy number aberrations. Samples came from 371 unique individuals: 263 were naive to treatment, and 108 were previously treated with targeted therapy (34, immunotherapy (54, or both (20. Models of all previously reported major melanoma subtypes (BRAF, NRAS, NF1, KIT, and WT/WT/WT were identified. Multiple minor melanoma subtypes were also recapitulated, including melanomas with multiple activating mutations in the MAPK-signaling pathway and chromatin-remodeling gene mutations. These well-characterized melanoma PDXs and cell lines can be used not only as reagents for a large array of biological studies but also as pre-clinical models to facilitate drug development. : Garman et al. have characterized melanoma PDXs and cell lines described in Krepler et al. (see the related paper in this issue of Cell Reports, identifying major and minor subtypes, some of which were previously not well defined, targeted and immunotherapy resistance, and tumor heterogeneity, creating a set of reagents for future drug discovery and biological studies. Keywords: melanoma, patient-derived xenografts, massively parallel sequencing, cell lines

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

Science.gov (United States)

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.

Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

Directory of Open Access Journals (Sweden)

P.A. De Sousa

2016-05-01

Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

Establishment and characterization of a unique 1 μm diameter liver-derived progenitor cell line

International Nuclear Information System (INIS)

Aravalli, Rajagopal N.; Behnan Sahin, M.; Cressman, Erik N.K.; Steer, Clifford J.

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 μm in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

Establishment and characterization of a unique 1 {mu}m diameter liver-derived progenitor cell line

Energy Technology Data Exchange (ETDEWEB)

Aravalli, Rajagopal N., E-mail: arava001@umn.edu [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Behnan Sahin, M. [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Cressman, Erik N.K. [Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)

2010-01-01

Liver-derived progenitor cells (LDPCs) are recently identified novel stem/progenitor cells from healthy, unmanipulated adult rat livers. They are distinct from other known liver stem/progenitor cells such as the oval cells. In this study, we have generated a LDPC cell line RA1 by overexpressing the simian virus 40 (SV40) large T antigen (TAg) in primary LDPCs. This cell line was propagated continuously for 55 passages in culture, after which it became senescent. Interestingly, following transformation with SV40 TAg, LDPCs decreased in size significantly and the propagating cells measured 1 {mu}m in diameter. RA1 cells proliferated in vitro with a doubling time of 5-7 days, and expressed cell surface markers of LDPCs. In this report, we describe the characterization of this novel progenitor cell line that might serve as a valuable model to study liver cell functions and stem cell origin of liver cancers.

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

Science.gov (United States)

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

2015-01-01

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

Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

Directory of Open Access Journals (Sweden)

Chao Yang

2014-01-01

Full Text Available Human mesenchymal stem cells (MSCs have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs and umbilical cord-derived mesenchymal stem cells (UC-MSCs. We found (1 MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2 MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3 real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4 furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

Directory of Open Access Journals (Sweden)

Susanne C. Hammer

2016-09-01

Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5

DEFF Research Database (Denmark)

Kjeldsen, Eigil; Nielsen, Christine J F; Roy, Amit

2018-01-01

-K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.......Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T......-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations...

Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

International Nuclear Information System (INIS)

Gu Yongpeng; Li Hongzhen; Miki, Jun; Kim, Kee-Hong; Furusato, Bungo; Sesterhenn, Isabell A.; Chu, Wei-Sing; McLeod, David G.; Srivastava, Shiv; Ewing, Charles M.; Isaacs, William B.; Rhim, Johng S.

2006-01-01

In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents

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

Directory of Open Access Journals (Sweden)

Víctor A. Solarte

2015-01-01

Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

International Nuclear Information System (INIS)

Youakim, A.; Herscovics, A.

1985-01-01

The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2- 3 H]mannose or L-[5,6- 3 H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2- 3 H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2- 3 H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6- 3 H]glucosamine and L-[1- 14 C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3 H-labeled N-acetylglucosamine and N-acetylgalactosamine

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

International Nuclear Information System (INIS)

Hashimoto, Tomoko; Furuyama, Jun-ichi; Nakano, Yoshiro; Owada, M. Koji; Kakunaga, Takeo

1986-01-01

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of γ-rays or ultraviolet irradiation was as high as those of their parental fibroblasts. (Auth.)

Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis

NARCIS (Netherlands)

Meng, X.; de rooij, D. G.; Westerdahl, K.; Saarma, M.; Sariola, H.

2001-01-01

We show with transgenic mice that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have

Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

Science.gov (United States)

Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

2010-04-01

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Differences between the genomes of lymphoblastoid cell lines and blood-derived samples

Directory of Open Access Journals (Sweden)

Joesch-Cohen LM

2017-02-01

Full Text Available Lena M Joesch-Cohen, Gustavo Glusman Institute for Systems Biology, Seattle, WA, USA Abstract: Lymphoblastoid cell lines (LCLs represent a convenient research tool for expanding the amount of biologic material available from an individual. LCLs are commonly used as reference materials, most notably from the Genome in a Bottle Consortium. However, the question remains how faithfully LCL-derived genome assemblies represent the germline genome of the donor individual as compared to the genome assemblies derived from peripheral blood mononuclear cells. We present an in-depth comparison of a large collection of LCL- and peripheral blood mononuclear cell-derived genomes in terms of distributions of coverage and copy number alterations. We found significant differences in the depth of coverage and copy number calls, which may be driven by differential replication timing. Importantly, these copy number changes preferentially affect regions closer to genes and with higher GC content. This suggests that genomic studies based on LCLs may display locus-specific biases, and that conclusions based on analysis of depth of coverage and copy number variation may require further scrutiny. Keywords: genomics, whole-genome sequencing, viral transformation, copy number changes, bioinformatics

Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions

Directory of Open Access Journals (Sweden)

Ana Sevilla

2018-05-01

Full Text Available The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

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.

Comparison of radiosensitivity between human hematopoietic cell lines derived from patients with Down's syndrome and from normal persons

International Nuclear Information System (INIS)

Huang, C.C.; Banerjee, A.; Tan, J.C.; Hou, Y.

1977-01-01

Seven hematopoietic cell lines, four derived from the peripheral blood of patients with Down's syndrome (DS) and three from normal persons, were irradiated with 100, 150, 300, and 500 rads from a 60 Co source and harvested for cell count and chromosome aberration studies every 12 hours for 72 hours post irradiation. Cell growth inhibition and an increase in chromosome aberration were observed in all the cell lines at each dose level and time interval. No significant difference was observed in the effects between DS and normal cell lines. The most common types of aberrations in the 12-hour samples were chromosome and/or chromatid breaks. In the later samples, chromatid exchanges were predominant. The results of the variance analyses on the induced chromosome aberrations in six lines (three DS and three normal lines) showed radiation dosage to be the largest component of total variance, following postirradiation duration and cell lines. The samples harvested 24 and 36 hours post irradiation generally showed greater effects than the samples of other harvest durations. The cell line variance could only be attributed to the differences among and between individual cell lines rather than the difference between DS and normal cell lines

Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

Science.gov (United States)

Bonin, L R; Madden, K; Shera, K; Ihle, J; Matthews, C; Aziz, S; Perez-Reyes, N; McDougall, J K; Conroy, S C

1999-03-01

The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2

Identification of two distinct chromosome 12-derived amplification units in neuroblastoma cell line NGP

NARCIS (Netherlands)

van Roy, N.; Forus, A.; Myklebost, O.; Cheng, N. C.; Versteeg, R.; Speleman, F.

1995-01-01

The neuroblastoma cell line NGP contains two homogeneously staining regions (hsr). One of these hsrs contains MYCN sequences. Reverse painting experiments demonstrated that the second HSR consisted of two chromosome 12-derived amplification units, located at 12q14-15 and 12q24. Southern blot and

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.

Cytotoxic effects of new synthesis heterocyclic derivatives of Amoxicillin on some cancer cell lines

Science.gov (United States)

Al-Rawi, M. S.; Hussei, D. F.; Al-Taie, A. F.; Al-Halbosiy, M. M.; Hameed, B. A.

2018-05-01

A new Schiff base [I] was prepared by refluxing Amoxicillin trihydrate and 4-Hydroxy- 3,5-dimethoxybenzaldehyde in aqueous methanol solution using glacial acetic acid as a catalyst. The new 1,3-oxazepine derivative [II] was obtained by Diels- Alder reaction of Schiff base [I] with phthalic anhydride in dry benzene. The reaction of Schiff base [I] with thioglycolic acid in dry benzene led to the formation of thiazolidin-4-one derivative [III]. While the imidazolidin-4-one [IV] derivative was produced by reacting the mentioned Schiff base [I] with glycine and triethylamine in ethanol for 9 hrs. Tetrazole derivative [V] was synthesized by refluxing Schiff base [I] with sodium azide in dimethylformamid DMF. The structure of synthesized compounds[I-V] was characterized by their melting points, elemental analysis CHN-S and by their spectral data; FTIR and 1H NMR spectroscopy. Two cancer cell lines include: (RD) human pelvic rhabdomyosarcoma and (L20B) the mice intestines carcinoma cell line (which expresses the genes for human cellular receptor for Polio viruses) were used in this study. The cytotoxic effect of different concentrations of all the synthesized compounds for 48 hrs was examined. All compounds except [IV] and [V] showed less than 50% inhibition for (L20B), while these compounds exhibit inhibition more than 50% inhibition for (RD).

Axon Guidance of Sympathetic Neurons to Cardiomyocytes by Glial Cell Line-Derived Neurotrophic Factor (GDNF)

NARCIS (Netherlands)

Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Hirabayashi, Masumi; Watabe, Kazuhiko; Jimbo, Yasuhiko; Kodama, Itsuo; Komuro, Issei

2013-01-01

Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal

Experimental infection of Leishmania (L. chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae

Directory of Open Access Journals (Sweden)

Felio J Bello

2005-10-01

Full Text Available The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5 cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6% and on day 4 in the J774 cells (51%. This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Influence of vitamin D on cisplatin sensitivity in testicular germ cell cancer-derived cell lines and in a NTera2 xenograft model

DEFF Research Database (Denmark)

Jørgensen, Anne; Blomberg Jensen, Martin; Nielsen, John Erik

2013-01-01

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has anti-proliferative, pro-apoptotic, and pro-differentiating effects in somatic cancer cells in vitro and in vivo. 1,25(OH)(2)D(3) also augments the anti-tumor effects of several chemotherapeutic agents, including...... cisplatin, which may have clinical relevance. Given the pro-differentiation effect of vitamin D recently demonstrated in testicular germ cell tumors (TGCTs), we hypothesized that 1,25(OH)(2)D(3) could be a beneficial adjunctive to existing chemotherapy regime used to treat these tumors. In this study, cell...... survival effects of 1,25(OH)(2)D(3), another pro-differentiation compound, retinoic acid and cisplatin were investigated in TGCT-derived cell lines in vitro. 1,25(OH)(2)D(3) augmented the effect of cisplatin in an embryonal carcinoma-derived cell line (NTera2), possibly through downregulation...

Proliferative and antiproliferative effects of interferon-gamma and tumor necrosis factor-alpha on cell lines derived from cervical and ovarian malignancies

International Nuclear Information System (INIS)

Mutch, D.G.; Massad, L.S.; Kao, M.S.; Collins, J.L.

1990-01-01

Four human cell lines derived from cervical carcinomas (ME-180, SiHa, HT-3, and MS751) and three human cell lines derived from ovarian carcinomas (SK-OV-3, Caov-3, and NIH:OVCAR-3) were analyzed in vitro to determine the effect of recombinant interferon-gamma and recombinant human tumor necrosis factor-alpha on cell growth and survival. The effects of interferon-gamma, tumor necrosis factor-alpha, and both interferon-gamma and tumor necrosis factor-alpha on cell growth were measured after 24 and 72 hours of incubation by the incorporation of chromium 51. The results of this analysis showed that all seven cell lines were resistant to the antiproliferative action of tumor necrosis factor-alpha, that the growth of most cell lines was inhibited by interferon-gamma by 72 hours of incubation, and that after 72 hours of incubation all cell lines demonstrated a synergistic antiproliferative response to the combination of interferon-gamma and tumor necrosis factor-alpha. However, the effects of these cytokines on cell growth were found to differ among cell lines and varied with the concentration and the duration of incubation. The growth of one cell line (Caov-3) was stimulated by both tumor necrosis factor-alpha and interferon-gamma. These results suggest that the clinical effects of these cytokines on the growth of gynecologic cancers may be more complex than previously supposed

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

International Nuclear Information System (INIS)

Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z.

2006-01-01

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

Science.gov (United States)

Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

Directory of Open Access Journals (Sweden)

Gina D Kusuma

Full Text Available Mesenchymal stem/stromal cells (MSCs exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25 human telomerase reverse transcriptase (hTERT transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs and decidua basalis (DMSCs, respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Differential responses to radiation and hyperthermia of cloned cell lines derived from a single human melanoma xenograft

International Nuclear Information System (INIS)

Rofstad, E.K.; Brustad, T.

