Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups.

PubMed

Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

2017-01-01

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Acetaminophen and Metamizole Induce Apoptosis in HT 29 and SW 480 Colon Carcinoma Cell Lines In Vitro.

PubMed

Bundscherer, Anika C; Malsy, Manuela; Gruber, Michael A; Graf, Bernhard M; Sinner, Barbara

2018-02-01

The perioperative phase is supposed to be a period with high vulnerability for cancer dissemination. Acetaminophen and metamizole are common analgesics administered during this phase. We investigated the effect of acetaminophen, metamizole and 4-methylaminoantipyrine (MAA) on proliferation and apoptosis of colon carcinoma cell lines (SW 480 and HT 29). Proliferation was detected by cell proliferation ELISA BrdU, and apoptosis by Annexin V staining. Cytochrome c and caspase 3, 8 and 9 expression levels were detected by western blot. Acetaminophen, metamizole or MAA caused slight changes in proliferation. Acetaminophen, metamizole or the combination increased apoptosis in both cell lines. All agents decreased caspase 3 and 8 expression in SW480. Acetaminophen decreased caspase 9 expression in both cell lines. In clinically relevant doses, acetaminophen and/or metamizole induce apoptosis in both colon cancer cell lines. Both mitochondrial and death receptor pathways might be involved in acetaminophen-induced apoptosis. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines].

PubMed

Ma, Li; Han, Xiao-hong; Wang, Shuai; Wang, Jian-fei; Shi, Yuan-kai

2012-09-25

To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

Sensor And Method For Detecting A Superstrate

NASA Technical Reports Server (NTRS)

Arndt, G. Dickey (Inventor); Cari, James R. (Inventor); Ngo, Phong H. (Inventor); Fink, Patrick W. (Inventor); Siekierski, James D. (Inventor)

2006-01-01

Method and apparatus are provided for determining a superstrate on or near a sensor, e.g., for detecting the presence of an ice superstrate on an airplane wing or a road. In one preferred embodiment, multiple measurement cells are disposed along a transmission line. While the present invention is operable with different types of transmission lines, construction details for a presently preferred coplanar waveguide and a microstrip waveguide are disclosed. A computer simulation is provided as part of the invention for predicting results of a simulated superstrate detector system. The measurement cells may be physically partitioned, nonphysically partitioned with software or firmware, or include a combination of different types of partitions. In one embodiment, a plurality of transmission lines are utilized wherein each transmission line includes a plurality of measurement cells. The plurality of transmission lines may be multiplexed with the signal from each transmission line being applied to the same phase detector. In one embodiment, an inverse problem method is applied to determine the superstrate dielectric for a transmission line with multiple measurement cells.

[Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue].

PubMed

Hrabal, V; Nekulová, M; Nenutil, R; Holčaková, J; Coates, P J; Vojtěšek, B

2017-01-01

PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

PubMed

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

[The expression of Cyclin D1 modulated by somatotropin on human pancreas cancer cell lines Bxpc-3].

PubMed

Li, Fei; Liu, Da-chuan; Sun, Jia-bang

2004-04-07

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

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

PubMed Central

Cheng, J; Haas, M

1990-01-01

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

Improved method increases sensitivity for circulating hepatocellular carcinoma cells

PubMed Central

Liu, Hui-Ying; Qian, Hai-Hua; Zhang, Xiao-Feng; Li, Jun; Yang, Xia; Sun, Bin; Ma, Jun-Yong; Chen, Lei; Yin, Zheng-Feng

2015-01-01

AIM: To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). METHODS: Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. RESULTS: CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+ selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR+ or/and CPS1+ CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. CONCLUSION: Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells. PMID:25780289

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

PubMed Central

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

2012-01-01

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

The transcriptional diversity of 25 Drosophila cell lines

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

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signalingmore » pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. In addition, we offer an initial analysis of previously unannotated transcripts in the cell lines.« less

Lining seam elimination algorithm and surface crack detection in concrete tunnel lining

NASA Astrophysics Data System (ADS)

Qu, Zhong; Bai, Ling; An, Shi-Quan; Ju, Fang-Rong; Liu, Ling

2016-11-01

Due to the particularity of the surface of concrete tunnel lining and the diversity of detection environments such as uneven illumination, smudges, localized rock falls, water leakage, and the inherent seams of the lining structure, existing crack detection algorithms cannot detect real cracks accurately. This paper proposed an algorithm that combines lining seam elimination with the improved percolation detection algorithm based on grid cell analysis for surface crack detection in concrete tunnel lining. First, check the characteristics of pixels within the overlapping grid to remove the background noise and generate the percolation seed map (PSM). Second, cracks are detected based on the PSM by the accelerated percolation algorithm so that the fracture unit areas can be scanned and connected. Finally, the real surface cracks in concrete tunnel lining can be obtained by removing the lining seam and performing percolation denoising. Experimental results show that the proposed algorithm can accurately, quickly, and effectively detect the real surface cracks. Furthermore, it can fill the gap in the existing concrete tunnel lining surface crack detection by removing the lining seam.

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

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

Infrequent detectable somatic mutations of the RET and glial cell line-derived neurotrophic factor (GDNF) genes in human pituitary adenomas.

PubMed

Yoshimoto, K; Tanaka, C; Moritani, M; Shimizu, E; Yamaoka, T; Yamada, S; Sano, T; Itakura, M

1999-02-01

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and tumors. RET is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF receptor-alpha (GDNFR-alpha). Activating mutations of the RET proto-oncogene were found in multiple endocrine neoplasia (MEN) 2 and in sporadic medullary thyroid carcinoma and pheochromocytoma of neuroendocrine origin. Mutations of the RET proto-oncogene and the glial cell line-derived neurotrophic factor (GDNF) gene were examined in human pituitary tumors. No mutations of the RET proto-oncogene including the cysteine-rich region or codon 768 and 918 in the tyrosine kinase domain were detected in 172 human pituitary adenomas either by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) or by PCR-restriction fragment length polymorphism (RFLP). Further, somatic mutations of the GDNF gene in 33 human pituitary adenomas were not detected by PCR-SSCP. One polymorphism of the GDNF gene at codon 145 of TGC or TGT was observed in a prolactinoma. The RET proto-oncogene message was detected in a normal human pituitary gland or 4 of 4 human pituitary adenomas with reverse transcription (RT)-PCR, and in rodent pituitary tumor cell lines with Western blotting. The expression of GDNF gene was detected in 1 of 4 human somatotroph adenomas, 1 of 2 corticotroph adenomas, and 2 of 6 rodent pituitary tumor cell lines with RT-PCR. Based on these, it is concluded that somatic mutations of the RET proto-oncogene or the GDNF gene do not appear to play a major role in the pituitary tumorigenesis in examined tumors.

Neurotensin is metabolized by endogenous proteases in prostate cancer cell lines.

PubMed

Moody, T W; Mayr, C A; Gillespie, T J; Davis, T P

1998-01-01

The formation and processing of neurotensin (NT) by three prostate cancer cell lines was investigated. Neurotensin (NT) immunoreactivity was detected in conditioned media and extracts of LNCaP cells. Using HPLC techniques, the immunoreactivity extracted from LNCaP cells coeluted with synthetic NT standard. Metalloendopeptidase 3.4.24.15 activity was detected in PC-3, DU-145 and LNCaP cells, whereas high levels of neutral endopeptidase 3.4.24.1 1 activity was detected only in LNCaP cells. NT was relatively stable when incubated with PC-3 or D-145 cells but was rapidly degraded by LNCaP cells to NT1-11 and NT1-10. Phosphoramidon inhibited the metabolism of NT by LNCaP cells. These data suggest that NT is present in and metabolized by LNCaP cellular enzymes.

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation.

PubMed

Dassanayake, Rohana P; Zhuang, Dongyue; Truscott, Thomas C; Madsen-Bouterse, Sally A; O'Rourke, Katherine I; Schneider, David A

2016-03-03

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP(Sc) accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP(res) isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP(Sc) accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

PubMed

Jia, Shiyu; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Gao, Peng; Han, Yanxi; Fu, Yu; Ding, Jiansheng; Wu, Qisheng; Zhang, Kuo; Xie, Jiehong; Li, Jinming

2018-06-01

KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material. © 2018 Wiley Periodicals, Inc.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

PubMed

Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

2015-01-01

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Establishment of a new human glioma cell line and analysis of its biological characteristics].

PubMed

Chen, Guilin; Li, Yanyan; Xie, Xueshun; Chen, Jinming; Wu, Tingfeng; Li, Xuetao; Wang, Hangzhou; Zhou, Youxin; Du, Ziwei

2015-02-01

To establish a new glioma cell line and analyze its biological characteristics, and to provide a useful cellular tool with new features for cancer research. Glioma tissue was taken from surgical specimen clinical of a clinical patient. Primary culture was carried out, and a cell line (SHG139) was established after 10 passages. Immunofluorescence staining was performed to detect the expression of proteins, and cell proliferation and cycle were detected by flow cytometry method (FCM). The biological characteristics of SHG139 cells were detected by chromosome karyotype analysis. SHG139s glioma cells derived from SHG139 glioma cell line were cultured with neural stem cell medium. Then stem cell markers were determined. SHG139s cells were induced with serum-containing medium, and their expression of A2B5, GFAP, β-III tubulin, and GalC was detected. Intracranial xenograft tumor of both SHG139 glioma cells and SHG139s glioma stem cell spheres was generated in rats. The expressions of A2B5, GalC, GFAP, S-100, and vimentin in the 20 and 60 passages of SHG139 cells were positive, consistent with the immunohistochemical results and pathological features. SHG139 cells proliferated significantly within 24 h after subculture, and their total number of chromosomes was 68 and mostly multiploid. They were positive for A2B5 (84.12±9.96)%, nestin (73.86±5.01)%, and NG2 (73.37±2.09)%. SHG139s cells were induced, and the ratio of positive cells of GFAP, β-III tubulin and GalC was (92.89±2.24)%, (64.85±4.09)% and (33.57±4.14)%, respectively. SHG139 is an astroglioma cell line, from which SHG139s cells can be successfully obtained by culture with NSCM. SHG139s cells are of A2B5(+)/CD133(-) GSCs subgroup cells, with potentials of self-renewal and multi-directional differentiation. Compared with the intracranial SHG139 xenograft tumor, the intracranial SHG139s xenograft tumor is more malignant and aggressive.

Specific PET Imaging Probes for Early Detection of Prostate Cancer Metastases

DTIC Science & Technology

2010-05-01

penetrating cell membranes. In one of our studies using such a peptide to deliver a therapeutic moiety to various prostate cancer cell lines, we...to exploit this group of peptides for the early detection of prostate tumor metastases. Promisingly, in our preliminary studies , th e peptide lab...cancer. Based on one of our studies using a polyarginine (NH2GR11) to deliver a therapeutic moiety to various prostate cancer cell lines, we hypothesize

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

PubMed

Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald

2015-02-01

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells.

PubMed

Ackerstaff, E; Pflug, B R; Nelson, J B; Bhujwalla, Z M

2001-05-01

In this study, a panel of normal human prostate cells (HPCs) and tumor cells derived from metastases were studied by (1)H NMR spectroscopy to determine whether the malignant transformation of HPCs results in the elevation of choline compounds. Although an elevated choline signal has been observed previously in clinical studies, the contribution of the different Cho compounds to this elevation, as well as their quantification, has not been established until now. Here we have shown that HPCs derived from metastases exhibit significantly higher phosphocholine as well as glycerophosphocholine levels compared with normal prostate epithelial and stromal cells. Thus the elevation of the choline peak observed clinically in prostate cancer is attributable to an alteration of phospholipid metabolism and not simply to increased cell density, doubling time, or other nonspecific effects. Androgen deprivation of the androgen receptor-positive cell lines resulted in a significant increase of choline compounds after chronic androgen deprivation of the LNCaP cell line and in a decrease of choline compounds after a more acute androgen deprivation of the LAPC-4 cell line. These data strongly support the use of proton magnetic resonance spectroscopic imaging to detect the presence of prostate cancer for diagnosis, to detect response subsequent to androgen ablation therapy, and to detect recurrence.

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Generation and characterization of a porcine endometrial endothelial cell line susceptible to porcine reproductive and respiratory syndrome virus.

PubMed

Feng, Lili; Zhang, Xinyu; Xia, Xiaoli; Li, Yangyang; He, Shan; Sun, Huaichang

2013-01-01

Previous studies on the underlying mechanism for porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure have been focused on the viral replication in the endothelial macrophages, and the susceptibility of porcine endometrial endothelial (PEE) cells to PRRSV has not yet been investigated. Therefore, in the present study we generated a PEE cell line by transfection of the primary cells with a SV40 large T antigen expression vector. The PEE cell line maintained the endothelial morphology with a significantly faster growth rate, shorter population doubling time and higher plating efficiency than the primary cells. The endothelial origination of the cell line was confirmed by detection of the endothelial cell-specific markers. The PEE cell line had been passed successively for 60 generations with an unlimited growth potential. To further characterize the PEE cell line, cells of different passages were infected with different PRRSV strains and analyzed for the viral antigen and replication. Overt cytopathic effect was observed from 36h postinfection (HPI) and the viral antigen detected as early as 12 HPI. The infectious virus was recovered from the infected PEE cells with a titer higher than that in MARC-145 cells. Since the data presented indicate a high susceptibility of PEE cells to PRRSV, we conclude that the PEE cell line generated will be useful for growth of PRRSV and further studies on the underlying mechanism for PRRSV infection of PEE cells. The finding of the susceptibility of PEE cells to PRRSV may provide an alternative explanation for PRRSV-induced reproductive failure. Copyright © 2012 Elsevier B.V. All rights reserved.

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

PubMed Central

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

1993-01-01

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

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

EPA Science Inventory

The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

PubMed

Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

2011-08-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

PubMed Central

Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

2012-01-01

Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

Uroepithelial cells are part of a mucosal cytokine network.

PubMed Central

Hedges, S; Agace, W; Svensson, M; Sjögren, A C; Ceska, M; Svanborg, C

1994-01-01

This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alpha), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1 beta mRNA was detected in J82 cells. IL-1 alpha induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha were not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-alpha mRNA was occasionally detected in the J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and beta-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-stimulated cells. beta-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1 alpha and TNF-alpha were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection. Images PMID:8188354

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

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

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

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

PubMed

Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

2016-08-01

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations.

PubMed

Lin, Yao-Cheng; Boone, Morgane; Meuris, Leander; Lemmens, Irma; Van Roy, Nadine; Soete, Arne; Reumers, Joke; Moisse, Matthieu; Plaisance, Stéphane; Drmanac, Radoje; Chen, Jason; Speleman, Frank; Lambrechts, Diether; Van de Peer, Yves; Tavernier, Jan; Callewaert, Nico

2014-09-03

The HEK293 human cell lineage is widely used in cell biology and biotechnology. Here we use whole-genome resequencing of six 293 cell lines to study the dynamics of this aneuploid genome in response to the manipulations used to generate common 293 cell derivatives, such as transformation and stable clone generation (293T); suspension growth adaptation (293S); and cytotoxic lectin selection (293SG). Remarkably, we observe that copy number alteration detection could identify the genomic region that enabled cell survival under selective conditions (i.c. ricin selection). Furthermore, we present methods to detect human/vector genome breakpoints and a user-friendly visualization tool for the 293 genome data. We also establish that the genome structure composition is in steady state for most of these cell lines when standard cell culturing conditions are used. This resource enables novel and more informed studies with 293 cells, and we will distribute the sequenced cell lines to this effect.

LINE-1 Cultured Cell Retrotransposition Assay

PubMed Central

Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

2016-01-01

Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

PubMed

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

[Establishment of fibroblast cell line and its biological characteristics in Matou goat].

PubMed

Li, Tianda; Liu, Chousheng; Wang, Zhigang; Zhang, Liping; Sun, Xiuzhu; Zhao, Junjin; Meng, Fei; Luo, Guihe; Zhu, Jinqing

2008-12-01

Taking Matou goat ear margin as the study material, we succeeded in established a fibroblast cell line by the method of explant culture directly. Observations on morphology, dynamic growth, determination of viability, analysis of karyotype, test of microorganism and other characteristics were detected. Results showed: Population Doubling Time (PDT) of cells was approximately 36 h; Cell viability was 96.7% after thawing; The status of cell After passage was constant; Analysis of chromosomal karyotyps indicated that diploid (2n=60) account for 98% in the cell line. Every index in the cell line met all the standard quality controls of ATCC in USA. The established of Matou goat ear fibroblast cell line has not only important genetic resources preserved at the cell level, but also valuable material for genome, postgenome and somatic cell nuclear transfer research.

Sensitive detection of EML4-ALK fusion oncoprotein of lung cancer by in situ proximity ligation assay.

PubMed

Rho, Jin Kyung; Lee, Hyangsin; Park, Chan-Sik; Choi, Chang-Min; Lee, Jae Cheol

2013-09-01

EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription. In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots. We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used. Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.

Enterovirus Species B Bias of RD Cell Line and Its Influence on Enterovirus Diversity Landscape.

PubMed

Faleye, Temitope Oluwasegun Cephas; Adeniji, Johnson Adekunle

2015-12-01

Despite its widespread use in poliovirus isolation, studies show that most RD cell line isolates are species B enteroviruses (EB), it was therefore employed to further catalogue the EB diversity in two different regions of Nigeria. Concentrates of 18 environmental samples were inoculated into RD cell line. Isolates were subjected to PCR assays to detect enteroviruses, species C and B members and partial VP1 gene which was subsequently sequenced and used for identification and phylogenetic analysis. Isolates were further passaged in L20B cell line to detect polioviruses. Sixty-eight isolates were recovered from the 18 concentrates, all of which were positive for the enterovirus 5'-UTR screen. Thirteen of the 68 isolates were positive for the species C screen and replicated in L20B cell line, eleven of which also contained species B enteroviruses. Some of the mixed isolates were successfully typed, but as species B members. In all, isolates recovered in this study were identified as CVB5, E6, E7, E11, E13, E19, E20, E33, EVB75 and WPV3, while some could not be typed. Alongside the ten different enterovirus serotypes confirmed, results of this study document for the first time in Nigeria, EVB75. It showed the EB bias of RD cell line might indicate something much more fundamental in its biology. Finally, the finding of WPV3 in a region considered low risk for poliovirus emphasizes the need to expand poliovirus environmental surveillance to enable early detection of poliovirus silent circulation before occurrence of clinical manifestations.

A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

PubMed

Goodrich, David; Tao, Xin; Bohrer, Chelsea; Lonczak, Agnieszka; Xing, Tongji; Zimmerman, Rebekah; Zhan, Yiping; Scott, Richard T; Treff, Nathan R

2016-11-01

A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

Tissue-specific expression of human CD4 in transgenic mice.

PubMed Central

Gillespie, F P; Doros, L; Vitale, J; Blackwell, C; Gosselin, J; Snyder, B W; Wadsworth, S C

1993-01-01

The gene for the human CD4 glycoprotein, which serves as the receptor for human immunodeficiency virus type 1, along with approximately 23 kb of sequence upstream of the translational start site, was cloned. The ability of 5' flanking sequences to direct tissue-specific expression was tested in cell culture and in transgenic mice. A 5' flanking region of 6 kb was able to direct transcription of the CD4 gene in NIH 3T3 cells but did not result in detectable expression in the murine T-cell line EL4 or in four lines of transgenic mice. A larger 5' flanking region of approximately 23 kb directed high-level CD4 transcription in the murine T-cell line EL4 and in three independent lines of transgenic mice. Human CD4 expression in all tissues analyzed was tightly correlated with murine CD4 expression; the highest levels of human CD4 RNA expression were found in the thymus and spleen, with relatively low levels detected in other tissues. Expression of human CD4 protein in peripheral blood mononuclear cells was examined by flow cytometry in these transgenic animals and found to be restricted to the murine CD4+ subset of lymphocytes. Human CD4 protein, detected with an anti-human CD4 monoclonal antibody, was present on the surface of 45 to 50% of the peripheral blood mononuclear cells from all transgenic lines. Images PMID:8474453

LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

PubMed

Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

2017-03-06

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

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

PubMed

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

2003-05-01

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

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

PubMed

Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

2015-08-01

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

PubMed

Easty, D J; Guthrie, B A; Maung, K; Farr, C J; Lindberg, R A; Toso, R J; Herlyn, M; Bennett, D C

1995-06-15

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

Replication of Syngrapha falcifera Multiple-Nuclear Polyhedrosis Virus-D in Different Insect Cells

NASA Astrophysics Data System (ADS)

Khalid Nessr Alhag, Sadeq; Xin, Peng Jian

Six insect cell lines were tested for susceptibility to Syngrapha falcifera multiple nucleocapsid nucleopolyhedrovirus-D (SfaMNPV-D) infection by use of a typical endpoint assay procedure. Cell lines from Trichoplusia ni (Tn5B1-4), (L105-clone), Spodoptera litura (SL-ZSU-1), Spodoptera frugiperda (IPLB-SF-21), Pieris rapaeb (Pr-E-HNU9) and Helicoverpa zea (BCIRL-HZ-AM1) in 96-well tissue culture plates were infected with dilutions of extra cellular virus suspensions of (SfaMNPV-D). Each cell/virus combination was incubated at temperatures 27°C and wells were scored for positive infection at 2 to 4 day intervals. The resulting data were analyzed by Reed and Muench method, providing virus titers for each combination of virus, cell line. The results were categorized by accuracy and by rapidity of maximum titer. Virus titer of Tn5B-4 was higher than other cell lines TCID50 8.7x108, the lowest level detected in infected was in (Pr-E-HNU9) cells TCID50 2.4x108. No Virions or polyhedral inclusion bodies were detected in infected SL-ZSU-1 cells.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

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

PubMed

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

2018-04-13

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

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

PubMed

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

1998-01-01

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

Aberrant expression of the PHF14 gene in biliary tract cancer cells

PubMed Central

AKAZAWA, TAKAKO; YASUI, KOHICHIROH; GEN, YASUYUKI; YAMADA, NOBUHISA; TOMIE, AKIRA; DOHI, OSAMU; MITSUYOSHI, HIRONORI; YAGI, NOBUAKI; ITOH, YOSHITO; NAITO, YUJI; YOSHIKAWA, TOSHIKAZU

2013-01-01

DNA copy number aberrations in human biliary tract cancer (BTC) cell lines were investigated using a high-density oligonucleotide microarray. A novel homozygous deletion was detected at chromosomal region 7p21.3 in the OZ cell line. Further validation experiments using genomic PCR revealed a homozygous deletion of a single gene, plant homeodomain (PHD) finger protein 14 (PHF14). No PHF14 mRNA or protein expression was detected, thus demonstrating the absence of PHF14 expression in the OZ cell line. Although the PHD finger protein is considered to be involved in chromatin-mediated transcriptional regulation, little is known about the function of PHF14 in cancer. The present study observed that the knock down of PHF14 using small interfering RNA (siRNA) enhanced the growth of the BTC cells. These observations suggest that aberrant PHF14 expression may have a role in the tumorigenesis of BTC. PMID:23833654

Glutathione determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response.

PubMed Central

Eady, J. J.; Orta, T.; Dennis, M. F.; Stratford, M. R.; Peacock, J. H.