1984-01-01

One uncloned and five cloned cell lines were derived from a single human melanoma xenograft. Cells from passages 7-12 were exposed to either radiation or hyperthermia (42.5 0 C, pH = 7.4) under aerobic conditions and the colony forming ability of the cells was assayed in soft agar. The five cloned lines showed individual and characteristic responses to radiation as well as to hyperthermia. The variation in the response to radiation was mainly reflected in the size of the shoulders of the survival curves rather than in the D 0 -values. The variation in the response to hyperthermia was mainly reflected in the terminal slopes of the survival curves. The survival curve of cells from the uncloned line, both when exposed to radiation and hyperthermia, was positioned in the midst of those of the cloned lines. The response of the cloned lines to radiation did not correlate with the response to hyperthermia, indicating that tumor cell subpopulations which are resistant to radiation may respond well to hyperthermia

Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

International Nuclear Information System (INIS)

Witasp, Erika; Gustafsson, Ann-Catrin; Cotgreave, Ian; Lind, Monica; Fadeel, Bengt

2005-01-01

Previous studies have suggested that 1,25(OH) 2 D 3 , the active form of vitamin D 3 , may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D 3 has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH) 2 D 3 induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in this model. Moreover, 1,25(OH) 2 D 3 failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D 3

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera).

Science.gov (United States)

Goodman, Cynthia L; Ringbauer, Joseph A; Li, Yao-Fa; Lincoln, Tamra Reall; Stanley, David

2017-05-01

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

Design, Synthesis and Evaluation of N13-Substituted Evodiamine Derivatives against Human Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Senchuan Song

2013-12-01

Full Text Available Attempting to improve the anticancer activity and solubility of evodiamine in simulated gastric fluid (SGF and simulated intestinal fluid (SIF solutions, thirty-eight N13-substituted evodiamine derivatives were designed, synthesized and tested for antitumor activities against six kinds of human cancer cell lines, namely prostate cancer (DU-145 and PC-3, lung cancer (H460, breast cancer (MCF-7, colon cancer (HCT-5 and glioblastoma (SF-268. The solubility of these compounds in SGF and SIF solutions was evaluated, and apoptosis induced by 2-2, 2-3, 2-16 and 3-2 was determined. The results showed: (1 among all compounds examined, 2-16 showed the highest antitumor activity and a broader spectrum of activity, with IC50 values ranging from 1–2 µM; (2 their solubility was obviously improved; (3 2-3, 2-16 and 3-2 had a significant impact inducing apoptosis in some cancer cell lines. The preliminary structure-activity relationships of these derivatives were discussed.

Lymphoblast-derived integration-free ISRM-CON9 iPS cell line from a 75 year old female

Directory of Open Access Journals (Sweden)

Soraia Martins

2018-01-01

Full Text Available Human lymphoblast cells were used to generate integration-free induced pluripotent stem cells (iPSCs employing episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The derived iPSCs were defined as pluripotent based on (i expression of pluripotency-associated markers, (ii embryoid body-based differentiation into cell types representative of the three germ layers and (iii the similarity between the transcriptomes of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95.

Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

Science.gov (United States)

Sitt, Tatjana; Pelle, Roger; Chepkwony, Maurine; Morrison, W Ivan; Toye, Philip

2018-05-06

The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Qian, Hui; Ding, Xiaoqing; Zhang, Jiao; Mao, Fei; Sun, Zixuan; Jia, Haoyuan; Yin, Lei; Wang, Mei; Zhang, Xu; Zhang, Bin; Yan, Yongmin; Zhu, Wei; Xu, Wenrong

2017-06-13

Mesenchymal stem cells (MSCs) transplantation has been used for therapeutic applications in various diseases. Here we report MSCs can malignantly transform in vivo. The novel neoplasm was found on the tail of female rat after injection with male rat bone marrow-derived MSCs (rBM-MSCs) and the new tumor cell line, K3, was isolated from the neoplasm. The K3 cells expressed surface antigens and pluripotent genes similar to those of rBM-MSCs and presented tumor cell features. Moreover, the K3 cells contained side population cells (SP) like cancer stem cells (CSCs), which might contribute to K3 heterogeneity and tumorigenic capacity. To investigate the metastatic potential of K3 cells, we established the nude mouse models of liver and lung metastases and isolated the corresponding metastatic cell lines K3-F4 and K3-B6. Both K3-F4 and K3-B6 cell lines with higher metastatic potential acquired more mesenchymal and stemness-related features. Epithelial-mesenchymal transition is a potential mechanism of K3-F4 and K3-B6 formation.

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.

A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer

International Nuclear Information System (INIS)

Oh, Sang Taek; Seo, Jung Seon; Moon, Uk Yeol; Kang, Kyeong Hee; Shin, Dong-Jik; Yoon, Sungjoo Kim; Kim, Woo Ho; Park, Jae-Gahb; Lee, Suk Kyeong

2004-01-01

In a process seeking out a good model cell line for Epstein-Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis

Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation

NARCIS (Netherlands)

Creemers, L. B.; Meng, X.; den Ouden, K.; van Pelt, A. M. M.; Izadyar, F.; Santoro, M.; Sariola, H.; de rooij, D. G.

2002-01-01

With a novel method of eliminating spermatogenesis in host animals, male germ cells isolated from mice with targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) were transplanted to evaluate their ability to reproduce the phenotype previously found in the transgenic animals.

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.

A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

Science.gov (United States)

Pérez-Campo, Flor M; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Zarrabeitia, María T; Riancho, José A

2014-08-01

Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

Directory of Open Access Journals (Sweden)

Svetlana Gavrilov

2011-01-01

Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

New Sorafenib Derivatives: Synthesis, Antiproliferative Activity Against Tumour Cell Lines and Antimetabolic Evaluation

Directory of Open Access Journals (Sweden)

Branka Zorc

2012-01-01

Full Text Available Sorafenib is a relatively new cytostatic drug approved for the treatment of renal cell and hepatocellular carcinoma. In this report we describe the synthesis of sorafenib derivatives 4a–e which differ from sorafenib in their amide part. A 4-step synthetic pathway includes preparation of 4-chloropyridine-2-carbonyl chloride hydrochloride (1, 4-chloro-pyridine-2-carboxamides 2a–e, 4-(4-aminophenoxy-pyridine-2-carboxamides 3a–e and the target compounds 4-[4-[[4-chloro-3-(trifluoromethylphenyl]carbamoylamino]-phenoxy]-pyridine-2-carboxamides 4a–e. All compounds were fully chemically characterized and evaluated for their cytostatic activity against a panel of carcinoma, lymphoma and leukemia tumour cell lines. In addition, their antimetabolic potential was investigated as well. The most prominent antiproliferative activity was obtained for compounds 4a–e (IC50 = 1-4.3 μmol·L−1. Their potency was comparable to the potency of sorafenib, or even better. The compounds inhibited DNA, RNA and protein synthesis to a similar extent and did not discriminate between tumour cell lines and primary fibroblasts in terms of their anti-proliferative activity.

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

Science.gov (United States)

Tansriratanawong, Kallapat; Ishikawa, Hiroshi; Toyomura, Junko; Sato, Soh

2017-10-01

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

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.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

Science.gov (United States)

Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

2011-08-01

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder

Energy Technology Data Exchange (ETDEWEB)

Lee, Young H. [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Apolo, Andrea B. [Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Agarwal, Piyush K.; Bottaro, Donald P., E-mail: dbottaro@helix.nih.gov [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

2014-11-25

There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.

Cytotoxicity of nano-hydroxyapatite on human-derived oral epithelium cell line: an in vitro study

Directory of Open Access Journals (Sweden)

Farid Abassi

2016-08-01

Full Text Available Background: Hydroxyapatite nanoparticles have a more surface contact and solubility than conventional hydroxyapatite. Hydroxynanoparticles enhances the biological and mechanical properties of new regenerated tissues. The hydroxyapatite nanoparticles have received attention as a new and effective osseous graft for using as scaffolds in bone regeneration. The reports on hydroxyapatite nanoparticles biocompatibility are controversial. It has been shown that hydroxyapatite nanoparticles induces inflammatory reaction and apoptosis. The aim of the present study was to evaluate the cytotoxicity of nano-hydroxyapatite on the human epithelial cells. Methods: The study was experimental and completed in vitro. The study was carried out in department of Immonulogy, Faculty of Medicine, Shahid Beheshti University of Medical Sciences in November 2014. The human-derived oral epithelium cell line (KB obtained from Pasteur Institute, Tehran, Iran were exposed to hydroxyapatite nanoparticles at 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2.5 and 5 mg/ml concentrations in 24, 48 and 72 hours. Rod-shaped hydroxyapatite nanoparticles with 99% purity and maximum 100 nm sized particles were used. Methylthiazol tetrazolium bromide (MTT method was employed for cell vitality evaluation. Enzyme-linked immunosorbent assay (ELISA was used for assessing the viability of cells. Distilled water and fetal bovine serum (FBS were positive and negative controls. ANOVA and Duncan tests were used for statistical analysis. Results: The cytotoxicity of different concentrations of hydroxyapatite nanoparticles on human-derived oral epithelium cell line in 24 (P< 0.001, 48 (P< 0.001 and 72 hours (P< 0.001 was significantly different. The nano-hydroxyapatite particles at 0.5 to 1 mg/ml had the highest cytotoxicity effect on human-derived oral epithelium cells in 24, 48 and 72 hours. Lower concentrations than 0.05 mg/ml had the best biocompatibility properties in 24, 48 and 72 hours. Conclusion

Photodynamic effects of a novel pterin derivative on a pancreatic cancer cell line

International Nuclear Information System (INIS)

Yamada, Hiroko; Arai, Toshiyuki; Endo, Nobuyuki; Yamashita, Kouhei; Nonogawa, Mitsuru; Makino, Keisuke; Fukuda, Kazuhiko; Sasada, Masataka; Uchiyama, Takashi

2005-01-01

6-Formylpterin (6FP) has the potential to produce singlet oxygen ( 1 O 2 ) under UV-A radiation. In order to apply this potential to anti-cancer photodynamic therapy (PDT), we prepared a novel variant of 6FP, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one (6FP-tBu-DMF), and examined its photodynamic effects on a pancreatic cancer cell line, Panc-1 cells. The study using laser scanning confocal microscopy showed that the drug uptake, the 1 O 2 generation, and cell death were observed in the 6FP-tBu-DMF-treated cells, while these phenomena were not observed in the 6FP-treated cells. The MTT assay also showed the decrease in cell viability only in the 6FP-tBu-DMF-treated cells. Since 6FP and 6FP-tBu-DMF generate 1 O 2 to the same extent under UV-A radiation in aqueous solutions, these results indicated that the differences in the photodynamic effects between 6FP and 6FP-tBu-DMF were entirely attributed to the differences in the cell permeability between them. The development of cell permeable pterin derivatives has the potential for application in PDT

Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

DEFF Research Database (Denmark)

Litvinov, Ivan V; Cordeiro, Brendan; Fredholm, Simon Mayland

2014-01-01

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype......R-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA....... by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines...

A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

Directory of Open Access Journals (Sweden)

Vinita Chauhan

2012-01-01

Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

Science.gov (United States)

Schatten, H.; Hedrick, J.; Chakrabarti, A.

1998-01-01

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Application of DNA fingerprints for cell-line individualization.

OpenAIRE

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

1990-01-01

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 d...

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

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

[Establishment and characterization of a cell line derived from human ovarian mucinous cystadenocarcinoma].

Science.gov (United States)

Wan, Q; Xu, D; Li, Z

2001-07-01

To establish a cell line of human ovarian cancer, and study its characterization. The cell line was established by the cultivation of subsides walls, and kept by freezing. The morphology was observed by microscope and electromicroscope. The authors studied its growth and propagation, the agglutination test of phytohemagglutinin (PHA), the chromosome analysis, heterotransplanting, immuno-histochemistry staining, the analysis of hormone, the pollution examination and the test of sensitivity to virus etc. A new human ovarian carcinoma cell line, designated ovarian mucinous cystadenocarcinoma 685 (OMC685), was established from mucinous cystadenocarcinoma. This cell line had subcultured to 91 generations, and some had been frozen for 8 years and revived, still grew well. This cell line possessed the feature of glandular epithelium cancer cell. The cells grew exuberantly, and the agglutinating test of PHA was positive. Karyotype was subtriploid with distortion. Heterotransplantations, alcian blue periobic acid-schiff (AbPAS), mucicarmine, alcian blue stainings, estradiol (E2) and progesterone were all positive. Without being polluted, it was sensitive to polivirus-I, adenovirus 7 and measles virus. OMC685 is a distinct human ovarian tumous cell line.

Influence of Spray-dried Hydroxyapatite-5-Fluorouracil Granules on Cell Lines Derived from Tissues of Mesenchymal Origin

Directory of Open Access Journals (Sweden)

Tim Scharnweber

2008-11-01

Full Text Available In our previous work we described the preparation and characterization of spray dried hydroxyapatite micro granules loaded with 5-fluorouracil (5-FU. These loaded particles are used as a model drug delivery system (DDS. In this study we examined the in vitro response of two cell lines derived from different tissues to 5-FU loaded granules (LG. Both cell lines, either L929 cells of a mouse fibroblast lineage or cells originating from a rat osteosarcoma (ROS 17/2.8 showed a dose dependent decrease in cell proliferation in response to 5-FU-, either dissolved in the culture medium or loaded onto particles. The response of the two cell lines to loaded and nonloaded particles was different. The effect of LG and of a corresponding concentration of free 5-FU was practically the same for the ROS 17/2.8 cells indicating that ROS 17/2.8 cells were not affected by the carrier material. In contrast, L929 cells showed a slight decrease in cell proliferation also in the presence of granules not loaded with 5-FU. This is thought to be attributed to the inhibition of mitogenesis by phosphocitrates, already demonstrated in fibroblasts. In summary, we found that the loaded 5-FU kept its effectivity after the spray drying process and that the response towards the granules varied with cell type. This is the first step towards a tissue specific DDS.

Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines.

Directory of Open Access Journals (Sweden)

Matthew G Permenter

Full Text Available Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.

Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

Directory of Open Access Journals (Sweden)

Margarita Chumarina

2017-03-01

Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

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

Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

Directory of Open Access Journals (Sweden)

Hui Peng

2012-01-01

Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

DEFF Research Database (Denmark)

Harkness, Linda; Rasmussen, Iben Anne; Erb, Karin

2010-01-01

The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analy...

High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines.

Science.gov (United States)

Danovi, Davide; Folarin, Amos A; Baranowski, Bart; Pollard, Steven M

2012-01-01

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries. Improvements in automated microscopy, desktop computational power, and freely available image processing tools, now means that such chemical screens are realistic to undertake in individual academic laboratories. Here we outline a cost effective and versatile time lapse imaging strategy suitable for chemical screening. Protocols are described for the handling and screening of human fetal Neural Stem (NS) cell lines and their malignant counterparts, Glioblastoma-derived neural stem cells (GNS). We focus on identification of cytostatic and cytotoxic "hits" and discuss future possibilities and challenges for extending this approach to assay lineage commitment and differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Patient-Derived Antibody Targets Tumor Cells

Science.gov (United States)

An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

The effect of the two epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) on cell lines established from patients with small cell carcinoma of the lung

DEFF Research Database (Denmark)

Roed, H; Vindeløv, Lars; Christensen, I J

1987-01-01

To determine whether there is any difference between the two epipodophyllotoxin derivatives etoposide and teniposide in their therapeutic effect in small cell carcinoma of the lung (SCCL), they were compared against five human SCCL cell lines in vitro. When the two were compared at equimolar...

Cabergoline decreases alcohol drinking and seeking behaviors via glial cell line-derived neurotrophic factor.

Science.gov (United States)

Carnicella, Sebastien; Ahmadiantehrani, Somayeh; He, Dao-Yao; Nielsen, Carsten K; Bartlett, Selena E; Janak, Patricia H; Ron, Dorit

2009-07-15

Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPgammaS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders.

Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity

Directory of Open Access Journals (Sweden)

Matthew eKruger

2016-03-01

Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

Dataset of curcumin derivatives for QSAR modeling of anti cancer against P388 cell line

Directory of Open Access Journals (Sweden)

Yum Eryanti

2016-12-01

Full Text Available The dataset of curcumin derivatives consists of 45 compounds (Table 1 with their anti cancer biological activity (IC50 against P388 cell line. 45 curcumin derivatives were used in the model development where 30 of these compounds were in the training set and the remaining 15 compounds were in the test set. The development of the QSAR model involved the use of the multiple linear regression analysis (MLRA method. Based on the method, r2 value, r2 (CV value of 0.81, 0.67 were obtained. The QSAR model was also employed to predict the biological activity of compounds in the test set. Predictive correlation coefficient r2 values of 0.88 were obtained for the test set.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

Energy Technology Data Exchange (ETDEWEB)

Wang, Meng-Yu [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no [Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo (Norway); Rekdal, Øystein [Department of Medical Biology, Faculty of Health Sciences, UiT The Arctic University of Norway, Tromsø (Norway); Kvalheim, Gunnar [Department of Cell Therapy, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway); Fodstad, Øystein [Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Oslo (Norway)

2017-03-15

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

International Nuclear Information System (INIS)

Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

2017-01-01

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.

Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

International Nuclear Information System (INIS)

Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S.; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia

2013-01-01

A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

Energy Technology Data Exchange (ETDEWEB)

Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S., E-mail: ccelso@dcm.ufrpe.br [Universidade Federal Rural de Pernambuco (LSCB/UFRPE), Recife, PE (Brazil). Dept. de Ciencias Moleculares. Lab. de Sintese de Compostos Bioativos; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia [Universidade Federal do Ceara (LOE/UFC), Fortaleza, CE (Brazil). Dept. de Fisiologia e Farmacologia. Lab. de Oncologia Experimental

2013-09-15

A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

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.

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

Directory of Open Access Journals (Sweden)

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.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

International Nuclear Information System (INIS)

Du Kejun; Chai Yubo; Hou Lichao; Chang Wenhui; Chen Suming; Luo Wenjing; Cai Tongjian; Zhang Xiaonan; Chen Nanchun; Chen Yaoming; Chen Jingyuan

2008-01-01

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2

Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

DEFF Research Database (Denmark)

Jakobsen, Jannik E; Li, Juan; Moldt, Brian

2011-01-01

We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

Transplantability of human lymphoid cell line, lymphoma, and leukemia in splenectomized and/or irradiated nude mice

International Nuclear Information System (INIS)

Watanabe, S.; Shimosato, Y.; Kuroki, M.; Sato, Y.; Nakajima, T.

1980-01-01

The effects of splenectomy and/or whole-body irradiation of nude mice before xenotransplantation of lymphoid cell lines, lymphoma, and leukemia were studied. Transplantation after whole-body irradiation resulted in the increased ''take'' rate of three cultured cell lines (two of T-cell-derived acute lymphocytic leukemia and one of B-cell derived acute lymphocytic leukemia) and in the tumorous growth of Burkitt-derived Raji and spontaneously transformed lymphoblastoid cell lines. With splenectomy plus irradiation as a pretreatment, tumorous growth occurred in four other cell lines which were not transplantable after irradiation only (two cell lines of Epstein-Barr virus-transformed cord blood cells and one each of null acute lymphocytic leukemia and nodular lymphoma-derived cell lines). Direct transplantation of leukemia and lymphoma cells into the pretreated mice was successful in 7 of 24 cases (29%). B-cell-derived diffuse large lymphoid lymphoma was transplantable in three of seven cases (43%). However, lymphoma and leukemia of peripheral T-cell origin was difficult to transplant even with pretreatment, and only one pleomorphic T-cell lymphoma grew to a significant size (2 cm). One tumor each of B-cell-derived diffuse large lymphoid and T-cell diffuse lymphoblastic lymphoma became transplantable

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

Science.gov (United States)

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

Low antigenicity of hematopoietic progenitor cells derived from human ES cells

Directory of Open Access Journals (Sweden)

Eun-Mi Kim

2010-02-01

Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

International Nuclear Information System (INIS)

Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

2010-01-01

Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated β-galactosidase (SA-β-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells ( WAF1/CIP1 was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

Generation of an iPS cell line via a non-integrative method using urine-derived cells from a patient with USH2A-associated retinitis pigmentosa

Directory of Open Access Journals (Sweden)

Yonglong Guo

2018-05-01

Full Text Available We have established an induced pluripotent stem (iPS cell line using urine-derived cells from a 27-year-old male patient with retinitis pigmentosa associated with point mutations in the USH2A gene. Feeder-free culture conditions and the integration-free CytoTune™-iPS 2.0 Sendai Reprogramming Kit were used.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

Directory of Open Access Journals (Sweden)

Marcela Cristina Correa de Freitas

2014-06-01

Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

Novel near-diploid ovarian cancer cell line derived from a highly aneuploid metastatic ovarian tumor.

Directory of Open Access Journals (Sweden)

Ester Rozenblum

Full Text Available A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY, and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found. A large number of focal copy number alterations (FCNAs were detected, including homozygous deletions (HDs targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements. Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and –negative bacteria, yeast and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

DEFF Research Database (Denmark)

Fiskesund, R.; Thomas, L.V.; Schobert, M.

2009-01-01

Aims: To prepare 1,5-anhydro-d-fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines. Methods and Results: Microthecin and nine other AF derivatives were synthesized...... both GP and GN bacteria, including Ps. aeruginosa. Further tests with three malignant blood cell lines (Mutu, Ramos, Raji) and one normal cell line indicated that microthecin was a cell toxin, with a cell mortality >85% at 50 ppm. The other nine AF derivatives demonstrated low or no antimicrobial...... activity. Conclusions: Microthecin was active 100–2000 ppm against GP and GN bacteria including Ps. aeruginosa, but was inactive against yeasts and moulds. Microthecin was also a cytotoxin to some mammalian cell lines. Significance and Impact of the Study: Microthecin might have potential for development...

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...

X-radiation-induced transformation in a C3H mouse embryo-derived cell line

International Nuclear Information System (INIS)

Terzaghi, M.; Little, J.B.

1976-01-01

Reproducible x-ray-induced oncogenic transformation has been demonstrated in an established cell line of mouse embryo fibroblasts. Cells derived from transformed foci formed malignant tumors when injected into syngeneic hosts. An exponential increase in the number of transformants per viable cell occurred with doses of up to 400 rads of x-radiation. The transformation frequency in exponentially growing cultures remained constant at 2.3 x 10 -3 following doses of 400 to 1500 rads. There was little change in survival following x-ray doses up to 300 rads. Doses greater than 300 rads were associated with an exponential decline in survival; the D 0 for the survival curve was 175 rads. Transformation frequency varied with changes in the number of viable cells seeded per dish. There was about a 10-fold decline in the transformation frequency when the number of cells was increased from 400 to 1000 viable cells/100-mm Petri dish. Below this density range there was little change in transformation frequency. The presence of lethally preirradiated cells was not associated with an enhancement of transformation in irradiated cells or with the induction of transformation in unirradiated cell cultures. Amphotericin B (Fungizone) inhibited the appearance of transformants when added to the culture medium within 2 to 3 weeks after initiation of the experiment

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

Energy Technology Data Exchange (ETDEWEB)

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.

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.

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

OpenAIRE

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

2014-01-01

Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes...

SYNTHESIS AND CYTOTOXIC ACTIVITY OF CHALCONE DERIVATIVES ON HUMAN BREAST CANCER CELL LINES

Directory of Open Access Journals (Sweden)

Nuraini Harmastuti

2012-12-01

Full Text Available Chalcone, an α,β-unsaturated ketone, has been shown have many biological activities such as anticancer and antifungi. This research was conducted to synthesize the chalcone derivatives and to obtain their cytotoxic activity on human cervix cancer cell lines. Synthesis of chalcone and its derivatives, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone was carried out using starting materials of benzaldehide and acetofenon, p-methylacetophenone, p-methoxyacetophenone, as well as m,p-dichloroacetophenone through Claisen Schmidt condensation catalized by NaOH in ethanol at 15 °C. The purity of synthesized compounds were analyzed by thin layer chromatography, melting range, and gas chromatography. Structure elucidations were conducted by UV spectrophotometer, IR spectrometer, 1H-NMR spectrometer, as well as mass spectrometer. Cytotoxic activities were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT microculture tetrazolium viability assay. The results showed that chalcone and derivatives compounds have been able to be synthesized and purified and had the same structure as a predicted structure. Chalcone had highest cytotoxic activity compared to that of its derivatives, with the IC50 values of chalcone, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone were 9.49, 14.79, 11.48, and 24.26 µg/mL respectively. It was concluded that methyl, methoxy as well as chlorine substitution at 3 II and 4II position decrease the cytotoxic activity of chalcone.

Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

Directory of Open Access Journals (Sweden)

Coppock Donald L

2008-12-01

Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

Science.gov (United States)

Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

1991-01-01

Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

Directory of Open Access Journals (Sweden)

Florence Wianny

2016-05-01

Full Text Available The imprinted genes of primate embryonic stem cells (ESCs often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs, SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum

Directory of Open Access Journals (Sweden)

Kohji Okamura

2015-12-01

Full Text Available Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP, which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

NANOS2 acts downstream of glial cell line-derived neurotrophic factor signaling to suppress differentiation of spermatogonial stem cells.

Science.gov (United States)

Sada, Aiko; Hasegawa, Kazuteru; Pin, Pui Han; Saga, Yumiko

2012-02-01

Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. Copyright © 2011 AlphaMed Press.

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

Science.gov (United States)

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

2008-02-15

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

Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Xiaoxiao Qin

2015-11-01

Full Text Available Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. “Radiant”, and their chemical structures were elucidated by UV, IR, ESI-MS, 1H-NMR and 13C-NMR analyses. These compounds, which include trilobatin (A1, phloretin (A2, 3-hydroxyphloretin (A3, phloretin rutinoside (A4, phlorizin (A5, 6′′-O-coumaroyl-4′-O-glucopyranosylphloretin (A6, and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin (A7, all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity.

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.

Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

OpenAIRE

Zhang Ping; Zhang Zhiyuan; Zhou Xiaojian; Qiu Weiliu; Chen Fangan; Chen Wantao

2006-01-01

Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differe...

Establishment and characterization of murine small cell lung carcinoma cell lines derived from HPV-16 E6/E7 transgenic mice.

Science.gov (United States)

Carraresi, Laura; Martinelli, Rosanna; Vannoni, Alessandro; Riccio, Massimo; Dembic, Maja; Tripodi, Sergio; Cintorino, Marcella; Santi, Spartaco; Bigliardi, Elisa; Carmellini, Mario; Rossini, Mara

2006-01-08

We have established two murine cell lines derived from Small Cell Lung Carcinomas (SCLCs) developed by HPV-E6/E7 transgenic mice. These cells named PPAP-9 and PPAP-10 were isolated from mice bearing tumors, 9 and 10 months old, respectively. The cells, 5 microm in diameter, express HPV oncoproteins and sustain tumor formation after subcutaneous injection in syngenic mice. A detailed analysis indicated the epithelial origin and the neuroendocrine differentiation of these cells. We showed by confocal immunofluorescence the expression of the epithelial marker cytokeratin 5, whose gene promoter was used to direct the expression of HPV E6/E. Cells express several neuroendocrine markers such as CGRP, MAP-2, Ash1, CgrA, Scg2. The neuroendocrine differentiation of these cells was further confirmed by electron microscopy demonstrating neuropeptides secreting granules in their cytoplasm. Furthermore, in agreement with the altered expression observed in the majority of human SCLC we showed in these cells the absence of both p53 and pRB and a dramatic reduction in the expression of Caveolin-1.

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

Science.gov (United States)

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

2012-07-01

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

Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes

DEFF Research Database (Denmark)

Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

2016-01-01

of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10...... with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P

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

Science.gov (United States)

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

2014-01-01

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

Warburg and Crabtree effects in premalignant Barrett's esophagus cell lines with active mitochondria.

Directory of Open Access Journals (Sweden)

Martin T Suchorolski

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

Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro

Directory of Open Access Journals (Sweden)

Heinrich Juliana K

2008-10-01

Full Text Available Abstract Background In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear. Methods This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability. Results Results showed that NG97(ht had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP and vimentin. In addition, β1 integrins were present in intermediate levels while α5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells

Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro

International Nuclear Information System (INIS)

Machado, Camila ML; Boetcher-Luiz, Fátima; Verinaud, Liana; Ikemori, Rafael Y; Zorzeto, Tatiana Q; Nogueira, Ana CMA; Barbosa, Suse DS; Savino, Wilson; Schenka, André A; Vassallo, José; Heinrich, Juliana K

2008-01-01

In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear. This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60 th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability. Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, β1 integrins were present in intermediate levels while α5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant. In

Comparison of Two Mouse Ameloblast-like Cell Lines for Enamel-specific Gene Expression

Directory of Open Access Journals (Sweden)

Juni eSarkar

2014-07-01

Full Text Available Ameloblasts are ectoderm-derived cells that produce an extracellular enamel matrix that mineralizes to form enamel. The development and use of immortalized cell lines, with a stable phenotype, is an important contribution to biological studies as it allows for the investigation of molecular activities without the continuous need for animals. In this study we compare the expression profiles of enamel-specific genes in two mouse derived ameloblast-like cell lines: LS8 and ALC cells. Quantitative PCR analysis indicates that, relative to each other, LS8 cells express greater mRNA levels for genes that define secretory-stage activities (Amelx, Ambn, Enam and Mmp20, while ALC express greater mRNA levels for genes that define maturation-stage activities (Odam and Klk4. Western blot analyses show that Amelx, Ambn and Odam proteins are detectable in ALC, but not LS8 cells. Unstimulated ALC cells form calcified nodules, while LS8 cells do not. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.

Synergistic Cytotoxicity Effect by Combination Treatment of Polyketide Derivatives from Annona muricata Linn Leaves and Doxorubicin as Potential Anticancer Material on Raji Cell Line

Science.gov (United States)

Artanti, A. N.; Astirin, O. P.; Prayito, A.; Fisma, R.; Prihapsara, F.

2018-03-01

Nasopharynx cancer is one of the most deadly cancer. The main priority of nasopharynx cancer treatment is the use of chemotherapeutic agents, especially doxorubicin. However, doxorubicin might also lead to diverse side effect. An approach recently develop to overcome side effect of doxorubicin is to used of combined chemotherapeutic agent. One of the compounds found effication as an anticancer agent on nasopharynx cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on raji cell line. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). Data analysis showed that combination of polyketide derivative from Annona muricata L. (14,4 µg/ml) and doxorubicin with all of concentration performed synergistic effect on raji cell line with CI value from 0.13 – 0.65.

Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I ifn.

Science.gov (United States)

Rodriguez Saint-Jean, S; González, C; Monrás, M; Romero, A; Ballesteros, N; Enríquez, R; Perez-Prieto, S

2014-11-01

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes. © 2014 The Fisheries Society of the British Isles.

Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

International Nuclear Information System (INIS)

Bronzert, D.A.; Pantazis, P.; Antoniades, H.N.; Kasid, A.; Davidson, N.; Dickson, R.B.; Lippman, M.E.

1987-01-01

The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of [ 3 H] thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 0 C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of ≅30 kDa on NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO 4 /polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth

Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

Science.gov (United States)

Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

2009-10-01

Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

Directory of Open Access Journals (Sweden)

Ana de la Cueva

Full Text Available Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

Directory of Open Access Journals (Sweden)

Aline Semblano Carreira Falcão

Full Text Available Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1 derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs and their tissue inhibitors (TIMPs was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1. Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG. Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

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

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

Derivation of hybrid ES cell lines from two different strains of mice

Directory of Open Access Journals (Sweden)

Ho-Tak Lau

2016-03-01

Full Text Available Parental origin-dependent expression of the imprinted genes is essential for mammalian development. Zfp57 maintains genomic imprinting in mouse embryos and ES cells. To examine the allelic expression patterns of the imprinted genes in ES cells, we obtained multiple hybrid ES clones that were directly derived from the blastocysts generated from the cross between mice on two different genetic backgrounds. The blastocyst-derived ES clones displayed largely intact DNA methylation imprint at the tested imprinted regions. These hybrid ES clones will be useful for future studies to examine the allelic expression of the imprinted genes in ES cells and their differentiated progeny.

Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD for monogenic disorder

Directory of Open Access Journals (Sweden)

Abdelkrim Hmadcha

2016-05-01

Full Text Available From 106 human blastocyts donate for research after in vitro fertilization (IVF and preimplantation genetic diagnosis (PGD for monogenetic disorder, 3 human embryonic stem cells (hESCs HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15(q34.3;q14 detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines.

Science.gov (United States)

Zhou, Wu-Xi; Cao, Jia-Qing; Wang, Xu-De; Guo, Jun-Hui; Zhao, Yu-Qing

2017-02-15

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation, isolation, and maintenance of rodent mast cells and mast cell lines

DEFF Research Database (Denmark)

Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko

2006-01-01

Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

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

Garlic-derived compound S-allylmercaptocysteine (SAMC) is active against anaplastic thyroid cancer cell line 8305C (HPACC).

Science.gov (United States)

Liu, Yuexin; Yan, Jinyin; Han, Xiaochen; Hu, Wanning

2015-01-01

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic have anticancer activity. In this study, the apoptotic effects of Garlic-derived compound S-allylmercaptocysteine (SAMC) were investigated in 8305C human anaplastic thyroid carcinoma cells. The cell line 8305C (HPACC) were treated with SAMC and the MTT assay, flow cytometry (FCM), electron microscope method were used to test cell cycle, inhibitory rate and morphologic changes respectively. HPACC-8305C cells were suppressed after exposure to SAMC of 0.02 mg/ml, 0.06 mg/ml, and 0.1 mg/ml for 48 h. Compared with the control, the difference was significant (P< 0.05). SAMC could induce apoptosis of the cells in a dose-dependent and non-linear manner and increase the proportion of cells in the G2/M phase. Compared with the control, the difference was significant in terms of the percentage of cells in the G2/M phase (P< 0.05). After exposure to SAMC at 0.02 mg/ml for 24 hours, HPACC-8305C cells showed typical morphologic change. SAMC inhibits the growth of HPACC-8305C cells by induction of apoptotic cell death and inhibit telomerase activity, which appears to account for its anti-cancer activity.

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

DEFF Research Database (Denmark)

Xu, Xun; Pan, Shengkai; Liu, Xin

2011-01-01

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder

OpenAIRE

Abdelkrim Hmadcha; Yolanda Aguilera; Maria Dolores Lozano-Arana; Nuria Mellado; Javier Sánchez; Cristina Moya; Luis Sánchez-Palazón; Jose Palacios; Guillermo Antiñolo; Bernat Soria

2016-01-01

From 106 human blastocyts donate for research after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for monogenetic disorder, 3 human embryonic stem cells (hESCs) HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15)(q34.3;q14) detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were r...

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

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.

Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

Science.gov (United States)

Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

2001-01-01

33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity.

Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Kyle J Hewitt

2011-02-01

Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines.

Science.gov (United States)

Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

2016-04-01

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

Science.gov (United States)

Bodel, P

1978-05-01

Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.

Science.gov (United States)

Barrett, Robert; Ornelas, Loren; Yeager, Nicole; Mandefro, Berhan; Sahabian, Anais; Lenaeus, Lindsay; Targan, Stephan R; Svendsen, Clive N; Sareen, Dhruv

2014-12-01

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblast-derived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications. ©AlphaMed Press.

Development and characterization of a bovine monocyte-derived macrophage cell line

Science.gov (United States)

Monocytes circulate in the blood, and later differentiate into macrophages in the tissues. They are components of the innate arm of the immune response and are one of the first lines of defense again invading pathogens. However, they also serve as host cells for intracellular pathogens such as Mycob...

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy

International Nuclear Information System (INIS)

Gioanni, J.; Courdi, A.; Lalanne, C.M.; Fischel, J.L.; Zanghellini, E.; Lambert, J.C.; Ettore, F.; Namer, M.

1985-01-01

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro

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.

Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

Directory of Open Access Journals (Sweden)

Clementina Sansone

Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

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.

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

Science.gov (United States)

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

Directory of Open Access Journals (Sweden)

Syed Mohmad Shah

2015-11-01

Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

Procedures for Derivation and Characterisation of Human Embryonic Stem Cells from Odense, Denmark

DEFF Research Database (Denmark)

Harkness, Linda; Kassem, Moustapha

2012-01-01

In 1998, a development occurred in stem cell biology with the fi rst report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolatio...

Derivation and characterization of monkey embryonic stem cells

Directory of Open Access Journals (Sweden)

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

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.

Synthesis of an anthraquinone derivative (DHAQC) and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line.

Science.gov (United States)

Yeap, SweeKeong; Akhtar, Muhammad Nadeem; Lim, Kian Lam; Abu, Nadiah; Ho, Wan Yong; Zareen, Seema; Roohani, Kiarash; Ky, Huynh; Tan, Sheau Wei; Lajis, Nordin; Alitheen, Noorjahan Banu

2015-01-01

Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2- carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the Friedel-Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line.

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.).

Guidelines for the use of cell lines in biomedical research.

Science.gov (United States)

Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

2014-09-09

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

International Nuclear Information System (INIS)

Beretta, L.; Boutterin, M.C.; Sobel, A.

1988-01-01

We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [ 32 P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins

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

Science.gov (United States)

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

2012-01-01

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

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

Science.gov (United States)

Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

2015-08-01

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

International Nuclear Information System (INIS)

Wieringa, Paul; Micera, Silvestro; Tonazzini, Ilaria; Cecchini, Marco

2012-01-01

The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds. (paper)

Generation of iPSC lines from primary human chorionic villi cells

Directory of Open Access Journals (Sweden)

Björn Lichtner

2015-11-01

Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

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

Science.gov (United States)

Rathore, Kusum; Cekanova, Maria

2015-01-01

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

The transcriptional diversity of 25 Drosophila cell lines

Energy Technology Data Exchange (ETDEWEB)

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

Incorporation study of 18FDG through its uptake into tumour-derived cell lines

International Nuclear Information System (INIS)

Costa, Flavia M.; Nascimento, Leonardo T.C.; Santos, Raquel G.