1995-01-01

Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines. PMID:7577452

Multimarker Quantitative Real-Time PCR Detection of Circulating Melanoma Cells in Peripheral Blood: Relation to Disease Stage in Melanoma Patients

PubMed Central

Koyanagi, Kazuo; Kuo, Christine; Nakagawa, Taku; Mori, Takuji; Ueno, Hideaki; Lorico, Arnulfo R.; Wang, He-Jing; Hseuh, Eddie; O’Day, Steven J.; Hoon, Dave S.B.

2010-01-01

Background Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable, multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients’ blood. Methods We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. Results All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 107 PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P <0.0001). Conclusions Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients. PMID:15817820

Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

PubMed

Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

2012-12-01

The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria. © 2011 Blackwell Publishing Ltd.

Simultaneous detection of MCF-7 and HepG2 cells in blood by ICP-MS with gold nanoparticles and quantum dots as elemental tags.

PubMed

Li, Xiaoting; Chen, Beibei; He, Man; Wang, Han; Xiao, Guangyang; Yang, Bin; Hu, Bin

2017-04-15

In this work, we demonstrate a novel method based on inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticles (Au NPs) and quantum dots (QDs) labeling for the simultaneous counting of two circulating tumor cell lines (MCF-7 and HepG2 cells) in human blood. MCF-7 and HepG2 cells were captured by magnetic beads coupled with anti-EpCAM and then specifically labeled by CdSe QDs-anti-ASGPR and Au NPs-anti-MUC1, respectively, which were used as signal probes for ICP-MS measurement. Under the optimal experimental conditions, the limits of detection of 50 MCF-7, 89 HepG2 cells and the linear ranges of 200-40000 MCF-7, 300-30000 HepG2 cells were obtained, and the relative standard deviations for seven replicate detections of 800 MCF-7 and HepG2 cells were 4.6% and 5.7%, respectively. This method has the advantages of high sensitivity, low sample consumption, wide linear range and can be extended to the simultaneous detection of multiple CTC lines in human peripheral blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting TMPRSS2-ERG in Prostate Cancer

DTIC Science & Technology

2017-11-01

phosphorylated proteins in ERG positive versus negative cell lines following suppression of kinases by shRNA or knockout using CRISPR /Cas9...the cofactor is below detection by silver staining. Future experiments are aimed at generating a stable cell line using CRISPR /Cas9 that has

Multiplex Detection of KRAS Mutations Using Passive Droplet Fusion.

PubMed

Pekin, Deniz; Taly, Valerie

2017-01-01

We describe a droplet microfluidics method to screen for multiple mutations of a same oncogene in a single experiment using passive droplet fusion. Genomic DNA from H1573 cell-line was screened for the presence of the six common mutations of the KRAS oncogene as well as wild-type sequences with a detection efficiency of 98 %. Furthermore, the mutant allelic fraction of the cell-line was also assessed correctly showing that the technique is quantitative.

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

PubMed Central

Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

2009-01-01

Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. Reviewers This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter. PMID:19715575

Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines.

PubMed

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

2003-03-20

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

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

A reevaluation of CD22 expression in human lung cancer.

PubMed

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

2014-01-01

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

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

PubMed Central

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

2015-01-01

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

Effect of dexamethasone on expression of glucocorticoid receptor in human monocyte cell line THP-1.

PubMed

Li, Bo; Bai, Xiangjun; Wanh, Haiping

2006-01-01

The effect of dexamethasone with different concentrations and different stimulating periods on the expression of glucocorticoid receptors (GRalpha, GRbeta) protein was investigated in human monocyte cell line THP-1. The cultured human monocyte line THP-1 cells were stimulated by dexamethasone with different concentrations and different periods. The expression of GRalpha and GRbeta protein was detected by Western blotting. The results showed that the expression of GRalpha and GRbeta was detected in the THP-1 cells. The quantity of GRalpha expression was reduced by dexamethasone under the same concentration with the prolongation of the stimulating periods. The quantity of GRbeta expression was increased by dexamethasone treatment in a time- and dose-dependent manner. It was concluded that dexamethasone stimulation time-dependently reduced the GRalpha expression in THP-1 cells. Dexamethasone stimulation time- and dose-dependently increased the GRbeta expression in THP-1 cells. The expression of GRalpha and GRbeta was regulated by glucocorticoid.

MicroRNA-145 Inhibits Cell Migration and Invasion and Regulates Epithelial-Mesenchymal Transition (EMT) by Targeting Connective Tissue Growth Factor (CTGF) in Esophageal Squamous Cell Carcinoma.

PubMed

Han, Qiang; Zhang, Hua-Yong; Zhong, Bei-Long; Wang, Xiao-Jing; Zhang, Bing; Chen, Hua

2016-10-23

BACKGROUND This study investigated the mechanism of miR-145 in targeting connective tissue growth factor (CTGF), which affects the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of ESCC cells. MATERIAL AND METHODS A total of 50 ESCC tissues and their corresponding normal adjacent esophageal tissue samples were collected. Then, miR-145 expression in both ESCC clinical specimens and cell lines was detected using quantitative real-time PCR. CTGF protein was detected using immunohistochemistry. Dual luciferase reporter gene assay was employed to assess the effect of miR-145 on the 3'UTR luciferase activity of CTGF. Eca109 cells were transfected with miR-145 mimics and CTGF siRNA, respectively, and changes in cellular proliferation, migration, and invasion were detected via MTT assay, wound-healing assay, and Transwell assay, respectively. Western blotting assay was used to detect the expression of marker genes related to EMT. RESULTS MiR-145 was significantly down-regulated in ESCC tissues and cell lines compared with normal tissues and cell lines (P<0.05). We found significantly more positively expressed CTGF protein in ESCC tissues was than in normal adjacent esophageal tissues (P<0.01). Dual luciferase reporter gene assay showed that miR-145 can specifically bind with the 3'UTR of CTGF and significantly inhibit the luciferase activity by 55% (P<0.01). Up-regulation of miR-145 or down-regulation of CTGF can suppress the proliferation, migration, invasion, and EMT process of ESCC cells. CONCLUSIONS MiR-145 was significantly down-regulated in ESCC tissues and cell lines, while the protein expression of CTGF exhibited the opposite trend. MiR-145 inhibited the proliferation, migration, invasiveness, and the EMT process of ESCC cells through targeted regulation of CTGF expression.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells.

PubMed

Chen, Jian; Mu, Qitian; Li, Xia; Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-06-20

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells

PubMed Central

Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-01-01

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT. PMID:28454099

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines.

PubMed

Abdi-Ali, A; Worobec, E A; Deezagi, A; Malekzadeh, F

2004-05-01

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.

Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

PubMed Central

Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

1999-01-01

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

Establishment and characterization of outer root sheath (ORS) cell line from Jining grey goat.

PubMed

Cui, Zhifeng; Hu, Yanxia; Wang, Hui; Zeng, Yongqing; Dong, Bin; Zhu, Houshun; Dong, Zhongdian; Liu, Zhiyuan

2012-03-01

A new line of outer root sheath (ORS) cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion. Cell morphology is described at different phases. The chromosome analysis of ORS cells, identification of the ORS cells and morphological reversion test were detected at the 4th and 40th passages. The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52 h. Chromosome analysis showed that >58% of cells were diploid. Test for ORS cell line CK19 expression was positive. This newly established ORS cell line not only lays the foundation for further studying on the growth, regeneration, development law of goat hair follicle but also provides a mirror for the research of human hair in medical field.

Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

PubMed

Dunn, Glynis

2016-01-01

The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence.

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas

USGS Publications Warehouse

Lu, Y.; Nerurkar, V.R.; Aguirre, A.A.; Work, Thierry M.; Balazs, G.H.; Yanagihara, R.

1999-01-01

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30A? C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Turner syndrome and 45,X/47,XXX mosaicism.

PubMed

Akbas, E; Mutluhan, H; Savasoglu, K; Soylemez, F; Ozturk, I; Yazici, G

2009-01-01

The occurrence of double aneuploidy is a relatively rare phenomenon. We report on a 17-year-old woman with short stature, minimal pubic and axillar hair and short hands. In cultured lymphocyte a double aneuploidy mosaicism was detected, consisting of a cell line with trisomy for X chromosome and a cell line with monosomy for the X-chromosome and no cell line with a normal karyotype. To our knowledge, this is the first case of mosaic 45,X/47,XXX in Turkey.

Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

PubMed Central

Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

2016-01-01

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

PubMed

Timm, Michael; Hansen, Erik W; Moesby, Lise; Christensen, Jens D

2006-02-01

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

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

Walker, C.; Nettesheim, P.; Barrett, J.C.

1987-04-01

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injectedmore » into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approx. = 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.« less

Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE).

PubMed

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

2013-06-01

Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it "S-TFE." The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma.

Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE)

PubMed Central

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

2013-01-01

Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it “S-TFE.” The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma. PMID:23760492

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

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

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Establishment, characterization, virus susceptibility and transfection of cell lines from cobia, Rachycentron canadum (L.), brain and fin.

PubMed

Cheng, T-C; Lai, Y-S; Lin, I-Y; Wu, C-P; Chang, S-L; Chen, T-I; Su, M-S

2010-02-01

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate-buffered saline containing 0.25% trypsin at 25 degrees C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 degrees C in Leibovitz-15 medium containing 10% foetal bovine serum. The cells have been sub-cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.

Sers Imaging of Nasopharyngeal Carcinoma Markers Using an Antibody-Conjugated Gold Nanoparticles Probe

NASA Astrophysics Data System (ADS)

Li, J.-H.; Du, Y.; Feng, G.-K.; Du, Y.-B.; Zhou, Y.-Q.; Zeng, M.-S.

2017-11-01

Surface-enhanced Raman scattering (SERS) nanotags as an ultrasensitive nanoprobe is becoming popular for the detection of biomarkers. Herein, antibody-conjugated gold nanoparticles (AuNPs) were used to target LMP2A in an LMP2A-infected CNE2 cell line. SERS maps showed that the LMP2A was distributed around the cell, which was consistent with the results of immunofl uorescence staining in the previous report. This location could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. However, the CNE2 cell line without LMP2A-infected showed no detectable signal at 1044 cm-1. The results demonstrated the potential feasibility of AuNPs nanotags as highly sensitive probes conjugated at the subcellular level for detection and localization of cancer markers in nasopharyngeal carcinoma (NPC).

A photoacoustic spectrometer for trace gas detection

NASA Astrophysics Data System (ADS)

Telles, E. M.; Bezerra, E.; Scalabrin, A.

2005-06-01

A high-resolution external laser photoacoustic spectrometer has been developed for trace gas detection with absorption transitions in coincidence with CO2 laser emission lines (9,2-10,9 μm: 920-1086 cm-1). The CO2 laser operates in 90 CW lines with power of up to 15 W. A PC-controlled step motor can tune the laser lines. The resonance frequency of first longitudinal mode of the photoacoustic cell is at 1600 Hz. The cell Q-factor and cell constant are measured close to 50 and 28 mVcmW-1, respectively. The spectrometer has been tested in preliminary studies to analyze the absorption transitions of ozone (O_3). The ethylene (C_2H_4) from papaya fruit is also investigated using N2 as carrier gas at a constant flow rate.

Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

PubMed

Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

2017-07-01

Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

Elucidation of proliferative capability of mononuclear tetraploid cells, emerging spontaneously from diploid cells, using image cytometry and fluorescence in situ hybridization.

PubMed

Ito, Hideaki; Oga, Atsunori; Furuya, Tomoko; Ikemoto, Kenzo; Amakawa, Genta; Chochi, Yasuyo; Kawauchi, Shigeto; Sasaki, Kohsuke

2013-06-01

Proliferation of tetraploid cells (TCs) emerging from diploid cells is considered to be a critical event toward tumourigenesis, or cancer progression. Recently, several studies have reported that binuclear TCs emerging from normal cells are capable of mitosis, however, it has not been confirmed directly whether mononuclear TCs emerging from normal cells could proliferate, even cancer cells. The aim of this study is to detect mononuclear TCs in vitro, spontaneously emerging from diploid cells and to elucidate their proliferative capability directly. For this purpose, we have developed a novel method. In this study, two completely disomic cell lines were used, TIG-7, a fibroblast cell line and CAL-51, a breast cancer cell line. Cells were cultured on microscope slides and their DNA content was determined using an image cytometer. On the same slides, chromosome numbers were scored using centromere fluorescence in situ hybridization (FISH). For evaluating proliferative capability of TCs, bromodeoxyuridine (BrdUrd) incorporation and colony-forming ability were examined. Using our method, spontaneous emergence of mononuclear TCs was detected in both TIG-7 and CAL-51. Colonies of TIG-7 TCs were not observed, but were observed of CAL-51 TCs. Our method enables detection of mononuclear TCs and elucidation of their proliferative capability, directly; this evidence reveals that mononuclear TIG-7 TCs do not proliferate but that mononuclear CAL-51 TCs are able to. © 2013 Blackwell Publishing Ltd.

There and Back Again: Development and Regeneration of the Zebrafish Lateral Line System

PubMed Central

Thomas, Eric D.; Cruz, Ivan A.; Hailey, Dale W.; Raible, David W.

2014-01-01

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish’s increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. PMID:25330982

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation

PubMed Central

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-01-01

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%–20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs. PMID:27270313

The impact of intermittent versus continuous exposure to EGFR tyrosine kinase inhibitor on selection of EGFR T790M-mutant drug-resistant clones in a lung cancer cell line carrying activating EGFR mutation.

PubMed

Lee, Youngjoo; Choi, Yu-Ra; Kim, Kyoung-Yeon; Shin, Dong Hoon

2016-07-12

Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs. We used PC9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 μM to 1.0 μM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined. T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC9/GRc) than in cells intermittently exposed to gefitinib (PC9/GRi) (0.04 μM vs 1.0 μM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC9/GRc and PC9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC9/GRi cells but not in the PC9/GRc cells. In the PC9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 μM to 1.0 μM of gefitinib. In the PC9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%-20.9%). In conclusion, compared with intermittent drug exposure, continuous exposure might select better minor drug-resistant clones when creating cell lines resistant to molecular-targeted drugs.

Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.

PubMed Central

Reeve, J. G.; Xiong, J.; Morgan, J.; Bleehen, N. M.

1996-01-01

As a first step towards elucidating the potential role(s) of bcl-2 and bcl-2-related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-x1-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl-2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8630278

Assembly And Initial Characterization Of A Panel Of 85 Genomically Validated Cell Lines From Diverse Head And Neck Tumor Sites

PubMed Central

Zhao, Mei; Sano, Daisuke; Pickering, Curtis R.; Jasser, Samar A.; Henderson, Ying C.; Clayman, Gary L.; Sturgis, Erich M.; Ow, Thomas J.; Lotan, Reuben; Carey, Thomas E.; Sacks, Peter G.; Grandis, Jennifer R.; Sidransky, David; Heldin, Nils Erik; Myers, Jeffrey N.

2011-01-01

Purpose Human cell lines are useful for studying cancer biology and pre-clinically modeling cancer therapy, but can be misidentified and cross contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma (HNSCC), thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium, was assembled from the collections of several individuals and institutions. Authenticity was verified by performing short tandem repeat (STR) analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination demonstrates a wide range of in vitro phenotypes. Conclusion This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be utilized for biological as well as preclinical studies. PMID:21868764

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

PubMed Central

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

2014-01-01

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

An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

PubMed

Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

2018-05-15

Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

[SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

PubMed

Wang, Lili; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Zhao, Song; Ma, Dongchu

2016-10-01

Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect of SP600125 on phosphorylation of S6K1 in cell lines at the different differentiation stages.

A Re-evaluation of CD22 Expression by Human Lung Cancer

PubMed Central

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

2014-01-01

CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B cell receptor and its co-receptor CD19. Recently it was reported that most human lung cancer cells and cell lines express CD22 making it an important new lung cancer therapeutic target (Can Res 72:5556, 2012). The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by qRT-PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200–60,000- fold lower than those observed in the human CD22+ Burkitt’s lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by CD22 antibodies or our highly potent anti-CD22 immunotoxin. By contrast, CD22+ Daudi cells expressed high levels of CD22 mRNA and protein and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from over 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells and that these cells can not be killed by anti-CD22 immunotoxins. PMID:24395821

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

NASA Astrophysics Data System (ADS)

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

2018-02-01

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

[Effect of cisplatin on the expression of Pokemon gene: experiment with different human lung cancer cells].

PubMed

Zhao, Zhi-Hong; Wang, Sheng-Fa; Yu, Liang; Wang, Ju; Cong, De-Gang; Chang, Hao; Wang, Xue-Feng; Zhang, Tie-Wa; Zhang, Jian; Fu, Kai; Jiang, Jiu-Yang

2008-04-29

To investigate the correlation between Pokemon gene and cisplatin mechanism. Human lung adenocarcinoma cells of the lines A549 and AGZY83-a, human lung squamous carcinoma cells of the line HE-99, and human giant cell lung cancer cells of the line 95D were cultured and cisplatin was added into the medium. Other lung cancer cells of the above mentioned lines were cultured in the medium without cisplatin and were used as control groups. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Pokemon. Pokemon mRNA and protein were expressed highly in all the 4 cell lines. The Pokemon gene expression did not changed significantly after cisplatin treatment groups. There were not significant differences in the mRNA and protein expression of Pokemon among the 4 experiment groups and the control groups (all P > 0.05). Cisplatin has no effect on the Pokemon gene expression of the human lung cancer cells.

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

PubMed

Lei, Lei; Spradling, Allan C

2013-05-21

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

PubMed

Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2016-09-01

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Epigenetic repression of HOXB cluster in oral cancer cell lines.

PubMed

Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

2014-08-01

Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transformation of primary chick embryo fibroblasts by Marek's disease virus.

PubMed

Buranathai, C; Rodriguez, J; Grose, C

1997-12-08

Marek's disease virus (MDV) is an alphaherpesvirus, which can mediate the malignant transformation of lymphocytes to form lymphomas in chickens. In this study, we demonstrate that MDV can transform primary chick embryo fibroblasts (CEF). The cell line derived from primary CEF infected with the GA strain of MDV was called CEM(MDV). The fibroblast nature of CEM(MDV) was verified by absence of cytokeratin type II. The CEM(MDV) phenotype differed from either primary CEF or MDV-infected CEF. CEM(MDV) were extensively vacuolated, with unusual multilamellar structures in the cytoplasm, The nuclei were considerably larger than those in primary CEF and were uniformly positive for proliferating cell nuclear antigen. The cell line was subcultured for more than 10 generations; however, CEM(MDV) did not support a fully productive MDV infection, because complete nucleocapsids were not detected and infectivity assays showed that cell line produced no infectious virus. PCR analyses demonstrated that this cell line carried both polypeptide 38 (pp38) and Meq DNA, MDV-specific genes associated with transformation. In addition, examination by laser scanning confocal microscopy revealed that CEM(MDV) constitutively produced MDV MEQ protein in nuclei and pp38 as well as glycoprotein B in the cytoplasm and on the plasma membrane. Growth in soft agar assay demonstrated that CEM(MDV) formed colonies, similar to HeLa and human melanoma cells. Retroviral insertion was not detected in DNA from the CEM(MDV) line.

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

PubMed

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

2011-04-01

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

Lack of differences in radiation-induced immunogenicity parameters between HPV-positive and HPV-negative human HNSCC cell lines.

PubMed

Schneider, Karolin; Bol, Vanesa; Grégoire, Vincent

2017-09-01

Clinical studies indicate that patients with HPV/p16-associated head & neck squamous cell carcinoma (HNSCC) represent a subgroup with a better prognosis and improved response to conventional radiotherapy. Involvement of immune-based factors has been hypothesized. In the present study, we investigated radiation-induced differences in release of damage associated molecular patterns (DAMPs), cytokines and activation of dendritic cells (DCs) in HPV-positive and negative HNSCC cancer cell lines. Calreticulin (CRT) exposure was detected on cancer cell surface. ATP, HMGB1 and cytokines were measured in culture supernatants. Maturation marker CD83 surface exposure was determined on DCs after co-incubation with irradiated tumor cells. There was no increase in DAMPs and cytokine profiles after radiation treatment and no difference between HPV+ and HPV- cell lines. The HPV/p16-positive SCC90 cells showed a trend for increased total CRT, HMGB1, and number of cytokines compared to all other cell lines. None of the irradiated cancer cell lines could affect DC maturation. Radiation treatment did not increase immunogenicity of HNSCC cell lines assessed by membrane CRT, ATP, HMGB1, cytokines production, and by activation of immature DCs. There was no difference between HPV-positive and HPV-negative cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

Unique volatolomic signatures of TP53 and KRAS in lung cells

PubMed Central

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

2014-01-01

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

Tectonic-1 contributes to the growth and migration of prostate cancer cells in vitro

PubMed Central

WANG, ZHIJUN; GAO, YI; LIU, YUSHAN; CHEN, JIE; WANG, JUNKAI; GAN, SISHUN; XU, DANFENG; CUI, XINGANG

2015-01-01

Tectonic-1 (TCTN1) is an upstream gene involved in embryonic development. The aim of the present study was to investigate the effect of the TCTN1 gene on the viability and migration of prostate cancer cells. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to silence the expression of TCTN1 in PC-3 and DU145 prostate cancer cells. Cell viability and proliferation were measured using MTT and colony formation assays, and the distribution of cells in phases of the cell cycle was determined using flow cytometry. Cell migration was detected using a Transwell assay. The results demonstrated that TCTN1 was widely expressed in several human prostate cancer cell lines. Knockdown of the TCTN1 gene by RNA interference markedly suppressed cell viability and colony formation in the PC-3 and DU145 cell lines. Cell cycle progression was also arrested by TCTN1 silencing. In addition, knockdown of the TCTN1 gene led to the inhibition of cell migration in the two cell lines. These findings confirmed the direct association between the TCTN1 gene and prostate cancer growth in vitro. With further understanding and clinical investigation, this indicates the potential for future development of a novel marker for early detection and gene therapy for prostate cancer. PMID:26310786

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

PubMed

Lahm, H; Hoeflich, A; Andre, S; Sordat, B; Kaltner, H; Wolf, E; Gabius, H J

2000-09-01

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

PubMed

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

PubMed

Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

2014-05-01

Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans. © 2014 Wiley Periodicals, Inc.

Anticancer activity of Astragalus polysaccharide in human non-small cell lung cancer cells.

PubMed

Wu, Chao-Yan; Ke, Yuan; Zeng, Yi-Fei; Zhang, Ying-Wen; Yu, Hai-Jun

2017-01-01

We have reported that Chinese herbs Astragalus polysaccharide (APS) can inhibit nuclear factor kappaB (NF-κB) activity during the development of diabetic nephropathy in mice. NF-κB plays important roles in genesis, growth, development and metastasis of cancer. NF-κB is also involved in the development of treatment resistance in tumors. Here we investigated the antitumor activity of APS in human non-small cell lung cells (A549 and NCI-H358) and the related mechanisms of action. The dose-effect and time-effect of antitumor of APS were determined in human lung cancer cell line A549 and NCI-H358. The inhibition effect of APS on the P65 mRNA and protein was detected by reverse transcriptase-PCR (RT-PCR) and Western blot in A549 cells respectively. The inhibition effect of APS on the p50, CyclinD1 and Bcl-xL protein was detected by Western blot in A549 cells respectively. The effect of APS on NF-κB transcription activity was measured with NF-κB luciferase detection. Finally, the nude mice A549 xenograft was introduced to confirm the antitumor activity of APS in vivo. Cell viability detection results indicated that APS can inhibit the proliferation of human lung cancer cell line A549 and NCI-H358 in the concentration of 20 and 40 mg/mL. NF-κB activator Phorbol 12-myristate13-acetate (PMA) can attenuate the antitumor activity of APS in both cell lines, but NF-κB inhibitor BAY 11-7082 (Bay) can enhance the effect of APS in both cell lines. In vivo APS can delay the growth of A549 xenograft in BALB/C nude mice. APS can down-regulate the expression of P65 mRNA and protein of A549 cells and decrease the expression of p50, CyclinD1 and Bcl-xL protein. The luciferase detection showed that the APS could reduce the P65 transcription activity in A549 cells. PMA can partially alleviate the inhibition activity of P65 transcription activity of APS in A549 cells, and Bay can enhance the down-regulation of the P65 transcription activity induced by APS in A549 cells. APS has a significant antitumor activity in human lung cancer cells A549 and NCI-H358. NF-κB inhibition may mediate the antitumor effect.