2013-01-01

In vitro tests of radiopharmaceuticals are an important instrument to study their mechanisms of action, binding and incorporation. 18 FDG is the most used radiopharmaceutical for diagnostics positron emission tomography (PET) on oncology, on the basis of accelerated rates of absorption of glucose in cell malignancies. This radiotracer has been routinely produced at CDTN; and therefore, it was selected for preliminary assays due to its availability. Nowadays, UPPR at CDTN produces routinely 18 FDG for the local PET Centers but others PET radiopharmaceuticals are in development such as 18 F-Fluorocholine and 18 F-Fluorothymidine. According to the Brazilian Health Regulatory Agency (ANVISA) it is necessary to validate and register these new radiopharmaceuticals in order to get the approval for their commercialization. Targeting efficacy is one of the important issues to be evaluated during radiopharmaceutical validation. The aim of this study was to develop a standard protocol to determine tumor targeting efficacy of PET radiopharmaceuticals in vitro. Therefore it was developed a protocol based on the incorporation og 18 FDG through the uptake in different tumor-derived cell lines. Three variables were investigated for the standardization of the test: the number of cells to be seeded in 96-well plates, the time of incubation with the radiopharmaceutical and the radiotracer concentration. The standardized protocol was considered suitable for 18 FDG incorporation assay and showed reproductive results. The protocol developed in this work will pave the way for the in vitro studies of incorporation of the new PET radiopharmaceuticals to be produced at UPPR-CDTN, such as: 18 F-Fluorocholine and 18 F-Fluorothymidine. (author)

MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

Directory of Open Access Journals (Sweden)

Vanita Vanas

Full Text Available Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin.

Global Conservation of Protein Status between Cell Lines and Xenografts

Directory of Open Access Journals (Sweden)

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.

Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM deficient blastocysts.

Directory of Open Access Journals (Sweden)

Duancheng Wen

Full Text Available Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs by injection of diploid (2n ESCs into tetraploid (4n blastocysts (ESC-derived mice. This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS cells. However, the underlying mechanism(s of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM. We designate these as type a (presence of ICM at blastocyst stage or type b (absence of ICM. ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

Cell Penetrating Capacity and Internalization Mechanisms Used by the Synthetic Peptide CIGB-552 and Its Relationship with Tumor Cell Line Sensitivity.

Science.gov (United States)

Astrada, Soledad; Fernández Massó, Julio Raúl; Vallespí, Maribel G; Bollati-Fogolín, Mariela

2018-03-30

CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization.

Generating a non-integrating human induced pluripotent stem cell bank from urine-derived cells.

Directory of Open Access Journals (Sweden)

Yanting Xue

Full Text Available Induced pluripotent stem cell (iPS cell holds great potential for applications in regenerative medicine, drug discovery, and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency, offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study, we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research, facilitating future applications of human iPS cells.

Synthesis of Chromonylthiazolidines and Their Cytotoxicity to Human Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Hoang Le Tuan Anh

2015-01-01

Full Text Available Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compounds 3a and 3b showed the most selective cytotoxic effects against human epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL and breast cancer (IC50 32.8 ± 1.4 μg/mL cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost, and selective anticancer agents.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

Science.gov (United States)

Goldoni, Dana; Zhao, YouYou; Green, Brian D; McDermott, Barbara J; Collins, Anthony

2010-11-01

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150  mV to 148 µM at -75  mV in 120  mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. © 2010 Wiley-Liss, Inc.

Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

Science.gov (United States)

Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

2005-10-01

Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

Data on the number and frequency of scientific literature citations for established medulloblastoma cell lines

Directory of Open Access Journals (Sweden)

D.P. Ivanov

2016-12-01

Full Text Available This article collates information about the number of scientific articles mentioning each of the established medulloblastoma cell lines, derived through a systematic search of Web of Science, Scopus and Google Scholar in 2016. The data for each cell line have been presented as raw number of citations, percentage share of the total citations for each search engine and as an average percentage between the three search engines. In order to correct for the time since each cell line has been in use, the raw citation data have also been divided by the number of years since the derivation of each cell line. This is a supporting article for a review of in vitro models of medulloblastoma published in “in vitro models of medulloblastoma: choosing the right tool for the job” (D.P. Ivanov, D.A. Walker, B. Coyle, A.M. Grabowska, 2016 [1].

Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation

International Nuclear Information System (INIS)

Droms, K.; Sueoka, N.

1987-01-01

This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP

Radiation of different human melanoma cell lines increased expression of RHOB. Level of this tumor suppressor gene in different cell lines

International Nuclear Information System (INIS)

Notcovich, C.; Molinari, B.; Duran, H.; Delgado González, D.; Sánchez Crespo, R.

2013-01-01

Previous results of our group show that a correlation exists between intrinsic radiosensitivity of human melanoma cells and cell death by apoptosis. RhoB is a small GTPase that regulates cytoskeletal organization. Besides, is related to the process of apoptosis in cells exposed to DNA damage as radiation. Also, RhoB levels decrease in a wide variety of tumors with the tumor stage, being considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. The aim of this study was to analyze the expression of RhoB in different human melanoma cell lines in relation to melanocytes, and evaluate the effect of gamma radiation on the expression of RhoB. We used the A375, SB2 and Meljcell lines, and the derived from melanocytes Pig1. It was found for all three tumor lines RhoB expression levels significantly lower than those of Pig1 (p <0.05), as assessed by semiquantitative RT-PCR . When tumor cells were irradiated to a dose of 2Gyinduction was observed at 3 hours RhoB irradiation. RhoB expression increased in all lines relative to non-irradiated control, showing a greater induction ( p< 0.05) for the more radiosensitive line SB2, consistent with apoptosis in response to radiation. The results allow for the first time in melanoma demonstrate that RhoB, as well as in other tumor types, has a lower expression in tumor cells than their normal counterparts. Moreover, induction in the expression of RhoB in irradiated cells may be associated with the process of radiation-induced apoptosis. The modulation of RhoB could be a new tool to sensitize radioresistant melanoma. (author)

Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation.

Science.gov (United States)

Yamaguchi, Kazunori; Takanashi, Tomoka; Nasu, Kentaro; Tamai, Keiichi; Mochizuki, Mai; Satoh, Ikuro; Ine, Shoji; Sasaki, Osamu; Satoh, Kennichi; Tanaka, Nobuyuki; Harigae, Hideo; Sugamura, Kazuo

2016-05-01

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

The effects of ultraviolet light on aspects of DNA metabolism in cell lines derived from plodia interpunctella and Trichoplusia ni

International Nuclear Information System (INIS)

Styer, S.C.

1991-01-01

Insect cells are significantly more resistant to the lethal effects of 254 nm ultraviolet light (UV) than mammalian cells. The predominant photoproduct produced by UV is the (5-6) cyclobutyl pyrimidine dimer. There is controversy whether this lesion, or another, the pyrimidine (6-4) pyrimidone, is responsible for the biological effects of UV. Insect cells contain a photolyase which selectively removes the (5-6), but not the (6-4) lesion, so that the relative roles of these lesions can be studied. Insect cell lines derived from the cabbage looper and the Indian meal moth were exposed to UV and analyzed for their ability to incorporate 3 H-thymidine. After exposure, cells from the Indian meal moth exhibited a rapid and prolonged depression in the rate of thymidine incorporation, whereas cells from the cabbage looper showed only a slight drop in incorporation and a rapid recovery. The extent of depression in thymidine incorporation was not correlated to the amount of cell killing by UV in these cell lines. Blockage of fork progression was correlated to the depression in thymidine incorporation. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation or cell killing, indicating that although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions, such as the (6-4) photoproduct, may play a role. In addition, activation of alternative sites of replicon initiation appeared to correlate with the depression in thymidine incorporation and the excision capabilities in these cells. The resistance to UV in these insect cells compared to mammalian cells may be due to their ability to rapidly remove the (5-6) lesion, which is the critical lesion in these insect cells

Sulforaphane Analogues with Heterocyclic Moieties: Syntheses and Inhibitory Activities against Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Ye-Hui Shi

2016-04-01

Full Text Available Recent studies have shown that sulforaphane (SFN selectively inhibits the growth of ALDH+ breast cancer stem-like cells.Herein, a series of SFN analogues were synthesized and evaluated against breast cancer cell lines MCF-7 and SUM-159, and the leukemia stem cell-like cell line KG-1a. These SFN analogues were characterized by the replacement of the methyl group with heterocyclic moieties, and the replacement of the sulfoxide group with sulfide or sulfone. A growth inhibitory assay indicated that the tetrazole analogs 3d, 8d and 9d were significantly more potent than SFN against the three cancer cell lines. Compound 14c, the water soluble derivative of tetrazole sulfide 3d, demonstrated higher potency against KG-1a cell line than 3d. SFN, 3d and 14c significantly induced the activation of caspase-3, and reduced the ALDH+ subpopulation in the SUM159 cell line, while the marketed drug doxrubicin(DOX increased the ALDH+ subpopulation.

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

OpenAIRE

Johnston, H. P.; Hawley, P.; White, S. E.; Gibson, I.; Tidd, D. M.

1985-01-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribo...

Expression and function of β-adrenergic receptors in human hematopoietic cell lines

International Nuclear Information System (INIS)

Maeki, T.; Andersson, L.C.; Kontula, K.K.

1992-01-01

We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

DEFF Research Database (Denmark)

Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike

2006-01-01

Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines ...

The transcriptomes of novel marmoset monkey embryonic stem cell lines reflect distinct genomic features.

Science.gov (United States)

Debowski, Katharina; Drummer, Charis; Lentes, Jana; Cors, Maren; Dressel, Ralf; Lingner, Thomas; Salinas-Riester, Gabriela; Fuchs, Sigrid; Sasaki, Erika; Behr, Rüdiger

2016-07-07

Embryonic stem cells (ESCs) are useful for the study of embryonic development. However, since research on naturally conceived human embryos is limited, non-human primate (NHP) embryos and NHP ESCs represent an excellent alternative to the corresponding human entities. Though, ESC lines derived from naturally conceived NHP embryos are still very rare. Here, we report the generation and characterization of four novel ESC lines derived from natural preimplantation embryos of the common marmoset monkey (Callithrix jacchus). For the first time we document derivation of NHP ESCs derived from morula stages. We show that quantitative chromosome-wise transcriptome analyses precisely reflect trisomies present in both morula-derived ESC lines. We also demonstrate that the female ESC lines exhibit different states of X-inactivation which is impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). The novel marmoset ESC lines will promote basic primate embryo and ESC studies as well as preclinical testing of ESC-based regenerative approaches in NHP.

The Genome of the Chicken DT40 Bursal Lymphoma Cell Line

DEFF Research Database (Denmark)

Molnar, Janos; Poti, Adam; Pipek, Orsolya

2014-01-01

The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell...... chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations...

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

Science.gov (United States)

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

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.

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.

Derivation of transplantable 7,12-dimethylbenz[a]anthracene-induced chicken fibrosarcoma lines: differences in metastasizing properties and organ specificity

International Nuclear Information System (INIS)

Galton, J.E.; Xue, B.; Hochwald, G.M.; Thorbecke, G.J.

1982-01-01

Transplantable 7,12-dimethylbenz[a]anthracene-induced SC chicken fibrosarcoma (CHCT-NYU) lines were studied for their ability to grow in internal organs after iv injection (artificial metastases) into 1- to 3-week-old chickens. Some tumor lines were recently derived, whereas others were studied after many serial subcutaneous transplantations. Artificial metastases were seen in the stomach, pancreas, lungs, heart, and muscle, and occasionally in the kidneys and liver. Agammaglobulinemic recipients showed more extensive organ involvement than normal recipients of the same age. Whole-body ν-irradiation enhanced the incidence of artificial metastases, particularly in lungs. Antibody from the serum of a primary tumor-bearing host reduced the growth of the corresponding tumor in many organs. The metastatic pattern of line CHCT-NYU4 was a relatively stable property. However, intravenous transplantation of tumor cells from line CHCT-NYU4 taken from the liver, lungs, and pancreas of a single recipient established sublines with changes in organ specificity. After a few such serial transplants of liver-derived tumor, a line was derived that grew virtually in the liver alone. A subline with preference for growth in lungs was also obtained, but its ability to grow in the pancreas persisted. A pancreas-derived tumor line also grew in the liver and lungs. Subcutaneous transplants of tissue fragments of the lung-derived tumor line caused the appearance of spontaneous metastases in lungs. The incidence of spontaneous metastases with the lung-derived line was much greater than that with the liver-derived line or with the original CHCT-NYU4 line

Cadmium-109 uptake by tumors derived from Balb C/3T3 cell lines with varying degrees of the transformed phenotype

International Nuclear Information System (INIS)

Morton, K.; Alazraki, N.; Winge, D.; Lynch, R.E.

1986-01-01

To determine if tumors are rich in metallothionein, the authors measured the vivo uptake of subcutaneously-injected carrier-free cadmium-109 in tumors and in normal tissues of Balb/C mice. The tumors were grown in the mice from cultured Balb/3T3 cells transformed by the Moloney murine sarcoma virus. Uptake of cadmium-109 per gram of tissue was greatest for liver, kidney, and spleen. However, tumor uptake of cadmium-109 was markedly greater than that in blood, skeletal muscle, bones, intestine or adipose tissue. B Sephadex G-75 chromatography, the radioactivity in tumor and in liver lysates eluted with cytochrome-C, a molecule similar in molecular weight to metal-lothionein. To determine if metallothionein levels are related to the degree of malignancy of tumors, cadmium-109 uptake in the tumors from the virally-transformed cells was compared to that in tumors from non-transformed Balb/3T3 cells and two derivative chemically transformed cell lines. There was strong correlation between the substrate-independent growth in soft agarose of the four cell lines, the rate of growth of the corresponding tumors, and the amount of cadmium-109 uptake in the tumors. The authors conclude that metallothionein levels may be elevated in tumors as a function of the degree of expression of the transformed phenotype

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

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.

A bovine cell line that can be infected by natural sheep scrapie prions.

Directory of Open Access Journals (Sweden)

Anja M Oelschlegel

Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

Radiosensitivity in lymphoblastoid cell lines derived from Shwachman-Diamond syndrome patients

International Nuclear Information System (INIS)

Morini, J.; Babini, G.; Mariotti, L.; Baiocco, G.; Ottolenghi, A.; Nacci, L.; Minelli, A.; Danesino, C.; Maccario, C.; Roessler, U.; Savio, M.; Gomolka, M.; Kulka, U.

2015-01-01

Shwachman-Diamond syndrome is an autosomal-recessive disorder characterised by bone marrow failure and a cumulative risk of progression to acute myeloid leukaemia. The Shwachman-Bodian-Diamond syndrome (SBDS) gene, the only gene known to be causative of the pathology, is involved in ribosomal biogenesis, stress responses and DNA repair, and the lack of SBDS sensitises cells to many stressors and leads to mitotic spindle destabilisation. The effect of ionising radiation on SBDS-deficient cells was investigated using immortalised lymphocytes from SDS patients in comparison with positive and negative controls in order to test whether, in response to ionising radiation exposure, any impairment in the DNA repair machinery could be observed. After irradiating cells with different doses of X-rays or gamma-rays, DNA repair kinetics and the residual damages using the alkaline COMET assay and the γ-H2AX assay were assessed, respectively. In this work, preliminary data about the comparison between ionising radiation effects in different patients-derived cells and healthy control cells are presented. (authors)

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

Science.gov (United States)

Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

2015-01-01

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

International Nuclear Information System (INIS)

Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

2005-01-01

The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Ebru YABAŞ

2017-08-01

Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

Science.gov (United States)

Knips, Jill; Czech-Sioli, Manja; Spohn, Michael; Heiland, Max; Moll, Ingrid; Grundhoff, Adam; Schumacher, Udo; Fischer, Nicole

2017-07-01

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation. © 2017 UICC.

Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

Directory of Open Access Journals (Sweden)

Silvia Dotti

Full Text Available The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor.

Directory of Open Access Journals (Sweden)

Simon Heidegger

Full Text Available Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.

Androgen responsiveness of the new human endometrial cancer cell line MFE-296.

Science.gov (United States)

Hackenberg, R; Beck, S; Filmer, A; Hushmand Nia, A; Kunzmann, R; Koch, M; Slater, E P; Schulz, K D

1994-04-01

MFE-296 endometrial cancer cells express androgen receptors in vitro. These cells, which are tumorigenic in nude mice, are derived from a moderately differentiated human endometrial adenocarcinoma. They express vimentin and the cytokeratins 7, 8, 18, and 19. Karyotyping revealed near-tetraploidy for most of the cells. No marker chromosomes were observed. DNA analyses confirmed the genetic identity of the cell line and the patient from whom the cell line was derived. Proliferation of MFE-296 cells was inhibited by the progestin R5020 and the androgen dihydrotestosterone (DHT). The inhibition of proliferation by DHT was antagonized by the antiandrogen Casodex, demonstrating the involvement of the androgen receptor. Androgen binding was determined at 22,000 binding sites per cell using a whole-cell assay (KD = 0.05 nM) and 30 fmol/mg protein with the dextran charcoal method; 7 fmol/mg protein of progesterone receptors were found, whereas estrogen receptors were below 5 fmol/mg protein. The androgen receptor was functionally intact, as demonstrated by transfection experiments with a reporter-gene construct, containing an androgen-responsive element. In MFE-296 cells the content of the androgen receptor was up-regulated by its own ligand.

Analysis of the regulation of fatty acid binding protein 7 expression in human renal carcinoma cell lines

Directory of Open Access Journals (Sweden)

Sugiyama Takayuki

2011-07-01

Full Text Available Abstract Background Improving the treatment of renal cell carcinoma (RCC will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7, also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system. Results We determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293 were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2 and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines. Conclusions Three putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key

Cancer Cell Cytotoxicities of 1-(4-Substitutedbenzoyl-4-(4-chlorobenzhydrylpiperazine Derivatives

Directory of Open Access Journals (Sweden)

Mine Yarim

2012-06-01

Full Text Available A series of novel 1-(4-substitutedbenzoyl-4-(4-chlorobenzhydrylpiperazine derivatives 5a–g was designed by a nucleophilic substitution reaction of 1-(4-chlorobenzhydrylpiperazine with various benzoyl chlorides and characterized by elemental analyses, IR and ¹H nuclear magnetic resonance spectra. Cytotoxicity of the compounds was demonstrated on cancer cell lines from liver (HUH7, FOCUS, MAHLAVU, HEPG2, HEP3B, breast (MCF7, BT20, T47D, CAMA-1, colon (HCT-116, gastric (KATO-3 and endometrial (MFE-296 cancer cell lines. Time-dependent cytotoxicity analysis of compound 5a indicated the long-term in situ stability of this compound. All compounds showed significant cell growth inhibitory activity on the selected cancer cell lines.

Marrow-derived mesenchymal stem cells: role in epithelial tumor cell determination.

Science.gov (United States)

Fierro, Fernando A; Sierralta, Walter D; Epuñan, Maria J; Minguell, José J

2004-01-01

Marrow stroma represents an advantageous environment for development of micrometastatic cells. Within the cellular structure of marrow stroma, mesenchymal stem cells (MSC) have been postulated as an interacting target for disseminated cancer cells. The studies reported here were performed to gain more information on the interaction of the human breast cancer cell line MCF-7 with human bone marrow-derived MSC cells and to investigate whether this interaction affects tumor cell properties. The results showed that after co-culture with MSC, changes were detected in the morphology, proliferative capacity and aggregation pattern of MCF-7 cells, but these parameters were not affected after the co-culture of MSC cells with a non-tumorigenic breast epithelial cell line, MCF-10. Since the indirect culture of MCF-7 with MSC or its products also resulted in functional changes in the tumor cells, we evaluated whether these effects could be attributed to growth factors produced by MSC cells. It was found that VEGF and IL-6 mimic the effects produced by MSC or its products on the proliferation and aggregation properties of MCF-7, cells, respectively. Thus, it seems that after entry of disseminated tumor cells into the marrow space, their proliferative and morphogenetic organization patterns are modified after interaction with distinct stromal cells and/or with specific signals from the marrow microenvironment.

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

Coelomic epithelium-derived cells in visceral morphogenesis.

Science.gov (United States)

Ariza, Laura; Carmona, Rita; Cañete, Ana; Cano, Elena; Muñoz-Chápuli, Ramón

2016-03-01

Coelomic cavities of vertebrates are lined by a mesothelium which develops from the lateral plate mesoderm. During development, the coelomic epithelium is a highly active cell layer, which locally is able to supply mesenchymal cells that contribute to the mesodermal elements of many organs and provide signals which are necessary for their development. The relevance of this process of mesenchymal cell supply to the developing organs is becoming clearer because genetic lineage tracing techniques have been developed in recent years. Body wall, heart, liver, lungs, gonads, and gastrointestinal tract are populated by cells derived from the coelomic epithelium which contribute to their connective and vascular tissues, and sometimes to specialized cell types such as the stellate cells of the liver, the Cajal interstitial cells of the gut or the Sertoli cells of the testicle. In this review we collect information about the contribution of coelomic epithelium derived cells to visceral development, their developmental fates and signaling functions. The common features displayed by all these processes suggest that the epithelial-mesenchymal transition of the embryonic coelomic epithelium is an underestimated but key event of vertebrate development, and probably it is shared by all the coelomate metazoans. © 2015 Wiley Periodicals, Inc.

Characterization of exosomes derived from ovarian cancer cells and normal ovarian epithelial cells by nanoparticle tracking analysis.

Science.gov (United States)

Zhang, Wei; Peng, Peng; Kuang, Yun; Yang, Jiaxin; Cao, Dongyan; You, Yan; Shen, Keng

2016-03-01

Cellular exosomes are involved in many disease processes and have the potential to be used for diagnosis and treatment. In this study, we compared the characteristics of exosomes derived from human ovarian epithelial cells (HOSEPiC) and three epithelial ovarian cancer cell lines (OVCAR3, IGROV1, and ES-2) to investigate the differences between exosomes originating from normal and malignant cells. Two established colloid-chemical methodologies, electron microscopy (EM) and dynamic light scattering (DLS), and a relatively new method, nanoparticle tracking analysis (NTA), were used to measure the size and size distribution of exosomes. The concentration and epithelial cellular adhesion molecule (EpCAM) expression of exosomes were measured by NTA. Quantum dots were conjugated with anti-EpCAM to label exosomes, and the labeled exosomes were detected by NTA in fluorescent mode. The normal-cell-derived exosomes were significantly larger than those derived from malignant cells, and exosomes were successfully labeled using anti-EpCAM-conjugated quantum dots. Exosomes from different cell lines may vary in size, and exosomes might be considered as potential diagnosis biomarkers. NTA can be considered a useful, efficient, and objective method for the study of different exosomes and their unique properties in ovarian cancer.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

Energy Technology Data Exchange (ETDEWEB)

Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

2013-02-15

Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

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

Science.gov (United States)

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

2016-11-01

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

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

Science.gov (United States)

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

2009-01-01

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

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

Inhibition of iron uptake is responsible for differential sensitivity to V-ATPase inhibitors in several cancer cell lines.

Directory of Open Access Journals (Sweden)

Sarah Straud

2010-07-01

Full Text Available Many cell lines derived from tumors as well as transformed cell lines are far more sensitive to V-ATPase inhibitors than normal counterparts. The molecular mechanisms underlying these differences in sensitivity are not known. Using global gene expression data, we show that the most sensitive responses to HeLa cells to low doses of V-ATPase inhibitors involve genes responsive to decreasing intracellular iron or decreasing cholesterol and that sensitivity to iron uptake is an important determinant of V-ATPase sensitivity in several cancer cell lines. One of the most sensitive cell lines, melanoma derived SK-Mel-5, over-expresses the iron efflux transporter ferroportin and has decreased expression of proteins involved in iron uptake, suggesting that it actively suppresses cytoplasmic iron. SK-Mel-5 cells have increased production of reactive oxygen species and may be seeking to limit additional production of ROS by iron.

The potential of induced pluripotent stem cell derived hepatocytes.

Science.gov (United States)

Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

2016-07-01

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Rathore K

2015-09-01

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

Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

Science.gov (United States)

Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

2014-10-01

NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society for Immunology.

A recurrent human papillomavirus integration site at chromosome region 12q14-q15 in SW756 and SK-v cell lines derived from genital tumors

International Nuclear Information System (INIS)

Sastre-Garau, X.; Couturier, J.; Favre, M.; Orth, G.

1995-01-01

The SW756 cell line, derived from an invasive cancer of the uterine cervix, harbours integrated human papillomavirus (HPV) 18 DNA sequences which have been located in chromosome band 12q13. By in situ hybridization experiments with tritiated and digoxigenin-labelled HPV18 probes on R-banded chromosomes, we now localize the integrated viral sequences in 12q14-q15. Interestingly, we have previously localized integrated HPV16 sequences in the same chromosomal region in SK-v cells, derived from a pre-invasive vulvar neoplasia. The chromosomal region 12q14-q15 could thus correspond to a preferential site for the integration of HPV DNA in genital tumors. (authors). 29 refs., 2 figs

Immune surveillance properties of human NK cell-derived exosomes.

Science.gov (United States)

Lugini, Luana; Cecchetti, Serena; Huber, Veronica; Luciani, Francesca; Macchia, Gianfranco; Spadaro, Francesca; Paris, Luisa; Abalsamo, Laura; Colone, Marisa; Molinari, Agnese; Podo, Franca; Rivoltini, Licia; Ramoni, Carlo; Fais, Stefano

2012-09-15

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

International Nuclear Information System (INIS)

Zhang, Ping; Zhang, Zhiyuan; Zhou, Xiaojian; Qiu, Weiliu; Chen, Fangan; Chen, Wantao

2006-01-01

Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in cisplatin resistance

Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

Directory of Open Access Journals (Sweden)

Zhang Ping

2006-09-01

Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

17-AAG and Apoptosis, Autophagy, and Mitophagy in Canine Osteosarcoma Cell Lines.