Nucleophosmin is overexpressed in thyroid tumors

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

Pianta, Annalisa; Puppin, Cinzia; Franzoni, Alessandra

2010-07-02

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed inmore » tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.« less

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

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Gfi1-Cre knock-in mouse line: A tool for inner ear hair cell-specific gene deletion

PubMed Central

Yang, Hua; Gan, Jean; Xie, Xiaoling; Deng, Min; Feng, Liang; Chen, Xiaowei; Gao, Zhiqiang; Gan, Lin

2010-01-01

Summary Gfi1encodes a zinc-finger transcription factor essential for the development and maintenance of haematopoiesis and the inner ear. In mouse inner ear, Gfi1 expression is confined to hair cells during development and in adulthood. To construct a genetic tool for inner ear hair cell-specific gene deletion, we generated a Gfi1-Cre mouse line by knocking-in Cre coding sequences into the Gfi1 locus and inactivating the endogenous Gfi1. The specificity and efficiency of Gfi1-Cre recombinase-mediated recombination in the developing inner ear was revealed through the expression of the conditional R26R-lacZ reporter gene. The onset of lacZ expression in the Gfi1Cre/+ inner ear was first detected at E13.5 in the vestibule and at E15.5 in the cochlea, coinciding with the generation of hair cells. Throughout inner ear development, lacZ expression was detected only in hair cells. Thus, Gfi1-Cre knock-in mouse line provides a useful tool for gene manipulations specifically in inner ear hair cells. PMID:20533399

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

NASA Astrophysics Data System (ADS)

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

2018-06-01

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

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

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

PubMed Central

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

2015-01-01

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

Heterogeneous expression and regulation of CD40 in human hepatocellular carcinoma.

PubMed

Holub, Margareta; Zakeri, Schaker M; Lichtenberger, Cornelia; Pammer, Johannes; Paolini, Pierre; Leifeld, Ludger; Rockenschaub, Susanne; Wolschek, Markus F; Steger, Günther; Willheim, Martin; Gangl, Alfred; Reinisch, Walter

2003-02-01

CD40, a member of the tumour necrosis factor receptor family, plays a major role in adaptive immune responses and contributes to cancer surveillance. Conflicting results have been reported recently on the expression and function of CD40 in carcinomas. The aim of the present study was to investigate the role of CD40 in human hepatoma. CD40 expression was examined in hepatomas and derived cell lines by immunohistochemistry, flow cytometry and reverse transcriptase polymerase chain reaction. We investigated in hepatoma cell lines the regulation of CD40 by pro-inflammatory cytokines and the effects of its ligation with soluble CD40L on the expression of co-stimulatory and pro-apoptotic cell-surface molecules and survival. CD40 was detected with a similar frequency of about 40% in hepatoma specimens and derived cell lines but not in normal hepatocytes. Tumour necrosis factor alpha and its combination with interferon gamma upregulated CD40 only in intrinsically positive cell lines. CD40 ligation had no effect on cell viability or surface expression of CD54, CD80, CD86 or CD95. CD40 is expressed variably in human hepatoma and enhanced by distinct pro-inflammatory cytokines. The lack of detectable effects of CD40 ligation does not support a major role of this molecule in hepatocellular carcinoma biology.

Quantitative analysis of cell columns in the cerebral cortex.

PubMed

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

2000-04-01

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

Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder.

PubMed

Bakali, Evangelia; Elliott, Ruth A; Taylor, Anthony H; Lambert, David G; Willets, Jonathon M; Tincello, Douglas G

2014-06-01

To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

PubMed

Pérez-Campo, Flor M; May, Tobias; Zauers, Jeannette; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Berciano, María T; Lafarga, Miguel; Riancho, José A

2017-03-01

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D 3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma.

PubMed

Mochizuki, Hiroyuki; Takahashi, Masashi; Nishigaki, Kazuo; Ide, Tetsuya; Goto-Koshino, Yuko; Watanabe, Shinya; Sato, Hirofumi; Sato, Masahiko; Kotera, Yukiko; Fujino, Yasuhito; Ohno, Koichi; Uchida, Kazuyuki; Tsujimoto, Hajime

2011-04-15

We established a novel feline B-cell line, MS4, from the neoplastic pleural effusion of a cat with cutaneous B-cell lymphoma. Immunophenotype staining of the MS4 cells was positive for CD20, CD79α, and IgA and negative for CD3, CD4, CD5, CD8α, CD18, CD21, CD22, IgM, IgG, Ig light chain, and MHC class II. PCR analysis for immunoglobulin heavy chain gene rearrangements revealed a monoclonal rearrangement, whereas no clonal rearrangement of the T-cell receptor γ gene was detected. Southern blotting with an exogenous feline leukemia virus (FeLV) U3 probe revealed no integration of exogenous FeLV provirus. The MS4 cell line is the first FeLV-negative feline B-cell lymphoma cell line, and may be used to investigate the pathogenesis of spontaneously occurring feline lymphoma and the development of new therapies. Copyright © 2011 Elsevier B.V. All rights reserved.

A Telomerase Immortalized Human Proximal Tubule Cell Line with a Truncation Mutation (Q4004X) in Polycystin-1

PubMed Central

Herbert, Brittney-Shea; Grimes, Brenda R.; Xu, Wei Min; Werner, Michael; Ward, Christopher; Rossetti, Sandro; Harris, Peter; Bello-Reuss, Elsa; Ward, Heather H.; Miller, Caroline; Gattone, Vincent H.; Phillips, Carrie L.; Wandinger-Ness, Angela; Bacallao, Robert L.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions [1], [2]. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X) in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells. PMID:23383103

Killing effect of TNF-mediated by conditionally replicating adenovirus on esophageal cancer and lung cancer cell lines.

PubMed

Jiang, Yue-Quan; Zhang, Zhi; Cai, Hua-Rong; Zhou, Hong

2015-01-01

The killing effect of TNF mediated by conditionally replicating adenovirus SG502 on human cancer cell lines was assessed by in vivo and in vitro experiments. The recombinant adenovirus SG502-TNF was used to infect human lung cancer cell line A549 and human esophageal cancer cell line TE-1. The expression of the exogenous gene and its inhibitory effect on the tumor cell lines were thus detected. Tumor transplantation experiment was performed in mice with the purpose of assessing the inhibitory effect of the adenovirus on tumor cells and tumor formation. The targeting of the adenovirus and the mechanism of tumor inhibition were discussed by in vivo imaging technology, HE staining and TUNEL assay. Recombinant adenovirus SG502-TNF targeted the tumor cells specifically with stable expression of TNF, which produced a killing effect on tumor cells by regulating the apoptotic signaling pathway. Recombinant adenovirus SG502-TNF possessed significant killing effect on TE-1 cells either in vivo or in vitro. This finding demonstrated the potential clinical application of adenovirus SG502.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

PubMed Central

Soares, Ricardo J; Maglieri, Giulia; Gutschner, Tony; Lund, Anders H; Nielsen, Boye S

2018-01-01

Abstract Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system. PMID:29059327

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-07-14

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

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

PubMed

Berndt, Benjamin; Haverkampf, Sonja; Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

PubMed Central

Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

Exogenous ACE2 Expression Allows Refractory Cell Lines To Support Severe Acute Respiratory Syndrome Coronavirus Replication

PubMed Central

Mossel, Eric C.; Huang, Cheng; Narayanan, Krishna; Makino, Shinji; Tesh, Robert B.; Peters, C. J.

2005-01-01

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression. PMID:15731278

Molecular characterization of immortalized normal and dysplastic oral cell lines.

PubMed

Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

2015-05-01

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

PubMed Central

Godlewska, Marlena; Krasuska, Wanda

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes. PMID:29513734

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

PubMed

Godlewska, Marlena; Krasuska, Wanda; Czarnocka, Barbara

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Molecular cloning and expression analysis of the canine chemokine receptor CCR9.

PubMed

Maeda, Shingo; Ohno, Koichi; Tsukamoto, Atsushi; Nakashima, Ko; Fukushima, Kenjiro; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

2012-01-15

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. Copyright © 2011 Elsevier B.V. All rights reserved.

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

PubMed

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

2009-01-01

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

Generation and Characterization of JCV Permissive Hybrid Cell Lines

PubMed Central

Sariyer, Ilker K.; Safak, Mahmut; Gordon, Jennifer; Khalili, Kamel

2009-01-01

JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents. PMID:19442856

Characterisation of the immune response to type I collagen in scleroderma

PubMed Central

Warrington, Kenneth J; Nair, Usha; Carbone, Laura D; Kang, Andrew H; Postlethwaite, Arnold E

2006-01-01

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSElow). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4+, activated (CD25+), memory (CD45RO+) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls. PMID:16879746

Immunohistochemical demonstration of epidermal growth factor in human gastric cancer xenografts of nude mice.

PubMed

Yoshiyuki, T; Shimizu, Y; Onda, M; Tokunaga, A; Kiyama, T; Nishi, K; Mizutani, T; Matsukura, N; Tanaka, N; Akimoto, M

1990-02-15

Thirty-two surgical specimens and three cell lines of human gastric cancers were used for subcutaneous transplantation into nude mice, resulting in the establishment of eight (25%) xenografts from the surgical specimens and two (67%) from the cell lines. The localization of epidermal growth factor (EGF) in the surgical specimens and cell lines of the gastric cancers and their xenografts in nude mice was then investigated immunohistochemically. Epidermal growth factor was stained in the cytoplasm of the cancer cells, being detected in 16 (50%) of the 32 surgical specimens and in all of the cell lines. Seven (44%) of the sixteen EGF-positive surgical specimens and one (6%) of the 16 EGF-negative ones were tumorigenic in nude mice. All of the xenografts in nude mice were positive for EGF. The tumorigenicity of human gastric cancer xenografts in nude mice may, therefore, be correlated with the presence of EGF in cancer cells.

[Anti-tumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line in vitro].

PubMed

Li, Maolan; Lu, Wei; Zhang, Fei; Ding, Qichen; Wu, Xiangsong; Tan, Zhujun; Wu, Wenguang; Weng, Hao; Wang, Xuefeng; Shi, Weibin; Dong, Ping; Gu, Jun; Liu, Yingbin

2014-11-04

To explore the antitumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line. DDP-PLLA-CNTs were prepared with the method of ultrasound emulsification. The morphology of DDP-PLLA-CNTs was determined by scanning electron microscope (SEM). And its drug loading and drug release curve in vitro was detected by UV-Vis-NIR spectrophotometer. CCK8 was used to test the cytotoxic effects of DDP-PLLA-CNTs at different concentrations on QBC939 cell proliferation.Flow cytometry was employed to measure the changes of apoptotic rate. With excellent controlled-release characteristic of in vitro drug release, DDP-PLLA-CNTs inhibited the proliferation and significantly increased the apoptotic rate of QBC939 cell line. DDP-PLLA-CNTs have drug sustained-release characteristics and can significantly inhibit the proliferation of QBC939 cell line.

Superhydrophobic surfaces allow probing of exosome self organization using X-ray scattering

NASA Astrophysics Data System (ADS)

Accardo, Angelo; Tirinato, Luca; Altamura, Davide; Sibillano, Teresa; Giannini, Cinzia; Riekel, Christian; di Fabrizio, Enzo

2013-02-01

Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates.Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34032e

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids

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

Yuan, Z.Q.; Gault, E.A.; Gobeil, P.

2008-04-10

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independentlymore » of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.« less

SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

PubMed Central

Lush, Mark E.; Piotrowski, Tatjana

2014-01-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer.

PubMed

Minker, Carole; Duban, Livine; Karas, Daniel; Järvinen, Päivi; Lobstein, Annelise; Muller, Christian D

2015-01-01

The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

[Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

PubMed

Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

2009-06-01

This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

[A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM].

PubMed

Yuan, W; Sui, C G; Ma, X; Ma, J

2018-05-23

Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day ( P <0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% ( P <0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field ( P <0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells ( P <0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level ( P <0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

Metabolic Imaging of Pancreatic Ductal Adenocarcinoma Detects Altered Choline Metabolism

PubMed Central

Penet, Marie-France; Shah, Tariq; Bharti, Santosh; Krishnamachary, Balaji; Artemov, Dmitri; Mironchik, Yelena; Wildes, Flonné; Maitra, Anirban; Bhujwalla, Zaver M.

2014-01-01

Purpose Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. The absence of early symptoms and effective treatments has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic targets. Experimental Design We investigated the metabolism of a panel of PDAC cell lines in culture and noninvasively in vivo with 1H magnetic resonance spectroscopic imaging (MRSI) to identify noninvasive biomarkers and uncover potential metabolic targets. Results We observed elevated choline-containing compounds in the PDAC cell lines and tumors. These elevated choline-containing compounds were easily detected by increased total choline (tCho) in vivo, in spectroscopic images obtained from tumors. Principal component analysis of the spectral data identified additional differences in metabolites between HPNE and neoplastic PDAC cells. Molecular characterization revealed overexpression of choline kinase (Chk)-α, choline transporter 1 (CHT1), and choline transporter-like protein 1 (CTL1) in the PDAC cell lines and tumors. Conclusions Collectively, these data identify new metabolic characteristics of PDAC and reveal potential metabolic targets. Total choline detected with 1H MRSI may provide an intrinsic, imaging-probe independent biomarker to complement existing techniques in detecting PDAC. The expression of Chk-α, CHT1, and CTL1 may provide additional molecular markers in aspirated cytological samples. PMID:25370468

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

PubMed

Franco, Marco E; Sutherland, Grace E; Lavado, Ramon

2018-04-01

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies. Copyright © 2018 Elsevier Inc. All rights reserved.

Improved detection of Bovine Viral Diarrhea Virus in Bovine lymphoid cell lines using PrimeFlow RNA assay

USDA-ARS?s Scientific Manuscript database

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

USDA-ARS?s Scientific Manuscript database

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. BVDV can be identified by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely di...

A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

Development and characterization of a new marine fish cell line from turbot (Scophthalmus maximus).

PubMed

Wang, N; Wang, X L; Sha, Z X; Tian, Y S; Chen, S L

2010-12-01

A new marine fish cell line, TK, derived from turbot (Scophthalmus maximus) kidney, was established by the method of trypsin digestion and subcultured for more than 50 passages over a period of 300 days. The TK cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with HEPES, antibiotics, fetal bovine serum (FBS), 2-Mercaptoethanol (2-Me), and basic fibroblast growth factor (bFGF). The suitable growth temperature for TK cells was 24°C, and microscopically, TK cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TK cell line has a normal diploid karyotype with 2n=44. Two fish viruses LCDV-C (lymphocystis disease virus from China) and TRBIV (turbot reddish body iridovirus) were used to determine the virus susceptibility of TK cell line. The TK cell line was found to be susceptible to TRBIV, and the infection was confirmed by cytopathic effect (CPE) and transmission electron microscopy, which detected the viral particles in the cytoplasm of virus-infected cells. Finally, significant green fluorescent signals were observed when the TK cells were transfected with pEGFP-N3 vector, indicating its potential utility for fish virus study and genetic manipulation.

JPRS Report, Science & Technology, China

DTIC Science & Technology

1991-01-04

B27 Recognizes an Allospecific Determinant of HLA - B27 Lymphoblastoid Cell Lines [Shi Bingiun, et al.; ZHONGHUA WEISHENGWUXUE HE MIANYIXUE ZAZHI, No 5...Antibody Produced by industrialization of bioengineering. Immunization With the Synthetic Peptide From HLA - B27 Recognizes an Allospecific Determinant...Direct Detection of Human Immunodeficiency of HLA - B27 Lymphoblastoid Cell Lines Virus (HIV) Gene by the Polymerase Chain 40091003C Beijing ZHONGHUA

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

PubMed Central

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

2012-01-01

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

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Pollak, A; Hengstschläger, M; Lubec, G

2006-10-01

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

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

PubMed

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

2004-05-01

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

The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture.

PubMed

Shioda, Setsuko; Kasai, Fumio; Ozawa, Midori; Hirayama, Noriko; Satoh, Motonobu; Kameoka, Yousuke; Watanabe, Ken; Shimizu, Norio; Tang, Huamin; Mori, Yasuko; Kohara, Arihiro

2018-02-01

Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

PubMed

Han, Xiang-ping; Shi, Gui-lan; Wang, Cheng-feng; Li, Jie; Zhang, Jian-wei; Zhang, Yu; Zhang, Shu-ren; Liu, Bin-lei

2012-12-01

To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

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

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

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

2012-06-29

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

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

PubMed

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

2000-07-31

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

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

PubMed Central

Knödgen, Eva; Göckeritz, Elisa; Vondey, Verena; Neumann, Lars; Herter, Sylvia; Klein, Christian; Hallek, Michael

2018-01-01

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism. PMID:29750146

Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

PubMed

Hoshino, Satoya; Kurotani, Reiko; Miyano, Yuki; Sakahara, Satoshi; Koike, Kanako; Maruyama, Minoru; Ishikawa, Fumio; Sakatai, Ichiro; Abe, Hiroyuki; Sakai, Takafumi

2014-06-01

We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

Differential regulation of interleukin-12 (IL-12), tumor necrosis factor alpha, and IL-1 beta production in human myeloid leukemia cell lines and peripheral blood mononuclear cells.

PubMed

Kubin, M; Chow, J M; Trinchieri, G

1994-04-01

Natural killer cell-stimulatory factor or interleukin-12 (NKSF/IL-12) was originally identified and purified from the conditioned medium of Epstein-Barr virus (EBV)-transformed B-cell lines. Phorbol diesters were observed to be potent stimulators of NKSF/IL-12 production from the B-cell lines. Although monocytes were found to be the major producers of NKSF/IL-12 in peripheral blood (PB) in response to lipopolysaccharide (LPS) or to Staphylococcus aureus, several myeloid leukemia cell lines tested did not produce detectable NKSF/IL-12 either constitutively or upon stimulation with phorbol diesters. However, three lines, ML-3, HL-60, and THP-1, responded to LPS with significant levels of NKSF/IL-12 production, whereas S aureus was effective only on THP-1 cells. When the cell lines were preincubated with compounds known to induce them to differentiate, production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta was in most cases maximal in cells with differentiated characteristics, whereas NKSF/IL-12 production in response to LPS in all three producing cell lines was significantly enhanced only by pretreatment with dimethylsulfoxide (DMSO) for 24 hours, or by costimulation with interferon gamma (IFN gamma). The efficiency of DMSO enhancement of NKSF/IL-12 production decreased after 2 to 5 days of incubation, when the cells acquired differentiated characteristics. Unlike DMSO, IFN gamma enhanced NKSF/IL-12 production, and IL-10 and dexamethasone inhibited it in cell lines and PB mononuclear cells stimulated by either LPS or S aureus. The ability of the cell lines to respond to these mediators of possibly physiologically relevant function provides a tissue-culture model for studying their mechanism of action.

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

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

PubMed Central

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

1992-01-01

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

Methylation Status of the RIZ1 Gene Promoter in Human Glioma Tissues and Cell Lines.

PubMed

Zhang, Chenran; Meng, Wei; Wang, Jiajia; Lu, Yicheng; Hu, Guohan; Hu, Liuhua; Ma, Jie

2017-08-01

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1), a strong tumor suppressor, is silenced in many human cancers. Our previous studies showed that RIZ1 expression was negatively correlated with the grade of glioma and was a key predictor of patient survival. Therefore, RIZ1 could be a potential tumor suppressor during glioma pathogenesis, although the mechanism underlying RIZ1 gene inactivation in gliomas is unknown. We investigated the methylation status of the RIZ1 promoter in human glioma tissues and four glioblastoma (GBM) cell lines, and verified the effect of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-CdR) on RIZ1 transcription and cell proliferation. Methylation-specific PCR (MSP) was performed to determine RIZ1 promoter methylation in human glioma specimens. The correlation between RIZ1 hypermethylation in tumors and clinicopathological features also was analyzed. 5-Aza-CdR treatment was used to reactivate gene expression silenced by hypermethylation in the U87 glioblastoma cell line, and real-time PCR was then used to measure RIZ1 expression. The ability of 5-aza-CdR to inhibit the proliferation of glioma cell lines whose RIZ1 promoters were hypermethylated was measured by bromodeoxyuridine (BrdU) incorporation. Among 51 human glioma specimens, RIZ1 promoter methylation was detected in 23 cases. Clinicopathological evaluation suggested that RIZ1 hypermethylation was negatively associated with tumor grade and patient age (P < 0.05). Hypermethylation of the RIZ1 promoter was detected in the U87 and U251 cell lines. RIZ1 mRNA expression in U87 cells was upregulated after treatment with 5-aza-Cdr, which correlated with inhibition of cell proliferation in a time- and concentration-dependent manner. Promoter hypermethylation may play an important role in the epigenetic silencing of RIZ1 expression in human glioma tissues and GBM cell lines.

Shock wave trauma leads to inflammatory response and morphological activation in macrophage cell lines, but does not induce iNOS or NO synthesis.

PubMed

Günther, Mattias; Plantman, Stefan; Gahm, Caroline; Sondén, Anders; Risling, Mårten; Mathiesen, Tiit

2014-12-01

Experimental CNS trauma results in post-traumatic inflammation for which microglia and macrophages are vital. Experimental brain contusion entails iNOS synthesis and formation of free radicals, NO and peroxynitrite. Shock wave trauma can be used as a model of high-energy trauma in cell culture. It is known that shock wave trauma causes sub-lytic injury and inflammatory activation in endothelial cells. Mechanical disruption of red blood cells can induce iNOS synthesis in experimental systems. However, it is not known whether trauma can induce activation and iNOS synthesis in inflammatory cell lines with microglial or macrophage lineage. We studied the response and activation in two macrophage cell lines and the consequence for iNOS and NO formation after shock wave trauma. Two macrophage cell lines from rat (NR8383) and mouse (RAW264.7) were exposed to shock wave trauma by the Flyer Plate method. The cellular response was investigated by Affymetrix gene arrays. Cell survival and morphological activation was monitored for 24 h in a Cell-IQ live cell imaging system. iNOS induction and NO synthesis were analyzed by Western blot, in cell Western IR-immunofluorescence, and Griess nitrite assay. Morphological signs of activation were detected in both macrophage cell lines. The activation of RAW264.7 was statistically significant (p < 0.05), but activation of NR8383 did not pass the threshold of statistical significance alpha (p > 0.05). The growth rate of idle cells was unaffected and growth arrest was not seen. Trauma did not result in iNOS synthesis or NO induction. Gene array analyses showed high enrichment for inflammatory response, G-protein coupled signaling, detection of stimulus and chemotaxis. Shock wave trauma combined with low LPS stimulation instead led to high enrichment in apoptosis, IL-8 signaling, mitosis and DNA-related activities. LPS/IFN-ɣ stimulation caused iNOS and NO induction and morphological activation in both cell lines. Shock wave trauma by the Flyer Plate method caused an inflammatory response and morphological signs of activation in two macrophage cell lines, while iNOS induction appeared to require humoral signaling by LPS/IFN-ɣ. Our findings indicated that direct energy transfer by trauma can activate macrophages directly without humoral mediators, which comprises a novel activation mechanism of macrophages.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-κB activation and promotes apoptosis in human leukaemia T-cell lines.

PubMed

Chen, Qian; Pang, Min-Hui; Ye, Xiao-Hong; Yang, Guang; Lin, Chen

2018-05-18

Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells. The Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting. Western blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively. Our data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

PubMed

da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R

2010-12-25

The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.

Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

DTIC Science & Technology

2001-08-01

Utilization of green fluorescent protein for the identification of metastasis in an in vivo breast cancer model system. In Preparation. REPRINTS OF ALL...phenotype. Utilizing the SUM-159PT cell line stably transfected with pEGFP-Ci (enhanced green fluorescent protein ) we have been able to successfully...accurately detected. To develop a model with enhanced resolution of micrometastases we created a stable cell line expressing green fluorescent protein

Development and characterization of an embryonic cell line from endangered endemic cyprinid Honmoroko Gnathopogon caerulescens (Sauvage, 1883).

PubMed

Higaki, Shogo; Shimada, Manami; Koyama, Yoshie; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

2015-09-01

Establishing a cell line from endemic species facilitates the cell biological research of these species in the laboratory. In this study, an epithelium-like cell line RME1 was established from the blastula-stage embryos of the critically endangered cyprinid Honmoroko Gnathopogon caerulescens, which is endemic to ancient Lake Biwa in Japan. To the best of our knowledge, this is the first embryonic cell line from an endangered fish species. This cell line is well adapted to grow at 28°C in the culture medium, which was successfully used for establishing testicular and ovarian cell lines of G. caerulescens, and has displayed stable growth over 60 passages since its initiation in June 2011. Although RME1 did not express the genes detected in blastula-stage embryos, such as oct4, sox2, nanog, and klf4, it showed a high euploidy rate (2n = 50; 67.2%) with normal diploid karyotype morphology, suggesting that RME1 retains the genomic organization of G. caerulescens and can prove to be a useful tool to investigate the unique properties of endangered endemic fishes at cellular level.

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

NASA Astrophysics Data System (ADS)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Parallel Monte Carlo Search for Hough Transform

NASA Astrophysics Data System (ADS)

Lopes, Raul H. C.; Franqueira, Virginia N. L.; Reid, Ivan D.; Hobson, Peter R.

2017-10-01

We investigate the problem of line detection in digital image processing and in special how state of the art algorithms behave in the presence of noise and whether CPU efficiency can be improved by the combination of a Monte Carlo Tree Search, hierarchical space decomposition, and parallel computing. The starting point of the investigation is the method introduced in 1962 by Paul Hough for detecting lines in binary images. Extended in the 1970s to the detection of space forms, what came to be known as Hough Transform (HT) has been proposed, for example, in the context of track fitting in the LHC ATLAS and CMS projects. The Hough Transform transfers the problem of line detection, for example, into one of optimization of the peak in a vote counting process for cells which contain the possible points of candidate lines. The detection algorithm can be computationally expensive both in the demands made upon the processor and on memory. Additionally, it can have a reduced effectiveness in detection in the presence of noise. Our first contribution consists in an evaluation of the use of a variation of the Radon Transform as a form of improving theeffectiveness of line detection in the presence of noise. Then, parallel algorithms for variations of the Hough Transform and the Radon Transform for line detection are introduced. An algorithm for Parallel Monte Carlo Search applied to line detection is also introduced. Their algorithmic complexities are discussed. Finally, implementations on multi-GPU and multicore architectures are discussed.

Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

PubMed

Schmitz, H

1981-01-01

An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

PubMed

Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

2016-10-18

BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

Convenient method of establishing permanent lines of xeroderma pigmentosum cells. [Ultraviolet radiation

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

Tohda, H.; Oikawa, A.; Katsuki, T.

Nine lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from four xeroderma pigmentosum (XP) patients, the parents of one XP patient, and three normal donors. All these cell lines proliferate as suspension in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal bovine serum, without detectable release of infectious Epstein-Barr virus. Some characteristics of these cell lines, such as growth rates, chromosome numbers, uv sensitivities, and activities of unscheduled DNA syntheses induced by uv, 4-nitroquinoline 1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine, were determined. Results confirm that the properties related to XP are not altered by transformation withmore » Epstein-Barr virus and are the same in degrees of defect as are those of dermal fibroblasts from the respective individuals. These XP and normal lymphoblastoid cell lines should be especially useful for biochemical studies on the mechanism of DNA repair, because they are easy to grow in mass culture.« less

Biometric assessment of prostate cancer's metastatic potential.

PubMed

Cooper, C R; Emmett, N; Harris-Hooker, S; Patterson, R; Cooke, D B

1994-01-01

Currently, no protocol exists that can assess the metastatic potential of prostate adenocarcinoma. The reason for this is partly due to the lack of information on cellular changes that result in a tumor cell's becoming metastatic. In this investigation, attempts were made to devise a method that correlated with the metastatic potential of AT-1, Mat-Lu, and Mat-LyLu cell lines of the Dunning R-3327 rat prostatic adenocarcinoma system. To accomplish this, we applied BioQuant biometric parameters, i.e., area, shape factor, and cell motility. AT-1 had a lower shape factor and a greater area as compared with the more highly metastatic Mat-Lu subline. No significant difference in area or shape factor was detected between the AT-1 cell line and the highly metastatic Mat-LyLu line. However, the lowly metastatic AT-1 line had less motility as compared with the Mat-Lu and Mat-LyLu lines. This study revealed that metastatic potential could be partially predicted via area and shape factor and accurately predicted via cell motility.

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

PubMed Central

2013-01-01

Background Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. PMID:23758893

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

PubMed

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

2004-11-18

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

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

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

Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk

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

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line

PubMed Central

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-01-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines. PMID:28810654

Roles of p38 and JNK protein kinase pathways activated by compound cantharidin capsules containing serum on proliferation inhibition and apoptosis of human gastric cancer cell line.

PubMed

Sun, Yonghao; Zhang, Dejuan; Mao, Mao; Lu, Yangping; Jiao, Ning

2017-08-01

The aim of the present study was to investigate the inhibitory effect of compound cantharides capsules (CCCs) on the viability and apoptosis of human gastric cancer cell lines, BGC-823 and SGC-7901, and to detect its regulation of gene expression levels, as well as its inhibition mechanisms. Each cell line was grouped into a control group, CCC serum group, 5-fluorouracil (5-FU) group, combination therapy group (CCC serum + 5-FU) and serum control group. Growth curves were measured and flow cytometry was used to detect cell apoptosis and cell viability. The mRNA expression level of proliferation-related C-MYC and p53 genes were assayed by reverse transcription-quantitative polymerase chain reaction. Protein phosphorylation levels of proliferating cell nuclear antigen, p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, c-Jun N-terminal kinase (JNK) and IκB were assayed by western blotting. The combined CCC serum and 5-FU group exhibited a higher inhibition rate in both cell lines and CCC serum therapy demonstrated a similar effect to 5-FU treatment, as demonstrated in the MTT and cell growth assay. Combined therapy significantly decreased the C-MYC mRNA expression levels and increased p53 mRNA expression levels (P<0.05). Combined therapy of 5-FU and CCC was more significant compared with CCC serum or 5-FU only (P<0.05). P38 and JNK-related protein phosphorylation are involved in apoptosis initiated by CCC combined 5-FU therapy. Combined therapy was able to significantly inhibit human gastric cancer cell growth (P<0.05), and advance cell apoptosis compared with CCC serum only. CCC serum resulted in downregulation of the c-Myc gene and upregulation of the p53 gene. p38 and JNK-related protein phosphorylation is involved in the inhibition of cell viability and apoptosis of human gastric cancer cell lines.

Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

PubMed Central

Carpenter, Erica L.; Rader, JulieAnn; Ruden, Jacob; Rappaport, Eric F.; Hunter, Kristen N.; Hallberg, Paul L.; Krytska, Kate; O’Dwyer, Peter J.; Mosse, Yael P.

2014-01-01

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients. PMID:25133137

BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma.

PubMed

Huang, Chen-Song; Zhai, Jing-Ming; Zhu, Xiao-Xu; Cai, Jian-Peng; Chen, Wei; Li, Jian-Hui; Yin, Xiao-Yu

2017-12-01

Our previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma (HCC) cell line. However, its clinical significances and biological impacts on HCC SP cells remained unclear. To investigate the prognostic value of BTG2 gene in HCC and its influences on cancer stem cells (CSCs)-like traits of HCC cell line SP cells. BTG2 expression in human HCC and adjacent non-cancerous tissues was detected by immunohistochemical staining and quantitative real-time PCR, and also obtained from GEO and TCGA data. Its prognostic values were assessed. Its biological influences on HCC cell line SP cells were evaluated using cell viability, cell cycle, plate clone-forming assay, and chemoresistance in vitro and tumorigenicity in vivo. BTG2 expression was significantly suppressed in human HCC compared to adjacent non-cancerous tissues. BTG2 expression was correlated with TNM stage, tumor size and vascular invasion. Lower expression of BTG2 was associated with poorer overall survival and disease-free survival. In vitro, overexpression of BTG2 substantially suppressed cell proliferation and accumulation of HCC cell line SP cells in G0/G1 phase. Colony formation ability was markedly suppressed by BTG2 overexpression. Moreover, sensitivity of HCC cell line SP cells to 5-fluorouracil was substantially increased by overexpression of BTG2. Furthermore, tumorigenicity of HCC cell line SP cells transfected with BTG2 plasmids was significantly reduced in vivo. BTG2 gene could regulate the CSC-like traits of HCC cell line SP cells, and it represented as a molecular prognostic marker for HCC.

Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines

PubMed Central

Zhao, Wei-Xiu; Wu, Zhen-Ming; Liu, Wei

2017-01-01

ABSTRACT Notch signaling pathways play important roles in cell fate and many diseases, including preeclampsia, the dysregulation of which may be the main cause of maternal mortality. This study aimed to investigate the roles of Notch2 and Notch3 in proliferation and invasion in trophoblast cell lines (BeWo and JAR). Small hairpin RNAs targeting Notch2/Notch3 and Notch2/Notch3-overexpression vectors were designed, constructed and transfected into BeWo and JAR cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were then used to detect Notch2 and Notch3 mRNA and protein levels, and confirm the efficiency of silence and overexpression. Flow cytometry assays were conducted to evaluate the cell cycle of the two cell lines, and transwell assays were used to detect migration and invasion. Western blot analysis was also performed to show the alteration of the cell lines' physiological activities at protein level. When Notch2 was downregulated in BeWo cells, proliferation was dramatically promoted, while migration and invasion were significantly inhibited. When Notch2 was upregulated in JAR cells, proliferation was inhibited, but migration and invasion were promoted. After overexpression of Notch3 in BeWo cells, proliferation was downregulated, but migration and invasion were both upregulated. By contrast, the silencing of Notch3 expression in JAR cells significantly enhanced proliferation, but suppressed migration and invasion. These data indicated that Notch2 and Notch3 mediate the invasion and migration of BeWo and JAR cells, and may play a potential role in early onset severe preeclampsia. PMID:28606936

Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines.

PubMed

Zhao, Wei-Xiu; Wu, Zhen-Ming; Liu, Wei; Lin, Jian-Hua

2017-08-15

Notch signaling pathways play important roles in cell fate and many diseases, including preeclampsia, the dysregulation of which may be the main cause of maternal mortality. This study aimed to investigate the roles of Notch2 and Notch3 in proliferation and invasion in trophoblast cell lines (BeWo and JAR). Small hairpin RNAs targeting Notch2/Notch3 and Notch2/Notch3-overexpression vectors were designed, constructed and transfected into BeWo and JAR cells. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were then used to detect Notch2 and Notch3 mRNA and protein levels, and confirm the efficiency of silence and overexpression. Flow cytometry assays were conducted to evaluate the cell cycle of the two cell lines, and transwell assays were used to detect migration and invasion. Western blot analysis was also performed to show the alteration of the cell lines' physiological activities at protein level.When Notch2 was downregulated in BeWo cells, proliferation was dramatically promoted, while migration and invasion were significantly inhibited. When Notch2 was upregulated in JAR cells, proliferation was inhibited, but migration and invasion were promoted. After overexpression of Notch3 in BeWo cells, proliferation was downregulated, but migration and invasion were both upregulated. By contrast, the silencing of Notch3 expression in JAR cells significantly enhanced proliferation, but suppressed migration and invasion. These data indicated that Notch2 and Notch3 mediate the invasion and migration of BeWo and JAR cells, and may play a potential role in early onset severe preeclampsia. © 2017. Published by The Company of Biologists Ltd.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

PubMed Central

Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wang, Hong; Fu, Hongyong; Zhou, Fan; Yao, Chencheng; Wang, Xiaobo; Li, Zheng; He, Zuping

2017-01-01

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine. PMID:28152522

Towards the elaboration of new gold-based optical theranostics.

PubMed

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

2015-03-21

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

[Establishment and characterization of a new carcinoma cell line from uterine cervix of Uyghur women].

PubMed

Zhang, Lu; Aerziguli, Tursun; Guzalnur, Abliz

2012-04-01

To establish a uterine cervical carcinoma cell line of Uyghur ethnical background and to evaluate the related biological characteristics for future biomedical investigations of diseases in the Uyghur population. Poorly-differentiated squamous cell carcinoma specimens of Uyghur patients were obtained and cultured in vitro by enzymatic digestion method, followed by continuous passaging to reach a stable growth determined by cell viability and growth curve. Morphological study, cell cycling and chromosomal analysis were performed. Tumorigenesis study was conducted by inoculation of nude mice. Biomarker (CK17, CD44, Ki-67, CK14 and vimentin) expression was detected by immunofluorescence and immunocytochemical techniques. A cervical carcinoma cell line was successfully established and maintained for 12 months through 70 passages. The cell line had a stable growth with a population doubling time of 51.9 h. Flask method and double agar-agar assay showed that the cell line had colony-forming rates of 32.5% and 15.6%, respectively. Ultrastructural evaluation demonstrated numerous cell surface protrusions or microvilli, a large number of rod-shape structures in cytoplasm, typical desmosomes and nuclear atypia. Chromosomal analysis revealed human karyotype with the number of chromosomes per cell varying from 32 - 97 with a majority of 54 - 86 (60.3%). Xenogeneic tumors formed in nude mice showed histological structures identical to those of the primary tumor. The cells had high expression of CK17, CD44, Ki-67 and vimentin but no CK14 expression. A cervical carcinoma cell line from a female Uyghur patient is successfully established. The cell line has the characteristics of human cervical squamous cell carcinoma, and it is stable with maintaining the characteristic biological and morphological features in vitro for more than 12 months, therefore, qualified as a stable cell line for further biomedical research.

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

PubMed

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.

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing

PubMed Central

Yaqoob, Zahid; Choi, Wonshik; Oh, Seungeun; Lue, Niyom; Park, Yongkeun; Fang-Yen, Christopher; Dasari, Ramachandra R.; Badizadegan, Kamran; Feld, Michael S.

2010-01-01

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell. PMID:19550464

Silencing of CXCR4 inhibits tumor cell proliferation and neural invasion in human hilar cholangiocarcinoma.

PubMed

Tan, Xin-Yu; Chang, Shi; Liu, Wei; Tang, Hui-Huan

2014-03-01

To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

Expression of gap junction genes connexin 32 and connexin 43 mRNAs and proteins, and their role in hepatocarcinogenesis

PubMed Central

Ma, Xiang-Dong; Ma, Xing; Sui, Yan-Fang; Wang, Wen-Liang

2002-01-01

AIM: To investigate the relationship between hepatocarcinogenesis and the expression of connexin32 (cx32), connexin43 (cx43) mRNAs and proteins in vitro. METHODS: Gap junction genes cx32 and cx43 mRNA in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal liver cell line QZG were detected by in situ hybridization (ISH) with digoxin-labeled cx32, and cx43 cDNA probes. Expression of Cx32 and Cx43 proteins in the cell lines was revealed by indirect immuno-fluorescence and flow cytometry (FCM). RESULTS: Blue positive hybridization signals of cx32 and cx43 mRNAs detected by ISH with cx32 and cx43 cDNA probes respectively were located in cytoplasm of cells of HHCC, SMMC-7721 and QZG. No significant difference of either cx32 mRNA or cx43 mRNA was tested among HHCC, SMMC-7721 and QZG (P = 2.673, HHCC vs QZG; P = 1.375, SMMC-7721 vs QZG). FCM assay showed that the positive rates of Cx32 protein in HHCC, SMMC-7721 and QZG were 0.7%, 1.7% and 99.0%, and the positive rates of Cx43 protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5% and 99.1% respectively. Significant differences of both Cx32 and Cx43 protein expression existed between hepatocellular carcinoma cell lines and normal liver cell line (P = 0.0069, HHCC vs QZG; P = 0.0087, SMMC-7721 vs QZG). Moreover, the fluorescent intensities of Cx32 and Cx43 proteins in HHCC, SMMC-7721 were lower than that in QZG. CONCLUSION: Hepatocellular carcinoma cell lines HHCC and SMMC-7721 exhibited lower positive rates and fluorescent intensities of Cx32, Cx43 proteins compared with that of normal liver cell line QZG. It is suggested that lower expression of both Cx32 and Cx43 proteins in hepatocellular carcinoma cells could play pivotal roles in the hepatocarcinogenesis. Besides, genetic defects of cx32 and cx43 in post-translational processing should be considered. PMID:11833073

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

PubMed

Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

2016-03-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

PubMed Central

KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

2016-01-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

Chromosome 2 short arm translocations revealed by M-FISH analysis of neuroblastoma cell lines.

PubMed

Van Roy, N; Van Limbergen, H; Vandesompele, J; Van Gele, M; Poppe, B; Laureys, G; De Paepe, A; Speleman, F

2000-12-01

M-FISH analysis was performed on 18 neuroblastoma cell lines, which were previously studied with cytogenetic, standard FISH and CGH data. One of the most striking findings of this study was the detection of chromosome 2 short arm rearrangements in 61% of the investigated cell lines. These rearrangements resulted from translocations with various partner chromosomes. All translocations, except one were unbalanced, leading to the consistent gain of chromosome segment 2pter-p22. A cryptic balanced translocation t(2;4) was observed with a breakpoint located in the vicinity of MYCN in cell line NBL-S. Combination of M-FISH results together with cytogenetic, standard FISH and CGH data yielded the most comprehensive description of chromosome 2 short arm rearrangements, leading to a consistent gain of chromosome 2 short arm material. Copyright 2000 Wiley-Liss, Inc.

[Effects of fasudil on bleomycin-induced pulmonary fibrosis in mice and on the biological behaviors in NIH3T3 mouse fibroblast cell line].

PubMed

Jiang, Chunguo; Huang, Hui; Liu, Jia; Wang, Yanxun; Zhao, Yuyue; Xu, Zuojun

2014-09-01

To determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line. The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue. NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell. The proliferation activity in NIH3T3 cells were detected by MTT assay and flat colony forming experiment. The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment. The expression of CyclinD1, MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR. The expression of CyclinD1, MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot. Compare to the mice administrated by bleomycin, the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil. Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells. The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2. Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

A high-throughput flow cytometry-on-a-CMOS platform for single-cell dielectric spectroscopy at microwave frequencies.

PubMed

Chien, Jun-Chau; Ameri, Ali; Yeh, Erh-Chia; Killilea, Alison N; Anwar, Mekhail; Niknejad, Ali M

2018-06-06

This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies. Compared with MHz impedance cytometers, operating at GHz frequencies offers direct intracellular permittivity probing due to electric fields penetrating through the cellular membrane. To overcome the detection challenges at high frequencies, the spectrometer employs on-chip oscillator-based sensors, which embeds simultaneous frequency generation, electrode excitation, and signal detection capabilities. By employing an injection-locking phase-detection technique, the spectrometer offers state-of-the-art sensitivity, achieving a less than 1 aFrms capacitance detection limit (or 5 ppm in frequency-shift) at a 100 kHz noise filtering bandwidth, enabling high throughput (>1k cells per s), with a measured cellular SNR of more than 28 dB. With CMOS/microfluidics co-design, we distribute four sensing channels at 6.5, 11, 17.5, and 30 GHz in an arrayed format whereas the frequencies are selected to center around the water relaxation frequency at 18 GHz. An issue in the integration of CMOS and microfluidics due to size mismatch is also addressed through introducing a cost-efficient epoxy-molding technique. With 3-D hydrodynamic focusing microfluidics, we perform characterization on four different cell lines including two breast cell lines (MCF-10A and MDA-MB-231) and two leukocyte cell lines (K-562 and THP-1). After normalizing the higher frequency signals to the 6.5 GHz ones, the size-independent dielectric opacity shows a differentiable distribution at 17.5 GHz between normal (0.905 ± 0.160, mean ± std.) and highly metastatic (1.033 ± 0.107) breast cells with p ≪ 0.001.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

PubMed

Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

2011-08-15

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

First laboratory detection of an absorption line of the first overtone electric quadrupolar band of N2 by CRDS near 2.2 μm

NASA Astrophysics Data System (ADS)

Čermák, P.; Vasilchenko, S.; Mondelain, D.; Kassi, S.; Campargue, A.

2017-01-01

The extremely weak 2-0 O(14) electric quadrupole transition of N2 has been detected by very high sensitivity Cavity Ring Down spectroscopy near 4518 cm-1. It is the first N2 absorption line in the first overtone band reported so far from laboratory experiments. By combining a feedback narrowed Distributed Feedback laser diode with a passive cell tracking technique, a limit of detection of αmin ∼ 1.2 × 10-11 cm-1 was achieved after one day of spectra averaging. The N2 2-0 O(14) line position and line intensity (about 1.5 × 10-30 cm/molecule) agree with calculated values provided in the HITRAN2012 database.

Radiosensitivity profiles from a panel of ovarian cancer cell lines exhibiting genetic alterations in p53 and disparate DNA-dependent protein kinase activities

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

Langland, Gregory T.; Yannone, Steven M.; Langland, Rachel A.

2009-09-07

The variability of radiation responses in ovarian tumors and tumor-derived cell lines is poorly understood. Since both DNA repair capacity and p53 status can significantly alter radiation sensitivity, we evaluated these factors along with radiation sensitivity in a panel of sporadic human ovarian carcinoma cell lines. We observed a gradation of radiation sensitivity among these sixteen lines, with a five-fold difference in the LD50 between the most radiosensitive and the most radioresistant cells. The DNA-dependent protein kinase (DNA-PK) is essential for the repair of radiation induced DNA double-strand breaks in human somatic cells. Therefore, we measured gene copy number, expressionmore » levels, protein abundance, genomic copy and kinase activity for DNA-PK in all of our cell lines. While there were detectable differences in DNA-PK between the cell lines, there was no clear correlation with any of these differences and radiation sensitivity. In contrast, p53 function as determined by two independent methods, correlated well with radiation sensitivity, indicating p53 mutant ovarian cancer cells are typically radioresistant relative to p53 wild-type lines. These data suggest that the activity of regulatory molecules such as p53 may be better indicators of radiation sensitivity than DNA repair enzymes such as DNAPK in ovarian cancer.« less

Construction of carbon nanotube based nanoarchitectures for selective impedimetric detection of cancer cells in whole blood.