Science.gov (United States)

Massimini, M; Palmieri, C; De Maria, R; Romanucci, M; Malatesta, D; De Martinis, M; Maniscalco, L; Ciccarelli, A; Ginaldi, L; Buracco, P; Bongiovanni, L; Della Salda, L

2017-05-01

Canine osteosarcoma is highly resistant to current chemotherapy; thus, clarifying the mechanisms of tumor cell resistance to treatments is an urgent need. We tested the geldanamycin derivative 17-AAG (17-allylamino-17-demethoxygeldanamycin) prototype of Hsp90 (heat shock protein 90) inhibitors in 2 canine osteosarcoma cell lines, D22 and D17, derived from primary and metastatic tumors, respectively. With the aim to understand the interplay between cell death, autophagy, and mitophagy, in light of the dual effect of autophagy in regulating cancer cell viability and death, D22 and D17 cells were treated with different concentrations of 17-AAG (0.5 μM, 1 μM) for 24 and 48 hours. 17-AAG-induced apoptosis, necrosis, autophagy, and mitophagy were assessed by transmission electron microscopy, flow cytometry, and immunofluorescence. A simultaneous increase in apoptosis, autophagy, and mitophagy was observed only in the D22 cell line, while D17 cells showed low levels of apoptotic cell death. These results reveal differential cell response to drug-induced stress depending on tumor cell type. Therefore, pharmacological treatments based on proapoptotic chemotherapy in association with autophagy regulators would benefit from a predictive in vitro screening of the target cell type.

Nuclear hormone receptor expression in mouse kidney and renal cell lines.

Directory of Open Access Journals (Sweden)

Daisuke Ogawa

Full Text Available Nuclear hormone receptors (NHRs are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN, the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m, and cell lines of mesangial (MES13, podocyte (MPC, proximal tubular epithelial (mProx24 and collecting duct (mIMCD3 origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77, nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

Parallel Evolution under Chemotherapy Pressure in 29 Breast Cancer Cell Lines Results in Dissimilar Mechanisms of Resistance

DEFF Research Database (Denmark)

Tegze, Balint; Szallasi, Zoltan Imre; Haltrich, Iren

2012-01-01

Background: Developing chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only...

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 cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

Science.gov (United States)

Wu, Shuai Shuai; Li, Dong Jiang; Lv, Chun Rong; Yang, Hong Yuan; Zhu, Lan; Li, Wei Juan; Quan, Guo Bo; Hong, Qiong Hua

2013-01-01

In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in this study. Additionally

The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

DEFF Research Database (Denmark)

Xu, Xun; Pan, Shengkai; Liu, Xin

2011-01-01

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most....... Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance...... property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production....

Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

Science.gov (United States)

Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

1988-02-01

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

Directory of Open Access Journals (Sweden)

Jian Ning

2011-01-01

Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

Science.gov (United States)

Osborn, Stephanie L; Kurzrock, Eric A

2018-01-01

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

Establishment, characterization and chemosensitivity of three mismatch repair deficient cell lines from sporadic and inherited colorectal carcinomas.

Directory of Open Access Journals (Sweden)

Claudia Maletzki

Full Text Available BACKGROUND: Colorectal cancer (CRC represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113 along with their corresponding xenografts. METHODOLOGY: MMR-D cell lines (HROC24, HROC87, and HROC113 were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total. Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo. PRINCIPAL FINDINGS: Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras(wt, B-raf(mut, HROC87: K-ras(wt, B-raf(mut, whereas the HROC113 cell line (K-ras(mut, B-raf(wt was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM(+ tumor cells, showing surface tumor marker expression (CEACAM(+. MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity. CONCLUSIONS: These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the

Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types

NARCIS (Netherlands)

Kessler, Jan H.; Mullauer, Franziska B.; de Roo, Guido M.; Medema, Jan Paul

2007-01-01

Betulinic acid (BA) is a widely available plant-derived triterpene with reported activity against cancer cells of neuroectodermal origin and leukaemias. Treatment with BA was shown to protect mice against transplanted human melanoma and led to tumor regression. In contrast, cells from healthy

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...

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

Energy Technology Data Exchange (ETDEWEB)

Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

2015-09-10

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

International Nuclear Information System (INIS)

Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing; Wang, Zehua

2015-01-01

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs

Plasma glial cell line-derived neurotrophic factor in patients with major depressive disorder: a preliminary study.

Science.gov (United States)

Lee, Bun-Hee; Hong, Jin-Pyo; Hwang, Jung-A; Na, Kyoung-Sae; Kim, Won-Joong; Trigo, Jose; Kim, Yong-Ku

2016-02-01

Some clinical studies have reported reduced peripheral glial cell line-derived neurotrophic factor (GDNF) level in elderly patients with major depressive disorder (MDD). We verified whether a reduction in plasma GDNF level was associated with MDD. Plasma GDNF level was measured in 23 healthy control subjects and 23 MDD patients before and after 6 weeks of treatment. Plasma GDNF level in MDD patients at baseline did not differ from that in healthy controls. Plasma GDNF in MDD patients did not differ significantly from baseline to the end of treatment. GDNF level was significantly lower in recurrent-episode MDD patients than in first-episode patients before and after treatment. Our findings revealed significantly lower plasma GDNF level in recurrent-episode MDD patients, although plasma GDNF levels in MDD patients and healthy controls did not differ significantly. The discrepancy between our study and previous studies might arise from differences in the recurrence of depression or the ages of the MDD patients.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

International Nuclear Information System (INIS)

Ji, S.Q.; Cao, J.; Zhang, Q.Y.; Li, Y.Y.; Yan, Y.Q.; Yu, F.X.

2013-01-01

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

Energy Technology Data Exchange (ETDEWEB)

Ji, S.Q.; Cao, J. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Zhang, Q.Y.; Li, Y.Y. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China); Yan, Y.Q. [Department of Liver Surgery I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai (China); Yu, F.X. [Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Wenzhou Medical College, Wenzhou (China)

2013-09-27

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitro and the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

Directory of Open Access Journals (Sweden)

Heema Hewitson

2016-03-01

Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

International Nuclear Information System (INIS)

Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

1979-01-01

Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

Basement membrane components secreted by mouse yolk sac carcinoma cell lines

DEFF Research Database (Denmark)

Damjanov, A; Wewer, U M; Tuma, B

1990-01-01

Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac...

Canine distemper virus induces apoptosis in cervical tumor derived cell lines

Directory of Open Access Journals (Sweden)

Rajão Daniela S

2011-06-01

Full Text Available Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi, by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

Antibacterial Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Cytotoxic Effect against MDA-MB-468 and MDA-MB-231 Breast Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Yerly Vargas Casanova

2017-09-01

Full Text Available Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B, containing the RRWQWR motif, were designed, synthesized, purified, and characterized using RP-HPLC chromatography and MALDI-TOF mass spectrometry. The antibacterial activity of the designed peptides against E. coli (ATCC 11775 and 25922 and their cytotoxic effect against MDA-MB-468 and MDA-MB-231 breast cancer cell lines were evaluated. Dimeric and tetrameric peptides showed higher antibacterial activity in both bacteria strains than linear peptides. The dimeric peptide (RRWQWR2K-Ahx exhibited the highest antibacterial activity against the tested bacterial strains. Furthermore, the peptides with high antibacterial activity exhibited significant cytotoxic effect against the tested breast cancer cell lines. This cytotoxic effect was fast and dependent on the peptide concentration. The tetrameric molecule containing RRWQWR motif has an optimal cytotoxic effect at a concentration of 22 µM. The evaluated dimeric and tetrameric peptides could be considered as candidates for developing new therapeutic agents against breast cancer. Polyvalence of linear sequences could be considered as a novel and versatile strategy for obtaining molecules with high anticancer activity.

Antibacterial Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Cytotoxic Effect against MDA-MB-468 and MDA-MB-231 Breast Cancer Cell Lines.

Science.gov (United States)

Vargas Casanova, Yerly; Rodríguez Guerra, Jorge Antonio; Umaña Pérez, Yadi Adriana; Leal Castro, Aura Lucía; Almanzar Reina, Giovanni; García Castañeda, Javier Eduardo; Rivera Monroy, Zuly Jenny

2017-09-29

Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B, containing the RRWQWR motif, were designed, synthesized, purified, and characterized using RP-HPLC chromatography and MALDI-TOF mass spectrometry. The antibacterial activity of the designed peptides against E. coli (ATCC 11775 and 25922) and their cytotoxic effect against MDA-MB-468 and MDA-MB-231 breast cancer cell lines were evaluated. Dimeric and tetrameric peptides showed higher antibacterial activity in both bacteria strains than linear peptides. The dimeric peptide (RRWQWR)₂K-Ahx exhibited the highest antibacterial activity against the tested bacterial strains. Furthermore, the peptides with high antibacterial activity exhibited significant cytotoxic effect against the tested breast cancer cell lines. This cytotoxic effect was fast and dependent on the peptide concentration. The tetrameric molecule containing RRWQWR motif has an optimal cytotoxic effect at a concentration of 22 µM. The evaluated dimeric and tetrameric peptides could be considered as candidates for developing new therapeutic agents against breast cancer. Polyvalence of linear sequences could be considered as a novel and versatile strategy for obtaining molecules with high anticancer activity.

Efficient differentiation of steroidogenic and germ-like cells from epigenetically-related iPSCs derived from ovarian granulosa cells.

Directory of Open Access Journals (Sweden)

Raymond Anchan

Full Text Available To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs, we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs was verified by demonstrating embryonic stem cell (ESC antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2 than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4 and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1 more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.

Establishment and characterization of a novel head and neck squamous cell carcinoma cell line USC-HN1.

Science.gov (United States)

Liebertz, Daniel J; Lechner, Melissa G; Masood, Rizwan; Sinha, Uttam K; Han, Jing; Puri, Raj K; Correa, Adrian J; Epstein, Alan L

2010-02-22

Head and neck squamous cell carcinoma (HNSCC) is an aggressive and lethal malignancy. Publically available cell lines are mostly of lingual origin, or have not been carefully characterized. Detailed characterization of novel HNSCC cell lines is needed in order to provide researchers a concrete keystone on which to build their investigations. The USC-HN1 cell line was established from a primary maxillary HNSCC biopsy explant in tissue culture. The immortalized cells were then further characterized by heterotransplantation in Nude mice; immunohistochemical staining for relevant HNSCC biomarkers; flow cytometry for surface markers; cytogenetic karyotypic analysis; human papillomavirus and Epstein-Barr virus screening; qRT-PCR for oncogene and cytokine analysis; investigation of activated, cleaved Notch1 levels; and detailed 35,000 gene microarray analysis. Characterization experiments confirmed the human HNSCC origin of USC-HN1, including a phenotype similar to the original tumor. Viral screening revealed no HPV or EBV infection, while western blotting displayed significant upregulation of activated, cleaved Notch1. USC-HN1, a novel immortalized cell line has been derived from a maxillary HNSCC. Characterization studies have shown that the cell line is of HNSCC origin and displays many of the same markers previously reported in the literature. USC-HN1 is available for public research and will further the investigation of HNSCC and the development of new therapeutic modalities.

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

International Nuclear Information System (INIS)

Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

2016-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

2016-08-05

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

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

Science.gov (United States)

Focosi, Daniele; Amabile, Giovanni

2017-12-27

Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

Whole-Genome Sequence of the Metastatic PC3 and LNCaP Human Prostate Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Inge Seim

2017-06-01

Full Text Available The bone metastasis-derived PC3 and the lymph node metastasis-derived LNCaP prostate cancer cell lines are widely studied, having been described in thousands of publications over the last four decades. Here, we report short-read whole-genome sequencing (WGS and de novo assembly of PC3 (ATCC CRL-1435 and LNCaP (clone FGC; ATCC CRL-1740 at ∼70 × coverage. A known homozygous mutation in TP53 and homozygous loss of PTEN were robustly identified in the PC3 cell line, whereas the LNCaP cell line exhibited a larger number of putative inactivating somatic point and indel mutations (and in particular a loss of stop codon events. This study also provides preliminary evidence that loss of one or both copies of the tumor suppressor Capicua (CIC contributes to primary tumor relapse and metastatic progression, potentially offering a treatment target for castration-resistant prostate cancer (CRPC. Our work provides a resource for genetic, genomic, and biological studies employing two commonly-used prostate cancer cell lines.

Whole-Genome Sequence of the Metastatic PC3 and LNCaP Human Prostate Cancer Cell Lines.

Science.gov (United States)

Seim, Inge; Jeffery, Penny L; Thomas, Patrick B; Nelson, Colleen C; Chopin, Lisa K

2017-06-07

The bone metastasis-derived PC3 and the lymph node metastasis-derived LNCaP prostate cancer cell lines are widely studied, having been described in thousands of publications over the last four decades. Here, we report short-read whole-genome sequencing (WGS) and de novo assembly of PC3 (ATCC CRL-1435) and LNCaP (clone FGC; ATCC CRL-1740) at ∼70 × coverage. A known homozygous mutation in TP53 and homozygous loss of PTEN were robustly identified in the PC3 cell line, whereas the LNCaP cell line exhibited a larger number of putative inactivating somatic point and indel mutations (and in particular a loss of stop codon events). This study also provides preliminary evidence that loss of one or both copies of the tumor suppressor Capicua ( CIC ) contributes to primary tumor relapse and metastatic progression, potentially offering a treatment target for castration-resistant prostate cancer (CRPC). Our work provides a resource for genetic, genomic, and biological studies employing two commonly-used prostate cancer cell lines. Copyright © 2017 Seim et al.

Molecular analysis of urothelial cancer cell lines for modeling tumor biology and drug response.