PubMed

Liu, Yang; Zhu, Fanjiao; Dan, Wangxia; Fu, Yu; Liu, Shaoqin

2014-10-21

A carbon nanotube (CNT) based nanoarchitecture is developed for rapid, sensitive and specific detection of cancer cells by using real time electrical impedance sensing. The sensor is constructed with carbon nanotube (CNT) multilayers and EpCAM (epithelial cell adhesion molecule) antibodies, which are assembled on an indium tin oxide (ITO) electrode surface. The binding of tumor cells to EpCAM antibodies causes increase of the electron-transfer resistance. The electrochemical impedance of the prepared biosensors is linear with the logarithm of concentration of the liver cancer cell line (HepG2) within the concentration range of 10 to 10(5) cells per mL. The detection limit for HepG2 cells is 5 cells per mL. The proposed impedimetric sensing devices allow for sensitive and specific detection of cancer cells in whole-blood samples without any sample pretreatment steps.

Sensory hair cell regeneration in the zebrafish lateral line.

PubMed

Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer

PubMed Central

Minker, Carole; Duban, Livine; Karas, Daniel; Järvinen, Päivi; Lobstein, Annelise; Muller, Christian D.

2015-01-01

Scope. The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. Methods and Results. Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. Conclusion. We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines. PMID:26180579

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Performance comparison of two androgen receptor splice variant 7 (AR-V7) detection methods.

PubMed

Bernemann, Christof; Steinestel, Julie; Humberg, Verena; Bögemann, Martin; Schrader, Andres Jan; Lennerz, Jochen K

2018-01-23

To compare the performance of two established androgen receptor splice variant 7 (AR-V7) mRNA detection systems, as paradoxical responses to next-generation androgen-deprivation therapy in AR-V7 mRNA-positive circulating tumour cells (CTC) of patients with castration-resistant prostate cancer (CRPC) could be related to false-positive classification using detection systems with different sensitivities. We compared the performance of two established mRNA-based AR-V7 detection technologies using either SYBR Green or TaqMan chemistries. We assessed in vitro performance using eight genitourinary cancer cell lines and serial dilutions in three AR-V7-positive prostate cancer cell lines, as well as in 32 blood samples from patients with CRPC. Both assays performed identically in the cell lines and serial dilutions showed identical diagnostic thresholds. Performance comparison in 32 clinical patient samples showed perfect concordance between the assays. In particular, both assays determined AR-V7 mRNA-positive CTCs in three patients with unexpected responses to next-generation anti-androgen therapy. Thus, technical differences between the assays can be excluded as the underlying reason for the unexpected responses to next-generation anti-androgen therapy in a subset of AR-V7 patients. Irrespective of the method used, patients with AR-V7 mRNA-positive CRPC should not be systematically precluded from an otherwise safe treatment option. © 2018 The Authors BJU International © 2018 BJU International Published by John Wiley & Sons Ltd.

Experimental verification of a predicted novel microRNA located in human PIK3CA gene with a potential oncogenic function in colorectal cancer.

PubMed

Saleh, Ali Jason; Soltani, Bahram M; Dokanehiifard, Sadat; Medlej, Abdallah; Tavalaei, Mahmoud; Mowla, Seyed Javad

2016-10-01

PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro.

PubMed

Griegel, S; Hong, C; Frötschl, R; Hülser, D F; Greger, V; Horsthemke, B; Rajewsky, M F

1990-07-15

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

A novel packaging system for the generation of helper-free oncolytic MVM vector stocks.

PubMed

Brandenburger, A; Russell, S

1996-10-01

MVM-based autonomous parvoviral vectors have been shown to target the expression of heterologous genes in neoplastic cells and are therefore of interest for cancer gene therapy. The traditional method for production of parvoviral vectors requires the cotransfection of vector and helper plasmids into MVM-permissive cell lines, but recombination between the cotransfected plasmids invariably gives rise to vector stocks that are heavily contaminated with wild-type MVM. Therefore, to minimise recombination between the vector and helper genomes we have utilised a cell line in which the MVM helper functions are expressed inducibly from a modified MVM genome that is stably integrated into the host cell chromosome. Using this MVM packaging cell line, we could reproducibly generate MVM vector stocks that contained no detectable helper virus.

Spectral line discriminator for passive detection of fluorescence

NASA Technical Reports Server (NTRS)

Kebabian, Paul L. (Inventor)

1996-01-01

A method and apparatus for detecting fluorescence from sunlit plants is based on spectral line discrimination using the A-band and B-band absorption of atmospheric oxygen. Light from a plant including scattered sunlight and the fluorescence from chlorophyll is passed through a chopper into a cell containing low-pressure, high-purity oxygen. A-band or B-band wavelengths present in the light are absorbed by the oxygen in the cell. When the chopper is closed, the absorbed light is remitted as fluorescence into a detector. The intensity of the fluorescence from the oxygen is proportional to the intensity of fluorescence from the plant.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach.

PubMed

Guruceaga, Elizabeth; Garin-Muga, Alba; Prieto, Gorka; Bejarano, Bartolomé; Marcilla, Miguel; Marín-Vicente, Consuelo; Perez-Riverol, Yasset; Casal, J Ignacio; Vizcaíno, Juan Antonio; Corrales, Fernando J; Segura, Victor

2017-12-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations.

Enhanced Missing Proteins Detection in NCI60 Cell Lines Using an Integrative Search Engine Approach

PubMed Central

2017-01-01

The Human Proteome Project (HPP) aims deciphering the complete map of the human proteome. In the past few years, significant efforts of the HPP teams have been dedicated to the experimental detection of the missing proteins, which lack reliable mass spectrometry evidence of their existence. In this endeavor, an in depth analysis of shotgun experiments might represent a valuable resource to select a biological matrix in design validation experiments. In this work, we used all the proteomic experiments from the NCI60 cell lines and applied an integrative approach based on the results obtained from Comet, Mascot, OMSSA, and X!Tandem. This workflow benefits from the complementarity of these search engines to increase the proteome coverage. Five missing proteins C-HPP guidelines compliant were identified, although further validation is needed. Moreover, 165 missing proteins were detected with only one unique peptide, and their functional analysis supported their participation in cellular pathways as was also proposed in other studies. Finally, we performed a combined analysis of the gene expression levels and the proteomic identifications from the common cell lines between the NCI60 and the CCLE project to suggest alternatives for further validation of missing protein observations. PMID:28960077

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

PubMed

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

2016-04-30

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

Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

PubMed

Cavnar, Peter; Inman, Kristina

2015-01-01

HS1-associated protein X-1 (Hax1) is a 35 kDa protein that is ubiquitously expressed. Hax1 is an anti-apoptotic protein with additional roles in cell motility, and autosomal recessive loss of Hax1 results in Kostmann syndrome, a form of severe congenital neutropenia. Because of the important role of Hax1 in neutrophils we demonstrate here validation of two commercially available research antibodies directed against human Hax1 in the human myeloid leukemia cell line PLB-985 cells. We show that both the mouse anti-Hax1 monoclonal IgG directed against amino acids 10-148 of Hax1 and a rabbit anti-Hax1 polyclonal IgG antibody directed against full-length Hax1 reliably and consistently detect Hax1 during immunoblotting of three different PLB-985 cell densities. Using shRNA mediated Hax1 knockdown, we demonstrate the specificity of both Hax1 antibodies. In addition, our results suggest that the rabbit anti-Hax1 polyclonal antibody provides a stronger intensity in detecting Hax1 protein, with detection in as few as 0.1 x 10 (6) cells in 6 total replicates we have performed.

Novel cell-based odorant sensor elements based on insect odorant receptors.

PubMed

Mitsuno, Hidefumi; Sakurai, Takeshi; Namiki, Shigehiro; Mitsuhashi, Hiroyuki; Kanzaki, Ryohei

2015-03-15

Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management. Copyright © 2014 Elsevier B.V. All rights reserved.

A long noncoding RNA UCA1 promotes proliferation and predicts poor prognosis in glioma.

PubMed

Zhao, W; Sun, C; Cui, Z

2017-06-01

Acting as a proto-oncogene, long noncoding RNAs (lncRNAs) urothelial carcinoembryonic antigen 1 (UCA1) plays a key role in the occurrence and development of several human tumors. However, the expression and biological functions of UCA1 in glioma are less known. This study discussed the expression of UCA1 in glioma and its effect on the proliferation and cell cycle of glioma cells. LncRNA UCA1 expressions in 64 glioma samples (Grade I-II in 22 cases and Grade III-IV in 42 cases, according to WHO criteria) and 10 normal brain samples were detected using real-time fluorescence quantitative PCR. On this basis, the correlations of UCA1 to clinicopathological characteristics and prognosis of glioma were assessed. Then, using qPCR, the lncRNA UCA1 expressions in glioma cell lines and astrocytes were detected. UCA1-overexpressing glioma cell lines U87 and U251 were further detected after siRNA transfection of these two cell lines, and the impact on cell proliferation and cell cycle was assessed with CCK-8 (cell counting kit-8) assay and flow cytometry method (FCM), respectively. The expression of cyclin D1, a cell cycle-related protein, was detected using Western Blot. LncRNA UCA1 expression in the glioma samples was obviously higher as compared with the normal brain samples (P < 0.001), and the expression was correlated significantly with grading of the tumors (P < 0.05). However, lncRNA UCA1 expression was not correlated with age, gender, tumor size and KPS score (P > 0.05). After interference of UCA1 expression by siRNA transfection, the proliferation of both U251 and SHG-44 cells was inhibited (P < 0.05), with more cells arrested in G0/G1 (P < 0.05). Moreover, cyclin D1 expression was also downregulated considerably. LncRNA UCA1 can promote the proliferation and cell cycle progression of glioma cells by upregulating cyclin D1 transcription. So UCA1 may serve as an independent prognostic indicator and a novel therapeutic target for glioma.

O-naphthoquinone isolated from Capraria biflora L. induces selective cytotoxicity in tumor cell lines.

PubMed

de S Wisintainer, G G N; Scola, G; Moura, S; Lemos, T L G; Pessoa, C; de Moraes, M O; Souza, L G S; Roesch-Ely, M; Henriques, J A P

2015-12-21

Biflorin is an o-naphthoquinone isolated from the roots of the plant Capraria biflora L. (Scrophulariaceae). In this study, the cytotoxic effects of biflorin were verified, and late apoptosis was detected in various cancer cell lines by in situ analysis. The cytotoxicity was further evaluated exclusively for 48 h of treatment in different tumor and non-tumor cell lines (Hep-2, HeLa, HT-29, A-375, and A-549, and HEK-293, respectively). The results indicated that biflorin induced selective cytotoxicity in tumor cells. HeLa cells were more susceptible to biflorin, followed by HT-29, A-549, A-375, and Hep-2 at all concentrations (range 5-50 μg/mL), and the highest half-maximal inhibitory concentration IC50 (56.01 ± 1.17 μg/mL) was observed in HEK-293 cells. Late apoptotic/necrotic events, observed by in situ immunostaining with Annexin V, varied with each cell line; an increase in late apoptotic events was observed corresponding to the increase in biflorin dosage. Hep-2 cells showed a greater percentage of late apoptotic events among the tumor cell lines when treated with higher concentrations of biflorin (69.63 ± 2.28%). The non-tumor HEK-293 line showed greater resistance to late apoptotic events, as well as a lower level of cytotoxicity (77.69 ± 6.68%) than the tested tumor lines. The data presented indicate that biflorin showed an important, possibly selective, cytotoxicity against tumor cell lines, thereby revealing a promising novel substance with potential anticancer activity for tumor therapy.

In vitro evaluation of a specific radiochemical compound based on 99mTc-labeled DARPinG3 for radionuclide imaging of tumors overexpressing Her-2/neu

NASA Astrophysics Data System (ADS)

Bragina, O.; Larkina, M.; Stasyuk, E.; Chernov, V.; Zelchan, R.; Medvedeva, A.; Sinilkin, I.; Yusubov, M.; Skuridin, V.; Deyev, S.; Buldakov, M.

2017-09-01

It is still necessary to search for new informative diagnostic methods to detect malignant tumors with overexpression of Her-2/neu, which are characterized by the aggressive course of the disease, rapid rate of tumor growth and low rates of relapse-free and overall survival. In recent years, the radioisotope techniques for detection of specific tumor targets have been developing actively. Purpose: to develop a chemically stable radiochemical compound for the targeted imaging of cells overexpressing Her-2/neu. Material and methods: The study was performed using 2 cell lines. The human breast adenocarcinoma HER2-overexpressing cell line BT-474 was chosen to detect specific binding. As a control, HER2-negative human breast adenocarcinoma MCF-7 was used. The human breast adenocarcinoma BT-474 and MCF-7 cell lines were seeded in chamber-slides at the density of 35,000 cells/ml in trypsin-EDTA (PanEco) medium and grown overnight at 37°C. After that both cell lines were washed with Phosphate buffered saline (PBS) and distributed into test tubes to 1 ml (5 millions cells in each). After adding 100 µl (70 MBq) studied complex of 99mTc-DPAH- DARPinG3 was incubated for 40 min at +4°C. Washing was performed three times with buffer PBS and 5% Bovine Serum Albumin (BSA). The characteristics of the binding specificity of the test set with the HER-2/neu receptor were determined by direct radiometric and planar scintigraphy. Nonparametric Mann-Whitney test was used to assess the differences in the quantitative characteristics between groups. Results: The output of the labeled complex was more than 91%, with a radiochemical purity of more than 94%. When carrying out a visual scintigraphic assessment much greater intensity accumulation of radiotracer was observed in the studied cell culture surface receptor overexpressing Her-2/neu. The results of direct radiometric also showed higher accumulation of the radiopharmaceutical in the adenocarcinoma cell line BT-474 human breast cancer overexpressing Her-2/neu compared to the control group. Conclusion: The preclinical studies demonstrated a high in vitro stability of the study compound, as well as its accumulation in the cell group overexpressing Her-2/neu.

Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity.

PubMed

Saupe, Madeleine; Rauschenberger, Lisa; Preuß, Melanie; Oswald, Stefan; Fussek, Sebastian; Zimmermann, Uwe; Walther, Reinhard; Knabbe, Cornelius; Burchardt, Martin; Stope, Matthias B

2015-10-01

The development of a drug-resistant phenotype is the major challenge during treatment of castration-resistant prostate cancer (PC). In solid cancer entities, one of the major contributors to chemoresistance is the multidrug resistance 1 (MDR1) protein. Believed to be involved in the induction of MDR1 expression is the presence of anticancer drugs as well as the Y box binding protein 1 (YB-1). Basal as well as drug-induced expression of MDR1 in established PC cell lines was assessed by Western blotting and mass spectrometry. Subsequently, the influence of YB-1 on MDR1 expression was examined via transient overexpression of YB-1. While LNCaP and PC-3 cells showed no detectable amounts of MDR1, the resistance factor was found to be expressed in 22Rv1 cells. Despite this difference, all three cell lines demonstrated similar growth behavior in the presence of the first-line chemotherapeutic agent docetaxel. Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and abiraterone did not significantly alter MDR1 expression levels. Furthermore, overexpression of the MDR1 controlling factor YB-1 showed no impact on MDR1 expression levels. MDR1 was detectable in the PC cell line 22Rv1. However, this study suggests that MDR1 is of less importance for drug resistance in PC cells than in other types of solid cancer. Furthermore, in contrast to YB-1 properties in other malignancies, MDR1 regulation through YB-1 seems to be unlikely.

Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

PubMed

Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

1996-03-01

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

PubMed

Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

2012-10-12

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties.

PubMed

Chiba, Tetsuhiro; Kita, Kaoru; Zheng, Yun-Wen; Yokosuka, Osamu; Saisho, Hiromitsu; Iwama, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2006-07-01

Recent advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs. In this study, we applied side population (SP) cell analysis and sorting to established hepatocellular carcinoma (HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis. Among four cell lines analyzed, SP cells were detected in Huh7 (0.25%) and PLC/PRF/5 cells (0.80%), but not in HepG2 and Huh6 cells. SP cells demonstrated high proliferative potential and anti-apoptotic properties compared with those of non-SP cells. Immunocytochemistry examination showed that SP fractions contain a large number of cells presenting characteristics of both hepatocyte and cholangiocyte lineages. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that only 1 x 10(3) SP cells were sufficient for tumor formation, whereas an injection of 1 x 10(6) non-SP cells did not initiate tumors. Re-analysis of SP cell-derived tumors showed that SP cells generated both SP and non-SP cells and tumor-initiating potential was maintained only in SP cells in serial transplantation. Microarray analysis discriminated a differential gene expression profile between SP and non-SP cells, and several so-called "stemness genes" were upregulated in SP cells in HCC cells. In conclusion, we propose that a minority population, detected as SP cells in HCC cells, possess extreme tumorigenic potential and provide heterogeneity to the cancer stem cell system characterized by distinct hierarchy.

Comparison of Normal and Breast Cancer Cell lines using Proteome, Genome and Interactome data

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

Patwardhan, Anil J.; Strittmatter, Eric F.; Camp, David G.

2005-12-01

Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of both protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled the confident identification of 2,235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multi-step search strategy was used to identify post-translational modifications and detected several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal andmore » cancer cell lines.« less

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

PubMed

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

2009-11-02

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

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Meerovich, Irina G.; Arslanbaeva, Lyaisan R.; Jerdeva, Viktoria V.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fiks, Ilya I.

2007-02-01

Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically. Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fix, Ilya I.; Popov, Vladimir O.

2007-07-01

Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Imiquimod activates p53-dependent apoptosis in a human basal cell carcinoma cell line.

PubMed

Huang, Shi-Wei; Chang, Shu-Hao; Mu, Szu-Wei; Jiang, Hsin-Yi; Wang, Sin-Ting; Kao, Jun-Kai; Huang, Jau-Ling; Wu, Chun-Ying; Chen, Yi-Ju; Shieh, Jeng-Jer

2016-03-01

The tumor suppressor p53 controls DNA repair, cell cycle, apoptosis, autophagy and numerous other cellular processes. Imiquimod (IMQ), a synthetic toll-like receptor (TLR) 7 ligand for the treatment of superficial basal cell carcinoma (BCC), eliminates cancer cells by activating cell-mediated immunity and directly inducing apoptosis and autophagy in cancer cells. To evaluate the role of p53 in IMQ-induced cell death in skin cancer cells. The expression, phosphorylation and subcellular localization of p53 were detected by real-time PCR, luciferase reporter assay, cycloheximide chase analysis, immunoblotting and immunocytochemistry. Using BCC/KMC1 cell line as a model, the upstream signaling of p53 activation was dissected by over-expression of TLR7/8, the addition of ROS scavenger, ATM/ATR inhibitors and pan-caspase inhibitor. The role of p53 in IMQ-induced apoptosis and autophagy was assessed by genetically silencing p53 and evaluated by a DNA content assay, immunoblotting, LC3 puncta detection and acridine orange staining. IMQ induced p53 mRNA expression and protein accumulation, increased Ser15 phosphorylation, promoted nuclear translocation and up-regulated its target genes in skin cancer cells in a TLR7/8-independent manner. In BCC/KMC1 cells, the induction of p53 by IMQ was achieved through increased ROS production to stimulate the ATM/ATR-Chk1/Chk2 axis but was not mediated by inducing DNA damage. The pharmacological inhibition of ATM/ATR significantly suppressed IMQ-induced p53 activation and apoptosis. Silencing of p53 significantly decreased the IMQ-induced caspase cascade activation and apoptosis but enhanced autophagy. Mutant p53 skin cancer cell lines were more resistant to IMQ-induced apoptosis than wildtype p53 skin cancer cell lines. IMQ induced ROS production to stimulate ATM/ATR pathways and contributed to p53-dependent apoptosis in a skin basal cell carcinoma cell line BCC/KMC1. Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

PubMed

Zhang, Li-Yong; Xing, Tao; Du, Li-Xin; Li, Qing-Min; Liu, Wei-Dong; Wang, Ji-Yue; Cai, Jing

2015-01-01

Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

Dihydroartemisinin increases temozolomide efficacy in glioma cells by inducing autophagy

PubMed Central

ZHANG, ZE-SHUN; WANG, JING; SHEN, YOU-BI; GUO, CHENG-CHENG; SAI, KE; CHEN, FU-RONG; MEI, XIN; HAN, FU; CHEN, ZHONG-PING

2015-01-01

Artemisinin, a powerful antimalarial medicine, is extracted from the Chinese herb, Artemisia annua L., and has the ability to inhibit the proliferation of cancer cells. Dihydroartemisinin (DHA), the major active metabolite of artemisinin, is able to inhibit the growth of a variety of types of human cancer. However, the effect of DHA on human glioma cells remains unclear. The aim of the present study was to investigate the effect of DHA on the proliferation of glioma cells, and whether DHA was able to enhance temozolomide (TMZ) sensitivity in vitro and in vivo. In total, 10 human glioma cell lines were used to analyze the growth inhibition ability of DHA by MTT assay. The typical autophagic vacuoles were monitored by the application of the autofluorescent agent, monodansylcadaverine. Western blotting was used to detect markers of apoptosis and autophagy, namely Caspase-3, Beclin-1 and LC3-B. The combination efficiency of DHA and TMZ was assessed in vitro and in vivo. The half maximal inhibitory concentration (IC50) of DHA differed among the ten human glioma cell lines. The number of autophagic vacuoles was higher in DHA-treated SKMG-4 cells; this was highest of all cell lines analyzed. The expression of autophagy molecular markers, Beclin-1 and LC3-B, was increased following DHA treatment, while no significant alteration was detected in the expression of apoptotic marker Caspase-3. When combined with DHA, the IC50 of TMZ decreased significantly in the four glioma cell lines analyzed. Furthermore, DHA enhanced the tumor inhibition ability of TMZ in tumor-burdened mice. The results of the present study demonstrated that DHA inhibited the proliferation of glioma cells and enhanced the tumor inhibition efficacy of TMZ in vitro and in vivo through the induction of autophagy. PMID:26171034

Hair cell heterogeneity and ultrasonic hearing: recent advances in understanding fish hearing.

PubMed Central

Popper, A N

2000-01-01

The past decade has seen a wealth of new data on the auditory capabilities and mechanisms of fishes. We now have a significantly better appreciation of the structure and function of the auditory system in fishes with regard to their peripheral and central anatomy, physiology, behaviour, sound source localization and hearing capabilities. This paper deals with two of the newest of these findings, hair cell heterogeneity and the detection of ultrasound. As a result of this recent work, we now know that fishes have several different types of sensory hair cells in both the ear and lateral line and there is a growing body of evidence to suggest that these hair cell types arose very early in the evolution of the octavolateralis system. There is also some evidence to suggest that the differences in the hair cell types have functional implications for the way the ear and lateral line of fishes detect and process stimuli. Behavioural studies have shown that, whereas most fishes can only detect sound to 1-3 kHz, several species of the genus Alosa (Clupeiformes, i.e. herrings and their relatives) can detect sounds up to 180 kHz (or even higher). It is suggested that this capability evolved so that these fishes can detect one of their major predators, echolocating dolphins. The mechanism for ultrasound detection remains obscure, though it is hypothesized that the highly derived utricle of the inner ear in these species is involved. PMID:11079414

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines.

PubMed

Zhang, Qi-Ya; Ruan, Hong-Mei; Li, Zhen-Qiu; Yuan, Xiu-Ping; Gui, Jian-Fang

2003-12-03

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.

Changes in cytoskeletal dynamics and nonlinear rheology with metastatic ability in cancer cell lines

NASA Astrophysics Data System (ADS)

Coughlin, Mark F.; Fredberg, Jeffrey J.

2013-12-01

Metastatic outcome is impacted by the biophysical state of the primary tumor cell. To determine if changes in cancer cell biophysical properties facilitate metastasis, we quantified cytoskeletal biophysics in well-characterized human skin, bladder, prostate and kidney cell line pairs that differ in metastatic ability. Using magnetic twisting cytometry with optical detection, cytoskeletal dynamics was observed through spontaneous motion of surface bound marker beads and nonlinear rheology was characterized through large amplitude forced oscillations of probe beads. Measurements of cytoskeletal dynamics and nonlinear rheology differed between strongly and weakly metastatic cells. However, no set of biophysical parameters changed systematically with metastatic ability across all cell lines. Compared to their weakly metastatic counterparts, the strongly metastatic kidney cancer cells exhibited both increased cytoskeletal dynamics and stiffness at large deformation which are thought to facilitate the process of vascular invasion.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

PubMed

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer.

PubMed

Scatena, Caroline D; Hepner, Mischa A; Oei, Yoko A; Dusich, Joan M; Yu, Shang-Fan; Purchio, Tony; Contag, Pamela R; Jenkins, Darlene E

2004-05-15

Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo. Copyright 2004 Wiley-Liss, Inc.

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

PubMed

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

2000-05-01

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

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

PubMed

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

PubMed

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

PubMed Central

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

Integrated HPV genomes tend to integrate in gene desert areas in the CaSki, HeLa, and SiHa cervical cancer cell lines.

PubMed

Diao, Ming-Kun; Liu, Chu-Yi; Liu, Hong-Wei; Li, Jin-Tao; Li, Fan; Mehryar, Mohammadreza Mohammadzad; Wang, Yang-Junqi; Zhan, Shao-Bing; Zhou, Yu-Bai; Zhong, Ru-Gang; Zeng, Yi

2015-04-15

The integration preferences of human papillomavirus (HPV) have been intensively studied and contested over recent years. To disclose the integration preferences of high-risk HPV in cervical cancer, HPV transcriptional sites and features in different cervical cancer cell lines were identified. In this study, three cervical cancer cell lines (CaSki, HeLa, and SiHa) were subjected for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. The numbers of viral copies in human genomes and numbers of viral-human fusion mRNAs in three HPV-integrated cervical cancer cell lines were measured and analysed. The results revealed that the gene desert region 8q24 of the HPV type 18 integrated HeLa cell line and the 13q21-22 region of the HPV type 16 integrated CaSki and SiHa cell lines were hotspots for HPV integration, and the numbers of viral copies in the human genomes of the three cell lines that we detected were not in accordance with those reported in previous studies. Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. This study provides information to benefit health care professionals seeking more comprehensive and accurate diagnostics for HPV-related disease"? Please check, and amend as necessary. Copyright © 2015 Elsevier Inc. All rights reserved.

Early development of the enteric nervous system visualized by using a new transgenic zebrafish line harboring a regulatory region for choline acetyltransferase a (chata) gene.

PubMed

Nikaido, Masataka; Izumi, Saki; Ohnuki, Honoka; Takigawa, Yuki; Yamasu, Kyo; Hatta, Kohei

2018-06-01

The enteric nervous system (ENS) is the largest part of the peripheral nervous system in vertebrates. Toward the visualization of the development of the vertebrate ENS, we report our creation of a new transgenic line, Tg(chata:GGFF2) which has a 1.5-kb upstream region of the zebrafish choline acetyltransferase a (chata) gene followed by modified green fluorescent protein (gfp). During development, GFP + cells were detected in the gut by 60 h post-fertilization (hpf). In the gut of 6- and 12-days post-fertilization (dpf) larvae, an average of 92% of the GFP + cells were positive for the neuronal marker HuC/D, suggesting that GFP marks enteric neurons in this transgenic line. We also observed that 66% of the GFP + cells were choline acetyltransferase (ChAT)-immunopositive at 1.5 months. Thus, GFP is expressed at the larval stages at which ChAT protein expression is not yet detected by immunostaining. We studied the spatiotemporal pattern of neural differentiation in the ENS by live-imaging of this transgenic line. We observed that GFP + or gfp + cells initially formed a pair of bilateral rows at 60 hpf or 53 hpf, respectively, in the migrating enteric neural crest cells. Most of the GFP + cells did not migrate, and most of the new GFP + cells were added to fill the space among the previously formed GFP + cells. GFP expression reached the anus by 72 hpf. New GFP + cells then also appeared in the dorsal and ventral sides of the initial GFP + rows, resulting in their distribution on the entire gut by 4 dpf. A small number of new GFP + cells were found to move among older GFP + cells just before the cells stopped migration, suggesting that the moving GFP + cells may represent neural precursor cells searching for a place for the final differentiation. Our data suggest that the Tg(chata:GGFF2) line could serve as a useful tool for studies of enteric neural differentiation and cell behavior. Copyright © 2018. Published by Elsevier B.V.

Enhanced electrochemical sensing of leukemia cells using drug/lipid co-immobilized on the conducting polymer layer.

PubMed

Gurudatt, N G; Naveen, M Halappa; Ban, Changill; Shim, Yoon-Bo

2016-12-15

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (Rct) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7-1.0V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10(3)-2.5×10(5) cells/mL and 1.0×10(3)-8.0×10(3) cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.

PubMed

Jaiswal, A K; Nebert, D W; Eisen, H W

1985-08-01

The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.

Establishment of an indicator cell line to quantify bovine foamy virus infection.

PubMed

Ma, Zhe; Hao, Peng; Yao, Xue; Liu, Chang; Tan, Juan; Liu, Li; Yang, Rongge; Geng, Yunqi; Chen, Qimin; Qiao, Wentao

2008-08-01

A cell line derived from baby hamster kidney (BHK-21) cells was transfected with the enhanced green fluorescent protein gene driven by the bovine foamy virus (BFV) long terminal repeat (LTR) to establish a BFV indicator cell line (BICL). Among 48 clones, one clone was chosen for its little constitutive enhanced green fluorescent protein (EGFP) expression and high level of EGFP expression after activation by BFV infection. By detecting the EGFP expression of the BFV indicator cell line, the titers of BFV were quantified by the end point method. As a result, the titer determined by the EGFP based assay 5-6 days post infection (d.p.i.) was 100 fold higher than traditional assays measuring cytopathic effects 8-9 d.p.i.. Moreover, the EGFP based assay was also used to determine the titer of those cells infected by BFV without inducing cytopathic effects. Using this simple and rapid assay, we examined the in vitro host range of BFV. It was found that BFV can productively infect various cell lines derived from bovine, human, rat and monkey. (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53

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

Hsieh Tzechen; Wang Zhirong; Hamby, Carl V.

2005-08-19

Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependentmore » increase in S phase and a concomitant reduction in the G{sub 1} phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.« less

Characterization of the camel skin cell line Dubca.

PubMed

Klopries, M; Wernery, U; Kaaden, O R

1995-01-01

A skin fibroblast cell culture was established from a 2-month-old dromedary foetus. The cells were transformed by infection with SV40 and cloned in soft agar. The established cell line is now designated Dubca cells (Dubai camel) and has been in permanent culture for 95 passages. The cell culture was examined morphologically, chromosome preparations made and DNA fingerprinting performed by hybridization with the oligonucleotide probe (GTG)5. SV40 large T antigen was detected by western blotting. The viral host range was determined by infection with viruses of different families. Camelpox virus (CaPV) bovine herpesvirus-1 (BHV-1), vesicular stomatitis virus (VSV) and border disease virus (BDV) could be propagated in these cells.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures.

PubMed

Costa, Michael Rodrigues; Fischer, Nicole; Gulich, Barbara; Tönjes, Ralf R

2014-01-01

Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. Porcine endogenous retroviruses-A/C in supernatants of human producer 293/5° cells was not able to infect huPBMC. Neither RT activity nor PERV copies were detected. Even provirus could not be detected displaying the inability of PERV-A/C to induce a productive infection in huPBMC. In cocultivation experiments only non-productive infection of huPBMC with PERV derived from 293/5° cell line and from PHA-activated poPBMC was observed by detection of provirus DNA in infected cells. Recombinant PERV-A/C in supernatants of producer cells failed to infect huPBMC, whereas coculture experiments with producer cell lines lead to non-productive infection of huPBMC. PERV in supernatants seem to have not sufficient infectious potential for huPBMC. However, extensive PERV exposure to huPBMC via cocultivation enabled at least virus cell entry as provirus was detected by nested PCR. Furthermore, results presented support previous data showing German landrace pigs as low producers with negligible infectious potential due to the absence of replication-competent PERV in the genome. The low PERV expression profile and the lack of significant replication competence of German landrace pigs raise hope for considering these animals as putative donor animals in future pig-to-human xenotransplantation. Nonetheless, data imply that PERV still represent a virological risk in the course of xenotransplantation, as the presence of PERV provirus in host cells may lead to a provirus integration resulting in insertional mutagenesis and chromosomal rearrangements. © 2014 John Wiley & Sons A/S.

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

PubMed Central

Laube, F; Göhring, B; Sann, H; Willhardt, I

2001-01-01

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. © 2001 Cancer Research Campaignhttp://www.bjcancer.com PMID:11556847

Case of 46,XX/47,XY, +21 chimerism in a newborn infant with ambiguous genitalia

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

Sawai, Tomoko; Yoshimoto, Masaaki; Kinoshita, Ei-ichi

The authors describe the whole-body chimerism in a newborn infant with small phallus, pseudo-vaginal perineal hypospadias, and a bifid scrotum containing gonads. The human testis determining factor gene (SRY) was detected by PCR amplification. GTG-banding chromosome analysis in peripheral blood lymphocytes and cultured fibroblasts derived from right cubital skin showed a 46,XX/47,XY, +21 karyotype. Their ratios in each cell line were 294:5 and 178:7, respectively. QFQ-banding chromosome analysis documented 3 heteromorphic satellites on trisomic chromsomes 21 in the 47,XY,+21 cell line and a homozygous satellite pattern in the 46,XX cell line. Heteromorphic patterns of chromsomes 4, 13, 14, and 22more » were also different between the two cell lines. To our knowledge, such disomy/trisomy chimeras have not been described previously. 10 refs., 3 figs.« less

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

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

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique

2013-04-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and themore » ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.« less

Hyperspectral Imaging, Flow cytometry and Microscopic Morphology of Silver Nanoparticle within Cells

EPA Science Inventory

The ability to detect and track silver nanoparticles (AgNP) that enter cells is important to understand the potential biological and toxicological actions of AgNP. The uptake and fate in cells of four different types of AgNP was studied in a retinal pigment epithelial cell line ...

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

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

PubMed

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

1993-09-01

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

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

PubMed

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

2000-11-01

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

Cdc20/p55 mediates the resistance to docetaxel in castration-resistant prostate cancer in a Bim-dependent manner.

PubMed

Wu, Fei; Lin, Yun; Cui, Peng; Li, Hongyun; Zhang, Lechao; Sun, Zeqiang; Huang, Shengliang; Li, Shun; Huang, Shiming; Zhao, Qingli; Liu, Qingyong

2018-06-01

At least to date, no effective treatment for advanced castration-resistant prostate cancer (CRPC) has been established. Recent studies indicated that cell division cycle 20 homolog (Cdc20) overexpression is associated with poor prognosis in patients with castration-resistant prostate cancer. However, the mechanism of Cdc20 in the development of docetaxel resistance in CRPC remains elusive. In this study, the transcription of Cdc20 was confirmed in three independent CRPC cell lines derived from different tissues, including LNCaP, PC3, and DU145. Docetaxel resistant (DR) cell lines were generated within the background of DU145 and PC3. The protein levels of Cdc20 and the biological phenotype were detected in both wild-type and DR cell lines. To further explore the mechanism of Cdc20 overexpression, stable cell lines with Cdc20 or Bcl-2 interacting mediator of cell death (Bim) deprivation were generated and examined for biological parameters. In addition, a specific Cdc20 inhibitor was used in DR cell lines to explore the potential solution for docetaxel resistant CRPC. Here, we identified Cdc20 is overexpressed in docetaxel resistant CRPC cell lines, including LNCaP, PC3, and DU145. We also reported that DR cell lines, which mimic the recurrent prostate cancer cells after docetaxel treatment, have higher levels of Cdc20 protein compared with the CRPC cell lines. Interestingly, the protein levels of Bim, an E3 ligase substrate of Cdc20, were decreased in DR cell lines compared with the wild-type, while the mRNA levels were similar. More importantly, in DR cell lines, the biological phenotype induced by Cdc20 deletion could be significantly reversed by the additional knockdown of Bim. As a result, docetaxel resistant prostate cancer cells treated with the pharmacological Cdc20 inhibitor became sensitive to docetaxel treatment. In conclusion, our data collectively demonstrated that Cdc20 overexpression facilitates the docetaxel resistant of the CRPC cell lines in a Bim-dependent manner. Furthermore, additionally targeting Cdc20 might be a promising solution for the treatment of the CRPC with docetaxel resistance.

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

PubMed Central

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

2017-01-01

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

FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

NASA Astrophysics Data System (ADS)

Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

2012-06-01

The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

Ames Test to Detect Mutagenicity of 2-Alkylcyclobutanones: A Review.

PubMed

Barbezan, Angélica B; Martins, Regiane; Bueno, Jennifer B; Villavicencio, Anna Lúcia C H

2017-07-01

Food irradiation is an effective and safe method for preservation and long-term storage, and it is approved for use in over 60 countries for various applications in a wide variety of food products. This process is performed by use of accelerated electron beams, X-rays, or gamma radiation ( 60 Co or 137 Cs). 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that have fatty acids (triglycerides) and are subjected to irradiation. Since the 1990s toxicological safety studies of 2-ACBs have been conducted extensively through synthetic compounds, then and tests to determine if the compounds have any mutagenic activity are strictly necessary. The Ames test was chosen by many researchers to assess the mutagenicity of 2-ACBs. The test uses distinct bacterial cell lines Salmonella typhimurium to detect point mutations at sites guanine-cytosine (G-C) and Escherichia coli to detect point mutations at sites adenine-thymine (A-T). This bibliographic research aims to bring together all the results obtained and a comparison and cell lines used, type of plates, and solvents. This research showed that no mutagenic activity was observed in any of the cell lines and concentrations evaluated by the works of authors, so the 2-ACBs compounds showed no mutagenic substance in concentrations detectable by the Ames test. © 2017 Institute of Food Technologists®.

Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

PubMed

Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

2014-02-01

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

PubMed

Player, A N; Shen, L P; Kenny, D; Antao, V P; Kolberg, J A

2001-05-01

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

Violation of Field Line Conservation and Associated Spatial Scales in Particle-in-Cell Simulations and MMS Data

NASA Astrophysics Data System (ADS)

Wendel, D. E.; Liu, Y. H.; Giles, B. L.; Torbert, R. B.

2017-12-01

For the first time, space flight technology exists to detect, in situ, violation of magnetic field line conservation. The violation of magnetic line conservation on scales smaller than the system size is a necessary and sufficient condition for magnetic reconnection. We demonstrate that violation of line conservation produces a detectable, structured signature in both particle-in-cell simulations of reconnection and in data from the Magnetospheric Multi-Scale mission. In particle-in-cell simulations of asymmetric reconnection, the quantity-which we call M-that identifies this violation achieves a significant value in electron skin depth-scale layers that extend from the electron diffusion region along the separatrices, with higher values emerging on the low density, high magnetic field side of the current sheet. In two MMS burst data intervals associated with detection of the electron diffusion region—one interval with antiparallel reconnecting fields and the other with a guide field-we determine the location and scale of M and of the diffusion region relative to electron outflows and the magnetic separatrices. We find that M exceeds measurement uncertainties both at the diffusion region and near the separatrices, where it attains its highest values in layered structures. The observed magnitude scales as the simulated magnitude after adjusting for the artificial parameters of the simulation. Bipolar forms of the quantity also appear further from the diffusion region, possibly associated with electron holes. The measure serves not only as a powerful diagnostic for magnetic reconnection, but reveals that electrons transport this signature of reconnection away from the x-line.

Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection.

PubMed

Scarano, Simona; Ermini, Maria Laura; Spiriti, Maria Michela; Mascini, Marco; Bogani, Patrizia; Minunni, Maria

2011-08-15

Surface plasmon resonance imaging (SPRi) was used as the transduction principle for the development of optical-based sensing for transgenes detection in human cell lines. The objective was to develop a multianalyte, label-free, and real-time approach for DNA sequences that are identified as markers of transgenosis events. The strategy exploits SPRi sensing to detect the transgenic event by targeting selected marker sequences, which are present on shuttle vector backbone used to carry out the transfection of human embryonic kidney (HEK) cell lines. Here, we identified DNA sequences belonging to the Cytomegalovirus promoter and the Enhanced Green Fluorescent Protein gene. System development is discussed in terms of probe efficiency and influence of secondary structures on biorecognition reaction on sensor; moreover, optimization of PCR samples pretreatment was carried out to allow hybridization on biosensor, together with an approach to increase SPRi signals by in situ mass enhancement. Real-time PCR was also employed as reference technique for marker sequences detection on human HEK cells. We can foresee that the developed system may have potential applications in the field of antidoping research focused on the so-called gene doping.

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

PubMed

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

2012-10-05

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

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.

PubMed

Hiatt, Joseph B; Pritchard, Colin C; Salipante, Stephen J; O'Roak, Brian J; Shendure, Jay

2013-05-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10(-6) in cell lines and 2.6 × 10(-5) in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

PubMed Central

Hiatt, Joseph B.; Pritchard, Colin C.; Salipante, Stephen J.; O'Roak, Brian J.; Shendure, Jay

2013-01-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10−6 in cell lines and 2.6 × 10−5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%–4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%–1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings. PMID:23382536

A 40 GHz fully integrated circuit with a vector network analyzer and a coplanar-line-based detection area for circulating tumor cell analysis using 65 nm CMOS technology

NASA Astrophysics Data System (ADS)

Nakanishi, Taiki; Matsunaga, Maya; Kobayashi, Atsuki; Nakazato, Kazuo; Niitsu, Kiichi

2018-03-01

A 40-GHz fully integrated CMOS-based circuit for circulating tumor cells (CTC) analysis, consisting of an on-chip vector network analyzer (VNA) and a highly sensitive coplanar-line-based detection area is presented in this paper. In this work, we introduce a fully integrated architecture that eliminates unwanted parasitic effects. The proposed analyzer was designed using 65 nm CMOS technology, and SPICE and MWS simulations were used to validate its operation. The simulation confirmed that the proposed circuit can measure S-parameter shifts resulting from the addition of various types of tumor cells to the detection area, the data of which are provided in a previous study: the |S 21| values for HepG2, A549, and HEC-1-A cells are -0.683, -0.580, and -0.623 dB, respectively. Additionally, the measurement demonstrated an S-parameters reduction of -25.7% when a silicone resin was put on the circuit. Hence, the proposed system is expected to contribute to cancer diagnosis.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma

PubMed Central

Tan, Xin-Yu; Chang, Shi; Liu, Wei

2014-01-01

Background/Aims To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. Methods An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. Results The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. Conclusions CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA. PMID:24672662

Application of recombinant fluorescent mammalian cells as a toxicity biosensor.

PubMed

Kim, E J; Lee, Y; Lee, J E; Gu, M B

2002-01-01

With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

In vivo delivery of recombinant human growth hormone from genetically engineered human fibroblasts implanted within Baxter immunoisolation devices.

PubMed

Josephs, S F; Loudovaris, T; Dixit, A; Young, S K; Johnson, R C

1999-01-01

Continuous delivery of therapeutic peptide to the systemic circulation would be the optimal treatment for a variety of diseases. The Baxter TheraCyte system is a membrane encapsulation system developed for implantation of tissues, cells such as endocrine cells or cell lines genetically engineered for therapeutic peptide delivery in vivo. To demonstrate the utility of this system, cell lines were developed which expressed human growth hormone (hGH) at levels exceeding 1 microgram per million cells per day. These were loaded into devices which were then implanted into juvenile nude rats. Significant levels of hGH of up to 2.5 ng/ml were detected in plasma throughout the six month duration of the study. In contrast, animals implanted with free cells showed peak plasma levels of 0.5 to 1.2 ng four days after implantation with no detectable hGH beyond 10 days. Histological examination of explanted devices showed they were vascularized and contained cells that were viable and morphologically healthy. After removal of the implants, no hGH could be detected which confirmed that the source of hGH was from cells contained within the device. The long term expression of human growth hormone as a model peptide has implications for the peptide therapies for a variety of human diseases using membrane encapsulated cells.

Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen.

PubMed

Banerjee, Arinjay; Rapin, Noreen; Miller, Megan; Griebel, Philip; Zhou, Yan; Munster, Vincent; Misra, Vikram

2016-11-01

It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture. Copyright © 2016 Elsevier B.V. All rights reserved.

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis.

PubMed

Kontostathi, Georgia; Zoidakis, Jerome; Makridakis, Manousos; Lygirou, Vasiliki; Mermelekas, George; Papadopoulos, Theofilos; Vougas, Konstantinos; Vlamis-Gardikas, Alexios; Drakakis, Peter; Loutradis, Dimitrios; Vlahou, Antonia; Anagnou, Nicholas P; Pappa, Kalliopi I

2017-01-01

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

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

PubMed

Yoshikawa, Rokusuke; Sato, Eiji; Miyazawa, Takayuki

2011-01-01

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

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis

PubMed Central

Zoidakis, Jerome; Makridakis, Manousos; Lygirou, Vasiliki; Mermelekas, George; Vougas, Konstantinos; Drakakis, Peter

2017-01-01

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis. PMID:28261610

Molecular characterization of variant alpha-subunit of electron transfer flavoprotein in three patients with glutaric acidemia type II--and identification of glycine substitution for valine-157 in the sequence of the precursor, producing an unstable mature protein in a patient.

PubMed Central

Indo, Y; Glassberg, R; Yokota, I; Tanaka, K

1991-01-01

In our previous study of eight glutaric acidemia type II (GAII) fibroblast lines by using [35S]methionine labeling and immunoprecipitation, three of them had a defect in the synthesis of the alpha-subunit of electron transfer flavoprotein (alpha-ETF) (Ikeda et al. 1986). In one of them (YH1313) the labeling of the mature alpha-ETF was barely detectable, while that of the precursor (p) was stronger. In another (YH605) no synthesis of immunoreactive p alpha-ETF was detectable. In the third cell line (YH1391) the rate of variant p alpha-ETF synthesis was comparable to normal, but its electrophoretic mobility was slightly faster than normal. In the present study, the northern blot analysis revealed that all three mutant cell lines contained p alpha-ETF mRNA and that their size and amount were comparable to normal. In immunoblot analysis, both alpha- and beta-ETF bands were barely detectable in YH1313 and YH605 but were detectable in YH1391 in amounts comparable to normal. Sequencing of YH1313 p alpha-ETF cDNA via PCR identified a transversion of T-470 to G. We then devised a simple PCR method for the 119-bp section (T-443/G-561) for detecting this mutation. In the upstream primer, A-466 was artificially replaced with C, to introduce a BstNI site into the amplified copies in the presence of G-470 from the variant sequence. The genomic DNA analysis using this method demonstrated that YH1313 was homozygous for T----G-470 transversion. It was not detected either in two other alpha-ETF-deficient GAII or in seven control cell lines. The alpha-ETF cDNA sequence in YH605 was identical to normal. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:1882842

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Surface glycoproteins of an African henipavirus induce syncytium formation in a cell line derived from an African fruit bat, Hypsignathus monstrosus.