Science.gov (United States)

Nickerson, M L; Witte, N; Im, K M; Turan, S; Owens, C; Misner, K; Tsang, S X; Cai, Z; Wu, S; Dean, M; Costello, J C; Theodorescu, D

2017-01-05

The utility of tumor-derived cell lines is dependent on their ability to recapitulate underlying genomic aberrations and primary tumor biology. Here, we sequenced the exomes of 25 bladder cancer (BCa) cell lines and compared mutations, copy number alterations (CNAs), gene expression and drug response to BCa patient profiles in The Cancer Genome Atlas (TCGA). We observed a mutation pattern associated with altered CpGs and APOBEC-family cytosine deaminases similar to mutation signatures derived from somatic alterations in muscle-invasive (MI) primary tumors, highlighting a major mechanism(s) contributing to cancer-associated alterations in the BCa cell line exomes. Non-silent sequence alterations were confirmed in 76 cancer-associated genes, including mutations that likely activate oncogenes TERT and PIK3CA, and alter chromatin-associated proteins (MLL3, ARID1A, CHD6 and KDM6A) and established BCa genes (TP53, RB1, CDKN2A and TSC1). We identified alterations in signaling pathways and proteins with related functions, including the PI3K/mTOR pathway, altered in 60% of lines; BRCA DNA repair, 44%; and SYNE1-SYNE2, 60%. Homozygous deletions of chromosome 9p21 are known to target the cell cycle regulators CDKN2A and CDKN2B. This loci was commonly lost in BCa cell lines and we show the deletions extended to the polyamine enzyme methylthioadenosine (MTA) phosphorylase (MTAP) in 36% of lines, transcription factor DMRTA1 (27%) and antiviral interferon epsilon (IFNE, 19%). Overall, the BCa cell line genomic aberrations were concordant with those found in BCa patient tumors. We used gene expression and copy number data to infer pathway activities for cell lines, then used the inferred pathway activities to build a predictive model of cisplatin response. When applied to platinum-treated patients gathered from TCGA, the model predicted treatment-specific response. Together, these data and analysis represent a valuable community resource to model basic tumor biology and to study

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

Science.gov (United States)

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.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

Science.gov (United States)

Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

2017-12-01

Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

Analysis of cell-cycle regulation following exposure of lung-derived cells to γ-rays

Science.gov (United States)

Trani, D.; Lucchetti, C.; Cassone, M.; D'Agostino, L.; Caputi, M.; Giordano, A.

Acute exposure of mammalian cells to ionizing radiation results in a delay of cell-cycle progression and/or augmentation of apoptosis. Following ionizing radiation-induced DNA damage, cell-cycle arrest in the G1- or G2-phase of the cell-cycle prevents or delays DNA replication or mitosis, providing time for the DNA repair machinery to exert its function. Deregulation or failing of cell-cycle checkpoints and/or DNA repair mechanisms may lead normal cells bearing chromosome mutations to acquire neoplastic autonomy, which in turn can trigger the onset of cancer. Existing studies have focused on the impact of p53 status on the radiation response of lung cancer (LC) cell lines in terms of both cell-cycle regulation and apoptosis, while no comparative studies have been performed on the radiation response of lung derived normal and cancerous epithelial cells. To investigate the radiation response in normal and cancerous phenotypes, along with the role and impact of p53 status, and possible correlations with pRb/p105 or other proteins involved in carcinogenesis and cell-cycle regulation, we selected two lung-derived epithelial cell lines, one normal (NL20, p53 wild-type) and one non-small cell lung cancer (NSCLC), H358 (known to be p53-deficient). We compared the levels of γ-induced cell proliferation ability, cell-cycle arrest, apoptotic index, and expression levels of cell-cycle regulating and regulated proteins. The different cell sensitivity, apoptotic response and protein expression profiles resulting from our study for NL20 and H358 cells suggest that still unknown mechanisms involving p53, pRb/p105 and their target molecules might play a pivotal role in determining cell sensitivity and resistance upon exposure to ionizing radiation.

Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

Science.gov (United States)

Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene

2015-01-01

Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

Science.gov (United States)

Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

2013-01-01

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

International Nuclear Information System (INIS)

Ramos-Solano, Moisés; Meza-Canales, Ivan D.; Torres-Reyes, Luis A.; Alvarez-Zavala, Monserrat

2015-01-01

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

Energy Technology Data Exchange (ETDEWEB)

Ramos-Solano, Moisés, E-mail: mrsolano84@gmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Meza-Canales, Ivan D., E-mail: imezacanales@ice.mpg.de [Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, 07745 Jena (Germany); Torres-Reyes, Luis A., E-mail: torres_reyes_88@hotmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Alvarez-Zavala, Monserrat, E-mail: monse_belan@hotmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); and others

2015-07-01

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.

Generation and properties of a new human ventral mesencephalic neural stem cell line

DEFF Research Database (Denmark)

Villa, Ana; Liste, Isabel; Courtois, Elise T

2009-01-01

. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal......Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro...... derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing....

Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3/Moat-D) Is Expressed in the SAE Squalus acanthias Shark Embryo–Derived Cell Line

Science.gov (United States)

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2008-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E. PMID:18284333

Multidrug resistance-associated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark embryo-derived cell line.

Science.gov (United States)

Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

2007-01-01

The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E.

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.

Generation and Characterization of Vascular Smooth Muscle Cell Lines Derived from a Patient with a Bicuspid Aortic Valve

Directory of Open Access Journals (Sweden)

Pamela Lazar-Karsten

2016-04-01

Full Text Available Thoracic aortic dilation is the most common malformation of the proximal aorta and is responsible for 1%–2% of all deaths in industrialized countries. In approximately 50% of patients with a bicuspid aortic valve (BAV, dilation of any or all segments of the aorta occurs. BAV patients with aortic dilation show an increased incidence of cultured vascular smooth muscle cell (VSMC loss. In this study, VSMC, isolated from the ascending aorta of BAV, was treated with Simian virus 40 to generate a BAV-originated VSMC cell line. To exclude any genomic DNA or cross-contamination, highly polymorphic short tandem repeats of the cells were profiled. The cells were then characterized using flow cytometry and karyotyping. The WG-59 cell line created is the first reported VSMC cell line isolated from a BAV patient. Using an RT2 Profiler PCR Array, genes within the TGFβ/BMP family that are dependent on losartan treatment were identified. Endoglin was found to be among the regulated genes and was downregulated in WG-59 cells following treatment with different losartan concentrations, when compared to untreated WG-59 cells.

Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

Directory of Open Access Journals (Sweden)

Kamalidehghan Behnam

2012-10-01

Full Text Available Abstract Background Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research. Methods Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping. Results MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER+, PR+, HER2+ and (ER+, PR-, HER2+, respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER-, PR-, HER2+ for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell. Conclusions Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER+/PR+/HER2+ and ER+/PR-/HER2+ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.

Isolation of Mature (Peritoneum-Derived Mast Cells and Immature (Bone Marrow-Derived Mast Cell Precursors from Mice.

Directory of Open Access Journals (Sweden)

Steffen K Meurer

Full Text Available Mast cells (MCs are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC or mucosal (MMC type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs and immature MC precursors from the bone marrow (BM. The latter are differentiated in vitro to yield BM-derived MCs (BMMC. These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

BRCA1/2 mutation analysis in 41 ovarian cell lines reveals only one functionally deleterious BRCA1 mutation.

LENUS (Irish Health Repository)

Stordal, Britta

2013-06-01

Mutations in BRCA1\\/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1\\/2 mutations occur in 8.6-13.7% of unselected epithelial ovarian cancers, somatic mutations are also frequent. BRCA1\\/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1\\/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors. The BRCA1\\/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1\\/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines. The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1\\/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1\\/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311. The incidence of BRCA1\\/2 deleterious mutations 1\\/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

Science.gov (United States)

Wakayama, Sayaka; Wakayama, Teruhiko

2010-01-01

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

HEK293T cell lines defective for O-linked glycosylation.

Directory of Open Access Journals (Sweden)

James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

Science.gov (United States)

Johnston, H P; Hawley, P; White, S E; Gibson, I; Tidd, D M

1985-04-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [dibut.MPRP] were tested for cytotoxic and/or growth inhibitory effects against MP-resistant sublines of V79 Chinese hamster lung fibroblasts (CH/TG) and L1210 mouse leukaemia cells (L1210/MPR) in which deficiencies of hypoxanthine-guanine phosphoribosyltransferase, and hence drug nucleotide forming capacity were the basis of resistance. L1210/MPR cells were totally resistant to 1 mM 6-mercaptopurine-9-beta-D-ribofuranoside [MPR] and 2 mM MPRP, but were inhibited by high concentrations (greater than 0.25 mM) of bis(MPR)P. These results suggested that bis(MPR)P was taken up by cells as the intact molecule since MPR and MPRP were its extracellular breakdown products. L1210/MPR cells were much more sensitive to the lipophilic bis(dibut.MPR)P derivative which had a predominantly cytotoxic action as judged by trypan blue staining and the ability of treated cells to produce macroscopic colonies in soft agar medium. However, cells killed by bis(dibut.MPR)P did not disintegrate appreciably over periods of up to 10 days. The effects of bis(dibut.MPR)P were probably the result of cellular uptake of the intact molecule. Dibut.MPRP showed minimal ability to inhibit L1210/MPR cells although this compound was a possible breakdown product of bis(dibut.MPR)P and a source of the same extracellular degradation products. The median cell size decreased in L1210/MPR cultures during exposure to both bis(MPR)P and bis(dibut.MPR)P. This effect was elicited more rapidly and

The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines.

Science.gov (United States)

Johnston, H. P.; Hawley, P.; White, S. E.; Gibson, I.; Tidd, D. M.

1985-01-01

6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug. 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [MPRP], bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(MPR)P], bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate [bis(dibut.MPR)P], and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate [dibut.MPRP] were tested for cytotoxic and/or growth inhibitory effects against MP-resistant sublines of V79 Chinese hamster lung fibroblasts (CH/TG) and L1210 mouse leukaemia cells (L1210/MPR) in which deficiencies of hypoxanthine-guanine phosphoribosyltransferase, and hence drug nucleotide forming capacity were the basis of resistance. L1210/MPR cells were totally resistant to 1 mM 6-mercaptopurine-9-beta-D-ribofuranoside [MPR] and 2 mM MPRP, but were inhibited by high concentrations (greater than 0.25 mM) of bis(MPR)P. These results suggested that bis(MPR)P was taken up by cells as the intact molecule since MPR and MPRP were its extracellular breakdown products. L1210/MPR cells were much more sensitive to the lipophilic bis(dibut.MPR)P derivative which had a predominantly cytotoxic action as judged by trypan blue staining and the ability of treated cells to produce macroscopic colonies in soft agar medium. However, cells killed by bis(dibut.MPR)P did not disintegrate appreciably over periods of up to 10 days. The effects of bis(dibut.MPR)P were probably the result of cellular uptake of the intact molecule. Dibut.MPRP showed minimal ability to inhibit L1210/MPR cells although this compound was a possible breakdown product of bis(dibut.MPR)P and a source of the same extracellular degradation products. The median cell size decreased in L1210/MPR cultures during exposure to both bis(MPR)P and bis(dibut.MPR)P. This effect was elicited more rapidly and

In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

Science.gov (United States)

Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

2016-12-06

The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2015-01-01

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

Chinese herb derived-Rocaglamide A is a potent inhibitor of pancreatic cancer cells.

Science.gov (United States)

Wang, Baochun; Li, Yixiong; Tan, Fengbo; Xiao, Zhanxiang

2016-01-01

Pancreatic cancer ranks No.1 in mortality rate worldwide. This study aims to identify the novel anti-pancreatic cancer drugs. Human pancreatic carcinoma cell lines were purchased from ATCC. CPE-based screening assay was used to examine the cell viability. Patient derived tumor xenografts in SCID mice was established. The Caspase-3 and 7 activities were measured using the Caspase Glo 3/7 Assay kit. Soft agar colony formation assay was used to evaluate the colony formation. Wound healing assay was employed to determine the cell migration. We screened a Chinese herbal product library and found three "hits" that kill cancer cells at nanomolar to micromolar concentrations. One of these compounds, rocaglamide, was found to be potent inhibitors of a wide spectrum of pancreatic cancer cell lines. Furthermore, Rocaglamide reduced the tumor size in a patient-derived pancreatic cancer xenograft mouse model without noticeable toxicity in vivo. Rocaglamide also inhibits pancreatic cancer cell migration and invasion. In conclusion, these data support that Rocaglamide may be a promising anti-pancreatic cancer drug.

Cytotoxicity and apoptosis induced by a plumbagin derivative in estrogen positive MCF-7 breast cancer cells

KAUST Repository

Sagar, Sunil; Esau, Luke; Moosa, Basem; Khashab, Niveen M.; Bajic, Vladimir B.; Kaur, Mandeep

2014-01-01

Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer. 2014 Bentham Science Publishers.

Cytotoxicity and apoptosis induced by a plumbagin derivative in estrogen positive MCF-7 breast cancer cells

KAUST Repository

Sagar, Sunil

2014-01-31

Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer. 2014 Bentham Science Publishers.