PubMed

Krüger, Nadine; Hoffmann, Markus; Weis, Michael; Drexler, Jan Felix; Müller, Marcel Alexander; Winter, Christine; Corman, Victor Max; Gützkow, Tim; Drosten, Christian; Maisner, Andrea; Herrler, Georg

2013-12-01

Serological screening and detection of genomic RNA indicates that members of the genus Henipavirus are present not only in Southeast Asia but also in African fruit bats. We demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional similarities to glycoproteins of Nipah virus.

Establishment of a new human pre-B acute lymphoblastic leukemia cell line (KMO-90) with 1;19 translocation carrying p53 gene alterations.

PubMed

Sotomatsu, M; Hayashi, Y; Kawamura, M; Yugami, S; Shitara, T

1993-10-01

A new human pre-B acute lymphoblastic leukemia cell line (KMO-90) was established from the bone marrow sample of a 12-year-old girl with acute lymphoblastic leukemia (ALL) carrying 1;19 chromosome translocation. KMO-90 cells expressed HLA-DR, CD10, CD19, and CD22 antigens. These cells had also cytoplasmic immunoglobulin lacking surface immunoglobulin, indicating that these had a pre-B phenotype. Chromosome analysis of this cell line showed 48, XX, +8, +19, t(1;19)(q23;p13). Southern blot analysis showed the same sized rearrangements of the E2A gene in KMO-90 cells as those in the original leukemic cells. By means of reverse transcriptase-polymerase chain reaction analysis, we detected E2A/PBX1 fusion transcripts in KMO-90 cells. KMO-90 is useful when studying the role of the 1;19 translocation in the etiology of pre-B ALL. Furthermore, we studied alterations of the p53 gene in this cell line by polymerase chain reaction, single-strand conformation polymorphism analysis. KMO-90 cells were identified to have a point mutation at codon 177 (CCC-->TCC) of the p53 gene, suggesting that alterations of the p53 gene may have an important role in the establishment of this cell line.

Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation.

PubMed

Xiao, M; Luo, Z; Mantel, C; Broxmeyer, H E; Lu, L

2000-02-01

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.

Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

PubMed

Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

2009-08-01

Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy.

PubMed

Ali, Eman Ali; Nakamura, Ryosuke; Falcone, Franco H

2017-01-01

Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

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

PubMed Central

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

2016-01-01

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

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

PubMed

Ogorevc, J; Dovč, P

2015-04-15

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

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation

PubMed Central

2013-01-01

The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

PubMed

Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

2013-08-09

: The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

PubMed Central

Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Véronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane

2011-01-01

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer. PMID:21935420

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

NASA Astrophysics Data System (ADS)

Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

2016-09-01

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

Substrate stiffness affects epithelial-mesenchymal transition of cervical cancer cells through miR-106b and its target protein DAB2.

PubMed

Piao, Jinlan; You, Ke; Guo, Yanli; Zhang, Youyi; Li, Zijian; Geng, Li

2017-06-01

The effects of different substrate stiffness were investigated on epithelial-mesenchymal transition (EMT) of cervical cancer cell lines and the role of miR-106b and its target protein DAB2 therein. Cervical cancer cell lines HeLa and SiHa were cultured on artificial substrates with different stiffness prepared using different ratios of acrylamide and bis-acrylamide. Changes of microRNA profiles were detected using microRNA chip analysis, and the expression levels of EMT-related markers E-cadherin and vimentin were detected using western blotting and real-time PCR. In addition, the effects of miR-106b overexpression as well as miR-106b and DAB2 knockdown on expression of E-cadherin and vimentin were also examined using western blotting and real-time PCR. The results showed that i) cervical cancer cell lines SiHa and HeLa cultured on substrate with stiffness of 20 kPa had the strongest EMT ability, showed the highest levels of vimentin and lowest levels of E-cadherin, compared with cells cultured on substrate with stiffness of 1 kPa; ii) miR-106b knockdown reversed the effects of substrate stiffness on EMT of cervical cancer cells, while miR-106 overexpression and DAB2 knockdown induced EMT of cervical cancer cells cultured on substrate with stiffness of 20 kPa. Overall, the results indicated that substrate stiffness could regulate EMT of cervical cancer cell lines HeLa and SiHa at least partially through miR-106b and its downstream target DAB2.

The chemokine receptor CXCR6 and its ligand CXCL16 are expressed in carcinomas and inhibit proliferation.

PubMed

Meijer, Joost; Ogink, Janneke; Kreike, Bas; Nuyten, Dimitry; de Visser, Karin E; Roos, Ed

2008-06-15

The chemokine receptor CXCR6 and its ligand CXCL16 are involved in inflammation. Thus far, they were known to be expressed mainly by T cells and macrophages, respectively. However, we detected both in all of 170 human primary mammary carcinomas and at similar levels in all 8 human mammary carcinoma cell lines tested by microarray analysis. Expression was confirmed by reverse transcription-PCR and for the cell lines also by fluorescence-activated cell sorting analysis. CXCR6 and CXCL16 were also detected in several mouse and human mammary, colon, and pancreatic carcinoma cell lines. CXCL16 is a transmembrane protein from which the soluble chemokine can be cleaved off. The transmembrane form is present on the surface of the carcinoma cells. Surprisingly, suppression of either CXCR6 or CXCL16 led to greatly enhanced proliferation in vitro as well as in vivo, indicating that their interaction inhibits proliferation. This notion was verified using inhibitory antibodies and by introduction of CXCL16 into a rare CXCL16-negative cell line. The effect was mediated by the G protein-coupled receptor CXCR6 because it was blocked by the G(i) protein inhibitor pertussis toxin. In contrast, the soluble CXCL16 chemokine enhanced proliferation, and this was also mediated by CXCR6 but not via G(i) protein. It is remarkable that both CXCR6 and CXCL16 are expressed by all mammary carcinomas because cells that lose either acquire a growth advantage and should be selected during tumor progression. This suggests an unknown important role in tumor formation. Proteases, possibly macrophage derived, might convert inhibitory transmembrane CXCL16 into the stimulatory chemokine.

Tools to study pathogen-host interactions in bats.

PubMed

Banerjee, Arinjay; Misra, Vikram; Schountz, Tony; Baker, Michelle L

2018-03-15

Bats are natural reservoirs for a variety of emerging viruses that cause significant disease in humans and domestic animals yet rarely cause clinical disease in bats. The co-evolutionary history of bats with viruses has been hypothesized to have shaped the bat-virus relationship, allowing both to exist in equilibrium. Progress in understanding bat-virus interactions and the isolation of bat-borne viruses has been accelerated in recent years by the development of susceptible bat cell lines. Viral sequences similar to severe acute respiratory syndrome corona virus (SARS-CoV) have been detected in bats, and filoviruses such as Marburg virus have been isolated from bats, providing definitive evidence for the role of bats as the natural host reservoir. Although viruses can be readily detected in bats using molecular approaches, virus isolation is far more challenging. One of the limitations in using traditional culture systems from non-reservoir species is that cell types and culture conditions may not be compatible for isolation of bat-borne viruses. There is, therefore, a need to develop additional bat cell lines that correspond to different cell types, including less represented cell types such as immune cells, and culture them under more physiologically relevant conditions to study virus host interactions and for virus isolation. In this review, we highlight the current progress in understanding bat-virus interactions in bat cell line systems and some of the challenges and limitations associated with cell lines. Future directions to address some of these challenges to better understand host-pathogen interactions in these intriguing mammals are also discussed, not only in relation to viruses but also other pathogens carried by bats including bacteria and fungi. Copyright © 2018 Elsevier B.V. All rights reserved.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

PubMed

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

2017-06-01

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

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

PubMed

Zhao, Zhenzhen; Liu, Wen; Liu, Jincheng; Wang, Jiayi; Luo, Bing

2017-10-01

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p. © 2017 Wiley Periodicals, Inc.

The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

PubMed

Yin, Haoyuan; Shao, Ying; Chen, Xuan

2017-01-01

To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P < 0.05). Downregulated the expression of CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

Assessment of the Tumorigenic Potential of Spontaneously Immortalized and hTERT-Immortalized Cultured Dental Pulp Stem Cells.

PubMed

Wilson, Ryan; Urraca, Nora; Skobowiat, Cezary; Hope, Kevin A; Miravalle, Leticia; Chamberlin, Reed; Donaldson, Martin; Seagroves, Tiffany N; Reiter, Lawrence T

2015-08-01

Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases. ©AlphaMed Press.

The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

PubMed

Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

2007-06-01

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

PubMed

Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

2017-07-01

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

EPA Science Inventory

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

PubMed

An, Jeung Hee; Kim, Seung U; Park, Mi-Kyung; Choi, Jeong Woo

2015-10-01

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

PubMed Central

Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

2012-01-01

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer. PMID:22745580

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

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

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

1983-09-01

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

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

PubMed

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

PubMed

Mestieri, Leticia Boldrin; Gomes-Cornélio, Ana Lívia; Rodrigues, Elisandra Márcia; Faria, Gisele; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mário

2017-01-01

The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

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

PubMed

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

2017-04-01

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

Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines.

PubMed

Shimada, Y; Kato, T; Ogami, K; Horie, K; Kokubo, A; Kudo, Y; Maeda, E; Sohma, Y; Akahori, H; Kawamura, K

1995-12-01

Recently, we purified rat thrombopoietin (TPO) from plasma of irradiated rats (XRP) by measuring its activity that stimulated the production of megakaryocytes from megakaryocyte progenitor cells (CFU-MK) in vitro. We then cloned the cDNAs for rat and human TPO. In this study, we found the production of TPO by hepatocytes isolated with the collagenase perfusion method from both normal and thrombocytopenic rats, by a two-step fractionation of hepatocyte culture medium (CM). Subsequently, CM of rat hepatoma cell lines was screened for the presence of TPO; three cell lines, H4-II-E, McA-RH8994, and HTC, were found to produce TPO. According to the purification procedure for TPO from XRP, TPO was partially purified from 2 L CM of each of three cell lines with a six-step procedure. In the final reverse-phase column, TPO from each cell line was eluted with the same retention time as that from XRP, and the TPO fraction exhibited megakaryocyte colony-stimulating activity (Meg-CSA). TPO-active fraction eluted from the final reverse-phase column was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), extracted from the gel, and assayed. TPO activity from each cell line was found in the respective molecular weight region, indicating the heterogeneity of the TPO molecule. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we detected the expression of TPO mRNA in hepatocytes, three hepatoma cell lines, normal rat liver, and X-irradiated rat liver. Northern blot analysis showed that TPO mRNA was expressed mainly in liver among the various organs tested. These data demonstrate that TPO is produced by rat hepatocytes and hepatoma cell lines and suggest that liver may be the primary organ that produces TPO.

[Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

PubMed

Li, Lixuan; Li, Jia

2015-05-01

To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

Multidrug resistance characterization in multicellular tumour spheroids from two human lung cancer cell lines.

PubMed

Barrera-Rodríguez, Raúl; Fuentes, Jorge Morales

2015-01-01

Most of the knowledge about the mechanisms of multidrug resistance in lung cancer has been achieved through the use of cell lines isolated from tumours cultivated either in suspensions of isolated cells or in monolayers and following exposition to different cytostatic agents. However, tumour cell lines growing as multicellular tumour spheroids (MTS) frequently develop multicellular resistance in a drug-independent form. The aim of this study was to characterize the phenotypic and functional differences between two human NSCLC cell lines (INER-37 and INER-51) grown as traditional monolayer cultures versus as MTS. After 72 hours treatment with anticancer drugs, chemosensitivity in monolayers and tumour spheroids cultures was assessed using MTT assay. Reverse transcription-polymerase chain reaction was employed to detect the mRNAs of multidrug resistance-related genes. The expression of P-gp was analyzed by immunohistochemical staining and cell cycle profiles were analyzed using FACS. The results indicate that when grown as MTS each lung cancer cell line had different morphologies as well as and abrogation of cell proliferation with decrease of the G2/M phase. Also, MTS acquired multicellular resistance to several chemotherapeutic agents in only a few days of culture which were accomplished by significant changes in the expression of MDR-related genes. Overall, the MTS culture changed the cellular response to drugs nevertheless each of the cell lines studied seems to implement different mechanisms to acquire multicellular resistance.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy

PubMed Central

Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.

2014-01-01

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853

Provirus Integration at the 3 Region of N‐myc in Cell Lines Established from Thymic Lymphomas Spontaneously Formed in AKR Mice and a [(BALB/c × B6)F1AKR] Bone Marrow Chimera

PubMed Central

Yano, Yoko; Kobayashi, Seiichi; Yasumizu, Ryoji; Tamaki, Junko; Kubo, Mitsumasa; Sasaki, Akio; Hasan, Shahid; Okuyama, Harue; Inaba, Muneo; Ikehara, Susumu; Hiai, Hiroshi; Kakinuma, Mitsuaki

1991-01-01

Among 18 thymic leukemia cell lines which have been established from spontaneous thymic lym‐phomas in AKR mice as well as in bone marrow chimeras which were constructed by transplanting allogeneic bone marrow cells into irradiated AKR mice, three proviral integration sites were identified; near c‐myc, N‐myc and pim‐l loci. No integration site specific for chimeric leukemia cell lines was found. In three thymic leukemia cell lines which contained rearranged N‐myc, genes, insertions of long terminal repeats (LTRs) of murine leukemia viruses were detected at 18 or 20 bp downstream of the translational termination codon. These results demonstrate that the 3’region of the N‐myc gene is one of the integration targets for murine leukemia viruses in spontaneous thymic lymphomas. In these three cell lines, N‐myc mRNA was stably transcribed and transcription of c‐myc mRNA was down‐regulated. The integrated murine leukemia viruses in AKR thymic leukemia were most likely AKV, though the DNA sequence of the LTR inserted in the genome of a leukemic cell line from [(BALB/c × B6)F1‐AKR], CAK20, was different from LTRs of murine leukemia viruses so far reported. PMID:1900822

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

PubMed

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

2018-03-01

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

L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation.

PubMed

Cui, Yan; Ying, Ying; van Hasselt, Andrew; Ng, Ka Man; Yu, Jun; Zhang, Qian; Jin, Jie; Liu, Dingxie; Rhim, Johng S; Rha, Sun Young; Loyo, Myriam; Chan, Anthony T C; Srivastava, Gopesh; Tsao, George S W; Sellar, Grant C; Sung, Joseph J Y; Sidransky, David; Tao, Qian

2008-08-20

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing. Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.

Alterations in ATR in nasal NK/T-cell lymphoma and chronic active Epstein-Barr virus infection.

PubMed

Liu, Angen; Takakuwa, Tetsuya; Luo, Wen-Juan; Fujita, Shigeki; Aozasa, Katsuyuki

2006-07-01

Nasal natural killer (NK)/T-cell lymphoma (NKTCL) and chronic active Epstein-Barr virus infection (CAEBV) are relatively frequent, especially in Asia, and are poor in prognosis. Both diseases are proliferative diseases of NK/T cells that show highly complicated karyotypes, suggesting the involvement of chromosomal instability. ATR is an important gene for DNA damage response and chromosomal stability. To evaluate the role of ATR gene alterations in the pathogenesis of NKTCL and CAEBV, the whole coding region of the ATR gene was examined in cell lines derived from NKTCL and CAEBV, as well as tumor samples from patients. ATR alterations were detected in two of eight NKTCL and in one of three CAEBV lines. Most aberrant transcripts observed were deletions resulting from aberrant splicing. ATR alterations were also detected in four of 10 NKTCL clinical samples. Both NKTCL and CAEBV cell lines with ATR alterations showed a delay or abrogation in repair of ionizing radiation-induced DNA double-strand breaks and ultraviolet-induced DNA single-strand breaks, and both exhibited a defect in p53 accumulation. These findings show that alterations in the ATR gene result in an abnormal response to DNA double-strand break and single-strand break repair, suggesting a role for ATR gene alterations in NKTCL lymphomagenesis.

Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer

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

Koukourakis, Michael I., E-mail: targ@her.forthnet.gr; Kalamida, Dimitra; Mitrakas, Achilleas

2015-05-29

Introduction: Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. Materials and methods: In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. Results: Using confocal microscopymore » on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a ‘highLC3A/lowLAMP2a’ phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. Conclusions: High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy. - Highlights: • High LC3A and low LAMP2a levels is a frequent expression pattern of prostate carcinoma. • This pattern of intensified autophagic flux relates with high relapse rates after radiotherapy. • The PC3 radio-resistant cell line sustains remarkably its autophagic flux ability after radiation. • Irradiation of the DU145 radio-sensitive cell line blocks the autophagic flux. • Intense autophagy activity defines prostate cancer radio-resistance, in vivo and in vitro.« less

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction

PubMed Central

Zhang, Li-Yong; Xing, Tao; Du, Li-Xin; Li, Qing-Min; Liu, Wei-Dong; Wang, Ji-Yue; Cai, Jing

2015-01-01

Background Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. Methods A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. Results This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. Conclusion This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice. PMID:25848224

[Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

PubMed

Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

2013-08-01

This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

Increased ERp57 Expression in HBV-Related Hepatocellular Carcinoma: Possible Correlation and Prognosis.

PubMed

Liu, Miao; Du, Lingyao; He, Zhiliang; Yan, Libo; Shi, Ying; Shang, Jin; Tang, Hong

2017-01-01

Aim. ERp57 is involved in virus induced endoplasmic reticulum stress (ERS) and plays an important role in tumorigenesis. This study aimed to find whether HBV infection altered ERp57 expression and whether ERp57 regulation was involved in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) genesis. Materials and Methods. HBV-HCC tissues, chronic hepatitis B (CHB) liver tissues, and normal liver tissues were acquired. ERp57 expressions in these tissues were detected through immunohistochemistry (IHC). And ERp57 expression in liver cell line L02, HBV replicative liver cell line L02-pHBV4.1, and HCC cell lines were detected through western blot for verification. Then medical data on patients providing HCC tissues were collected and analyzed along with ERp57 expression. Results. Higher ERp57 expression was found in HCC and CHB tissues ( p < 0.001). And HCC cell lines and L02-pHBV4.1 presented higher ERp57 expression as well. In patients, ERp57 expression showed significant differences between death and survival groups ( p = 0.037). And cumulative survival in patients with higher ERp57 (score ⩾ 8.75) is significantly lower ( p = 0.009). Conclusion. Our study found increased expression of ERp57 in HBV-HCC. Such altered expression could be related to HBV infection and high ERp57 expression may lead to poor prognosis of HBV-HCC patients.

A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

PubMed Central

Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

2014-01-01

In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 μM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ∼300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 μM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (μTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings. PMID:24967604

Field validation of protocols developed to evaluate in-line mastitis detection systems.

PubMed

Kamphuis, C; Dela Rue, B T; Eastwood, C R

2016-02-01

This paper reports on a field validation of previously developed protocols for evaluating the performance of in-line mastitis-detection systems. The protocols outlined 2 requirements of these systems: (1) to detect cows with clinical mastitis (CM) promptly and accurately to enable timely and appropriate treatment and (2) to identify cows with high somatic cell count (SCC) to manage bulk milk SCC levels. Gold standard measures, evaluation tests, performance measures, and performance targets were proposed. The current study validated the protocols on commercial dairy farms with automated in-line mastitis-detection systems using both electrical conductivity (EC) and SCC sensor systems that both monitor at whole-udder level. The protocol for requirement 1 was applied on 3 commercial farms. For requirement 2, the protocol was applied on 6 farms; 3 of them had low bulk milk SCC (128×10(3) cells/mL) and were the same farms as used for field evaluation of requirement 1. Three farms with high bulk milk SCC (270×10(3) cells/mL) were additionally enrolled. The field evaluation methodology and results were presented at a workshop including representation from 7 international suppliers of in-line mastitis-detection systems. Feedback was sought on the acceptance of standardized performance evaluation protocols and recommended refinements to the protocols. Although the methodology for requirement 1 was relatively labor intensive and required organizational skills over an extended period, no major issues were encountered during the field validation of both protocols. The validation, thus, proved the protocols to be practical. Also, no changes to the data collection process were recommended by the technology supplier representatives. However, 4 recommendations were made to refine the protocols: inclusion of an additional analysis that ignores small (low-density) clot observations in the definition of CM, extension of the time window from 4 to 5 milkings for timely alerts for CM, setting a maximum number of 10 milkings for the time window to detect a CM episode, and presentation of sensitivity for a larger range of false alerts per 1,000 milkings replacing minimum performance targets. The recommended refinements are discussed with suggested changes to the original protocols. The information presented is intended to inform further debate toward achieving international agreement on standard protocols to evaluate performance of in-line mastitis-detection systems. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

KS-Detect – Validation of Solar Thermal PCR for the Diagnosis of Kaposi’s Sarcoma Using Pseudo-Biopsy Samples

PubMed Central

Snodgrass, Ryan; Gardner, Andrea; Jiang, Li; Fu, Cheng; Cesarman, Ethel; Erickson, David

2016-01-01

Resource-limited settings present unique engineering challenges for medical diagnostics. Diagnosis is often needed for those unable to reach central healthcare systems, making portability and independence from traditional energy infrastructure essential device parameters. In 2014, our group presented a microfluidic device that performed a solar-powered variant of the polymerase chain reaction, which we called solar thermal PCR. In this work, we expand on our previous effort by presenting an integrated, portable, solar thermal PCR system targeted towards the diagnosis of Kaposi’s sarcoma. We call this system KS-Detect, and we now report the system’s performance as a diagnostic tool using pseudo-biopsy samples made from varying concentrations of human lymphoma cell lines positive for the KS herpesvirus (KSHV). KS-Detect achieved 83% sensitivity and 70% specificity at high (≥10%) KSHV+ cell concentrations when diagnosing pseudo-biopsy samples by smartphone image. Using histology, we confirm that our prepared pseudo-biopsies contain similar KSHV+ cell concentrations as human biopsies positive for KS. Through our testing of samples derived from human cell lines, we validate KS-Detect as a viable, portable KS diagnostic tool, and we identify critical engineering considerations for future solar-thermal PCR devices. PMID:26799834

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

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

Jeang, K.T.; Cho, M.S.; Hayward, G.S.

1984-10-01

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably bymore » a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.« less

Methylation at Global LINE-1 Repeats in Human Blood Are Affected by Gender but Not by Age or Natural Hormone Cycles

PubMed Central

El-Maarri, Osman; Singer, Heike; Diaz-Lacava, Amalia; Nüsgen, Nicole; Niemann, Barbara; Watzka, Matthias; Reinsberg, Jochen; van der Ven, Hans; Wienker, Thomas; Stoffel-Wagner, Birgit; Schwaab, Rainer; Oldenburg, Johannes

2011-01-01

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor. PMID:21311577

Antitumor activity of resveratrol is independent of Cu(II) complex formation in MCF-7 cell line.

PubMed

Andrade Volkart, Priscylla; Benedetti Gassen, Rodrigo; Mühlen Nogueira, Bettina; Nery Porto, Bárbara; Eduardo Vargas, José; Arigony Souto, André

2017-08-01

Resveratrol (Rsv) is widely reported to possess anticarcinogenic properties in a plethora of cellular and animal models having limited toxicity toward normal cells. In the molecular level, Rsv can act as a suppressive agent for several impaired signaling pathways on cancer cells. However, Fukuhara and Miyata have shown a non-proteic reaction of Rsv, which can act as a prooxidant agent in the presence of copper (Cu), causing cellular oxidative stress accompanied of DNA damage. After this discovery, the complex Rsv-Cu was broadly explored as an antitumor mechanism in multiples tumor cell lines. The aim of the study is to explore the anticarcinogenic behavior of resveratrol-Cu(II) complex in MCF-7 cell line. Selectivity of Rsv binding to Cu ions was analyzed by HPLC and UV-VIS. The cells were enriched with concentrations of 10 and 50µM CuSO 4 solution and treated with 25µM of Rsv. Copper uptake after enrichment of cells, as its intracellular distribution in MCF-7 line, was scanned by ICP-MS and TEM-EDS. Cell death and intracellular ROS production were determined by flow cytometry. Different from the extracellular model, no relationship of synergy between Rsv-Cu(II) and reactive oxidative species (ROS) production was detected in vitro. ICP-MS revealed intracellular copper accumulation to both chosen concentrations (0.33±0.09 and 1.18±0.13ppb) but there is no promotion of cell death by Rsv-Cu(II) complex. In addition, significant attenuation of ROS production was detected when cells were exposed to CuSO 4 after Rsv treatment, falling from 7.54% of ROS production when treated only with Rsv to 3.07 and 2.72% with CuSO 4 . Based on these findings antitumor activity of resveratrol when in copper ions presence, is not mediated by Rsv-Cu complex formation in MCF-7 human cell line, suggesting that the antitumoral reaction is dependent of a cancer cellular model. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dynamics of human cancer cell lines monitored by electrical and acoustic fluctuation analysis.

PubMed

Tarantola, Marco; Marel, Anna-Kristina; Sunnick, Eva; Adam, Holger; Wegener, Joachim; Janshoff, Andreas

2010-03-01

Early determination of the metastatic potential of cancer cells is a crucial step for successful oncological treatment. Besides the remarkable progress in molecular genomics- or proteomics-based diagnostics, there is a great demand for in vitro biosensor devices that allow rapid and selective detection of the invasive properties of tumor cells. Here, the classical cancer cell motility in vitro assays for migration and invasion relying on Boyden chambers are compared to a real-time biosensor that analyzes the dynamic properties of adherent cells electro-acoustically with a time resolution on the order of seconds. The sensor relies on the well-established quartz crystal microbalance technique (QCM) that measures the shift in resonance frequency and damping of an oscillating quartz crystal when adsorption, desorption or changes in material properties close to the quartz surface occur. In addition, the QCM is capable of detecting the rather subtle fluctuations of the cell bodies as an indicator for their micromotility. QCM-based micromotility readings of three different cancer cell lines (HT-29, HSC-4, FaDu) are compared with the well-known electrical cell-substrate impedance sensing (ECIS) revealing collective stochastic motion that corresponds to the malignancy of the cells.

Integration of Breast Cancer Secretomes with Clinical Data Elucidates Potential Serum Markers for Disease Detection, Diagnosis, and Prognosis.

PubMed

Ziegler, Yvonne S; Moresco, James J; Yates, John R; Nardulli, Ann M

2016-01-01

Cancer cells secrete factors that influence adjacent cell behavior and can lead to enhanced proliferation and metastasis. To better understand the role of these factors in oncogenesis and disease progression, estrogen and progesterone receptor positive MCF-7 cells, triple negative breast cancer MDA-MB-231, DT22, and DT28 cells, and MCF-10A non-transformed mammary epithelial cells were grown in 3D cultures. A special emphasis was placed on triple negative breast cancer since these tumors are highly aggressive and no targeted treatments are currently available. The breast cancer cells secreted factors of variable potency that stimulated proliferation of the relatively quiescent MCF-10A cells. The conditioned medium from each cell line was subjected to mass spectrometry analysis and a variety of secreted proteins were identified including glycolytic enzymes, proteases, protease inhibitors, extracellular matrix proteins, and insulin-like growth factor binding proteins. An investigation of the secretome from each cell line yielded clues about strategies used for breast cancer proliferation and metastasis. Some of the proteins we identified may be useful in the development of a serum-based test for breast cancer detection, diagnosis, prognosis, and monitoring.

Triterpenoid Acids as Important Antiproliferative Constituents of European Elderberry Fruits.

PubMed

Gleńsk, Michał; Czapińska, Elżbieta; Woźniak, Marta; Ceremuga, Ireneusz; Włodarczyk, Maciej; Terlecki, Grzegorz; Ziółkowski, Piotr; Seweryn, Ewa

2017-01-01

In Europe, both the fruits and flowers of Sambucus nigra L. have been used against cold, as well as laxative, diaphoretic, and diuretic remedies. There are also a number of commercially available food products that contain elderberry juice, puréed or dried elderberries. Recent comprehensive literature data on pharmacology and chemistry of Sambuci fructus have encouraged us to screen extracts with different polarities from this plant material against cancer cell lines. The cytotoxic activity of the ethyl acetate and aqueous acetone extracts from elderberries as well as detected triterpenoids on human colon adenocarcinoma cell line (LoVo) and human breast cancer cell line (MCF-7) was investigated by sulforhodamine B assay. Moreover, cell migration assay was conducted for triterpenoid fraction and pure compounds. Aqueous acetone extract possessed much lower IC 50 value in cancer cell lines compared to ethyl acetate extract. The latter manifested high cytotoxicity against studied cell lines, suggesting that nonpolar compounds are responsible for the cytotoxic activity. Indeed, the phytochemical analysis revealed that ursolic and oleanolic acids are the main triterpenoids in the mentioned extract of which ursolic acid showed the highest activity with IC 50 values of 10.7 µg/mL on MCF-7 and 7.7 µg/mL on LoVo cells.

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

PubMed Central

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

2009-01-01

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

Expression of prostaglandin- and vitamin D-metabolising enzymes in benign and malignant breast cells.

PubMed

Thill, Marc; Hoellen, Friederike; Becker, Steffi; Dittmer, Christine; Fischer, Dorothea; Kümmel, Sherko; Salehin, Darius; Friedrich, Michael; Köster, Frank; Diedrich, Klaus; Cordes, Tim

2012-01-01

Cyclooxygenase-2 (COX-2) plays a crucial role in prognosis of malignancy and has been associated with carcinogenesis, particularly neoangiogenesis and tumor progression. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is described as a tumour suppressor in cancer. The antiproliferative effects of calcitriol [1,25(OH)(2)D(3)] mediated via the vitamin D receptor (VDR) render vitamin D a promising target in breast cancer therapy. The expression of prostaglandin (PG)-metabolizing enzymes, vitamin D-metabolising enzymes and VDR were determined in benign and malignant breast cell lines using western blot analysis. We detected an inverse correlation between the two types of metabolism, a reduced VDR expression in the malignant breast cell lines, and therefore an insufficient induction of 24-hydroxylase in the malignant cells. We suggest the possibility of dysregulation of vitamin D-metabolizing enzymes in malignant breast cell lines.

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

PubMed

Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus.

PubMed

Kim, Ji Woo; Kim, Hye-Min; Lee, Sang Mi; Kang, Man-Jong

2012-10-01

The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.

Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method.

PubMed

Zhang, Yanni; Tang, En-Tzu; Du, Zhiqiang

2016-01-01

The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision. The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively. In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 (95% CI, 0.498-1.000; P <0.001). The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.

Using mass spectrometry to detect, differentiate, and semiquantitate closely related peptide hormones in complex milieu: measurement of IGF-II and vesiculin.

PubMed

Lee, Kate L; Middleditch, Martin J; Williams, Geoffrey M; Brimble, Margaret A; Cooper, Garth J S

2015-03-01

The search for an islet β-cell growth factor has been a key objective in recent diabetes research, because the ability to regenerate and/or protect the functioning β-cell population in patients could result in a great advancement for diabetes treatment. IGF-I and IGF-II are known to play crucial roles in fetal growth and prenatal development, and there is growing evidence that IGF-II increases β-cell proliferation and survival in vitro and in vivo. A search for the source of IGF-II-like immunoreactivity in isolated β-cell secretory granules from the murine cell line βTC6-F7 revealed a novel 2-chain IGF-II-derived peptide, which we named vesiculin and which has been shown to be a full insulin agonist. Here, we present a liquid chromatography-tandem mass spectrometry method that enables selective detection and semiquantitation of the highly related IGF-II and vesiculin molecules. We have used this method to measure these 2 peptides in conditioned media from 2 β-cell lines, produced under increasing glucose concentrations. This technique detected both IGF-II and vesiculin in media conditioned by MIN6 and βTC6-F7 cells at levels in the range of 0 to 6 μM (total insulin, 80-450 μM) and revealed a glucose-stimulated increase in insulin, IGF-II, and vesiculin. IGF-II was detected in adult human and neonatal mouse serum in high levels, but vesiculin was not present. The methodology we present herein has utility for detecting and differentiating active peptides that are highly related and of low abundance.

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

NASA Technical Reports Server (NTRS)

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

2001-01-01

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

Immortalisation of a human diploid fibroblast cell strain: a DT-diaphorase paradox.

PubMed Central

Kuehl, B. L.; Brezden, C. B.; Traver, R. D.; Siegel, D.; Ross, D.; Renzing, J.; Rauth, A. M.

1996-01-01

Transfection of a normal human diploid fibroblast cell strain, GM38, with a simian virus 40 (SV40) large T antigen containing plasmid, yielded an immortal cell line, G38-8X, which had a similar sensitivity as the parental cell strain to the quinone-containing chemotherapeutic agent mitomycin C (MMC), under both aerobic and hypoxic exposure conditions. The activity level of DT-diaphorase was similar in both the parental GM38 and G38-8X cells. Although DT-diaphorase could be detected by Western blot analysis, using two mouse anti-human monoclonal antibodies, in GM38 cells, it was not detected in the G38-8X cells. G38-8X cells have a slightly increased P450R activity (2-fold), and have elevated P-glycoprotein levels compared with the parental GM38 cell strain. The immortal G38-8X cell line is 2-fold more resistant to ionising radiation than the parental GM38 cell strain (D10 approximately 5 Gy). Although these SV40 large T antigen immortalised human diploid fibroblasts behaved similarly to their parental cell strain in terms of MMC sensitivity and DT-diaphorase activity, careful characterisation revealed that these cells had enhanced P-glycoprotein activity and had a decreased sensitivity to ionising radiation. Images Figure 3 PMID:8763839

[Lentivirus-mediated RNA interference of CD133 inhibits the proliferation of CD133(+) liver cancer stem cells and increases their cisplatin chemosensitivity].

PubMed

Lan, Xi; Wang, Yong; Cao, Shu; Zou, Dongling; Li, Fang; Li, Shaolin

2012-12-01

To study the effects of CD133 suppression by lentivirus-mediated RNA interference (RNAi) on the proliferation and chemosensitivity of CD133(+) cancer stem cells (CSCs) sorted from HepG2 cell line. CD133(+) and CD133- cells were sorted from HepG2 cell line by flow cytometry, and the expression of CD133 before and after cell sorting were detected. The stem cell property of sorted CD133(+) cells were validated by sphere-forming assay in vitro and xenograft experiments in vivo. Lentivirus-mediated short hairpin RNA (shRNA) targeting CD133 were transfected into CD133(+) cells, and CD133 mRNA and protein expressions of the transfected cells were detected by RT-PCR and Western blotting, respectively. Before and after the transfection, the proliferative ability of CD133(+) cells was evaluated by colony formation assay, and the cell growth inhibition rate and apoptosis following cisplatin exposure were detected using CCK-8 assay and flow cytometry. The sorted CD133(+) cells showed a high purity of (88.74∓3.19)%, as compared with the purity of (3.36∓1.80)% before cell sorting. CD133(+) cells showed a high tumor sphere formation ability and tumorigenesis capacity compared with CD133- cells. CD133 shRNA transfection significantly inhibited CD133 mRNA and protein expressions in CD133(+) cells (P<0.01), resulting also in a significantly lowered cell proliferative ability (P<0.01) and an increased growth inhibition rate (P<0.01) and obviously increased cell apoptosis (P<0.05) after cisplatin exposure. Lentivirus-mediated RNAi for CD133 suppression inhibits the proliferation of CD133(+) liver cancer stem cells and increases their chemosensitivity to cisplatin.

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

PubMed

Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

2016-02-02

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. Copyright © 2015 Elsevier B.V. All rights reserved.

Influence of calcitriol on prostaglandin- and vitamin D-metabolising enzymes in benign and malignant breast cell lines.

PubMed

Thill, Marc; Cordes, Tim; Hoellen, Friederike; Becker, Steffi; Dittmer, Christine; Kümmel, Sherko; Salehin, Darius; Friedrich, Michael; Diedrich, Klaus; Köster, Frank

2012-01-01

Cyclooxygenase-2 (COX-2) is a potential molecular prognostic factor for breast cancer, and calcitriol [1,25(OH)(2)D(3)], the biologically active form of vitamin D, is a promising target in breast cancer therapy. The influence of calcitriol on the proliferation and the effects of calcitriol on the expression of prostaglandin- and vitamin D-metabolising enzymes were examined in benign and malignant breast cells. Calcitriol inhibited the proliferation of MCF-10F and MCF-7 cells but not of invasive MDA-MB-231 cells and reduced the expression of COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in the benign breast cell line MCF-10F. Furthermore, dysregulation in vitamin D-metabolising proteins was detected, especially in MDA-MB-231 cells. These results suggest dysregulation of vitamin D metabolism and a lack of a possible influence of calcitriol on the metabolism of prostaglandins in the malignant breast cell lines.

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

PubMed

Hovsépian, S; Aouani, A; Fayet, G

1986-05-01

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

Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells.

PubMed

Daugherty, D F; Dickinson, C J; Takeuchi, T; Bachwich, D; Yamada, T

1991-05-01

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

Cytotoxicity and apoptosis of ovarian and breast cancer cell lines induced by OVS1 monoclonal antibody and paclitaxel.

PubMed

Moongkarndi, Primchanien; Kaslungka, Sineenart; Kosem, Nuttavut; Junnu, Sarawut; Jongsomboonkusol, Suna; Theptaranon, Yodsaward; Neungton, Neelobol

2003-03-01

OVS1 monoclonal antibody (MAb) produced against ovarian cancer is currently used to identify mucinous cystadenocarcinoma antigen as a tumor marker secreted in serum. The potential of OVS1 MAb in ovarian cancer treatment was studied by evaluating the induction of cytotoxicity and apoptosis of SKOV3 ovarian cancer and BT549 breast cancer cell lines induced by OVS1. Paclitaxel, an antitumor drug, was used as positive control and applied as a combined drug together with OVS1 MAb. OVS1 MAb and paclitaxel were found by MTT assay to induce cytotoxicity against both cell lines. The ED50 of OVS1 MAb were 26.25 and 25.00 microg/ml and of paclitaxel were 21.88 and 9.20 nM against SKOV3 and BT549 cell lines, respectively. The quantitative amount of cells determined by fluorimetric assay was correlated to the results of the MTT assay. The combined application of OVS1 MAb and paclitaxel on these two cell lines resulted in a greater cytotoxicity than observed by either agent alone. OVS1 MAb and paclitaxel applied against both cell lines induced the morphological changes of apoptotic cell death at 24 hours visualized by two color fluorescence dyes, Ho33342 and propidium iodide. Combination of the two substances enhanced the rate of apoptosis compared to either OVS1 MAb or paclitaxel given alone. DNA fragmentation was detected in an agarose gel electrophoresis after treating cells with OVS1 MAb and paclitaxel at 24 hours. These findings on the induction of cytotoxicity and apoptosis by OVS1 MAb on cancer cell lines have implications on the potential application of OVS1 MAb for clinical therapy.

The novel ependymin related gene UCC1 is highly expressed in colorectal tumor cells.

PubMed

Nimmrich, I; Erdmann, S; Melchers, U; Chtarbova, S; Finke, U; Hentsch, S; Hoffmann, I; Oertel, M; Hoffmann, W; Müller, O

2001-04-10

Normal cells differ from malignant tumor cells in the transcription levels of many different genes. Two colorectal tumor cell lines were compared with a normal colorectal cell line by differential display reverse transcription PCR to screen for tumor cell specific differentially transcribed genes. By this strategy the upregulation of a novel gene was detected designated as 'upregulated in colorectal cancer gene-1' (UCC1). The UCC1 gene transcript level is increased in cultured tumor cells and in two out of three analyzed colorectal tumor tissue specimens compared to normal cultured cells and to corresponding normal tissue samples. Remarkably, the UCC1 protein shows significant sequence similarity to the highly divergent piscine glycoproteins termed ependymins which are synthesized by leptomeningeal fibroblasts and secreted into the cerebrospinal fluid.

Acute Exposure to High Dose γ-Radiation Results in Transient Activation of Bone Lining Cells

PubMed Central

Turner, Russell T.; Iwaniec, Urszula T.; Wong, Carmen P.; Lindenmaier, Laurence B.; Wagner, Lindsay A.; Branscum, Adam J.; Menn, Scott A.; Taylor, James; Zhang, Ye; Wu, Honglu; Sibonga, Jean D.

2014-01-01

The present studies investigated the cellular mechanisms for the detrimental effects of high dose whole body γ-irradiation on bone. In addition, radioadaptation and bone marrow transplantation were assessed as interventions to mitigate the skeletal complications of irradiation. Increased trabecular thickness and separation and reduced fractional cancellous bone volume, connectivity density, and trabecular number were detected in proximal tibia and lumbar vertebra 14 days following γ-irradiation with 6 Gy. To establish the cellular mechanism for the architectural changes, vertebrae were analyzed by histomorphometry 1, 3, and 14 days following irradiation. Marrow cell density decreased within 1 day (67% reduction, p<0.0001), reached a minimum value after 3 days (86% reduction, p<0.0001), and partially rebounded by 14 days (30% reduction, p=0.0025) following irradiation. In contrast, osteoblast-lined bone perimeter was increased by 290% (1 day, p=0.04), 1230% (3 days, p<0.0001), and 530% (14 days, p=0.003), respectively. There was a strong association between radiation-induced marrow cell death and activation of bone lining cells to express the osteoblast phenotype (Pearson correlation −0.85, p<0.0001). An increase (p=0.004) in osteoclast-lined bone perimeter was also detected with irradiation. A priming dose of γ-radiation (0.5 mGy), previously shown to reduce mortality, had minimal effect on the cellular responses to radiation and did not prevent detrimental changes in bone architecture. Bone marrow transplantation normalized marrow cell density, bone turnover, and most indices of bone architecture following irradiation. In summary, radiation-induced death of marrow cells is associated with 1) a transient increase in bone formation due, at least in part, to activation of bone lining cells, and 2) an increase in bone resorption due to increased osteoclast perimeter. Bone marrow transplantation is effective in mitigating the detrimental effects of acute exposure to high dose whole body γ-radiation on bone turnover. PMID:23954507

Detection and proteomic characterization of extracellular vesicles in human pancreatic juice.

PubMed

Osteikoetxea, Xabier; Benke, Márton; Rodriguez, Marta; Pálóczi, Krisztina; Sódar, Barbara W; Szvicsek, Zsuzsanna; Szabó-Taylor, Katalin; Vukman, Krisztina V; Kittel, Ágnes; Wiener, Zoltán; Vékey, Károly; Harsányi, László; Szűcs, Ákos; Turiák, Lilla; Buzás, Edit I

2018-04-30

The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer. In addition, we also studied blood plasma and pancreatic cancer cell line-derived EVs. Comparative proteomic analysis was performed of 102 EV preparations from human pancreatic juices, blood, and pancreatic cancer cell lines Capan-1 and MIA PaCa-2. EV preparations were also characterized by electron microscopy, tunable resistive pulse sensing, and flow cytometry. Here we describe the presence of EVs in human pancreatic juice samples. Pancreatic juice EV-associated proteins that we identified as possible candidate markers for pancreatic cancer included mucins, such as MUC1, MUC4, MUC5AC, MUC6 and MUC16, CFTR, and MDR1 proteins. These candidate biomarkers could also be detected by flow cytometry in EVs found in pancreatic juice and those secreted by pancreatic cancer cell lines. Together our data show that detection and characterization of EVs directly in pancreatic juice is feasible and may prove to be a valuable source of potential biomarkers of pancreatic cancer. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1.

PubMed

Bakos, Agnes; Banati, Ferenc; Koroknai, Anita; Takacs, Maria; Salamon, Daniel; Minarovits-Kormuta, Susanna; Schwarzmann, Fritz; Wolf, Hans; Niller, Hans Helmut; Minarovits, Janos

2007-10-01

Transcripts for the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNAs) are initiated at alternative promoters (Wp, Cp, for EBNA 1-6 transcripts and Qp, for EBNA 1 transcripts only) located in the BamHI W, C or Q fragment of the viral genome. To understand the host-cell dependent expression of EBNAs in EBV-associated tumors (lymphomas and carcinomas) and in vitro transformed cell lines, it is necessary to analyse the regulatory mechanisms governing the activity of the alternative promoters of EBNA transcripts. Such studies focused mainly on lymphoid cell lines carrying latent EBV genomes, due to the lack of EBV-associated carcinoma cell lines maintaining latent EBV genomes during cultivation in tissue culture. We took advantage of the unique nasopharyngeal carcinoma cell line, C666-1, harboring EBV genomes, and undertook a detailed analysis of CpG methylation patterns and in vivo protein-DNA interactions at the latency promoters Qp and Cp. We found that the active, unmethylated Qp was marked with strong footprints of cellular transcription factors and the viral protein EBNA 1. In contrast, we could not detect binding of relevant transcription factors to the methylated, silent Cp. We concluded that the epigenetic marks at Qp and Cp in C666-1 cells of epithelial origin resemble those of group I Burkitt's lymphoma cell lines.

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

PubMed

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

2018-06-14

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

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

PubMed Central

McKinnon, R D; Danielson, P; Brow, M A; Bloom, F E; Sutcliffe, J G

1987-01-01

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture. Images PMID:2439903

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Integration of High-Risk Human Papillomavirus into Cellular Cancer-Related Genes in Head and Neck Cancer Cell Lines

PubMed Central

Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-01-01

Background HPV-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. This study evaluates viral oncogene expression and viral integration sites in HPV16 and HPV18-positive squamous carcinoma cell lines. Methods E6-E7 alternate transcripts were assessed by RT-PCR. Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. Results All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 HNSCC lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. Conclusions HPV integration into cancer-related genes occurred in 7/9 HPV-positive cell lines and of these six were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. PMID:28236344

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

PubMed

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

2015-01-01

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

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

PubMed

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

2017-10-01

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

Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging.

PubMed

Mallett, Christiane L; McFadden, Catherine; Chen, Yuhua; Foster, Paula J

2012-07-01

A novel cell line of cytotoxic natural killer (NK) cells, KHYG-1, was examined in vivo for immunotherapy against prostate cancer. The feasibility of using magnetic resonance imaging (MRI) tracking to monitor the fate of injected NK cells following intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) administration was assessed. PC-3M human prostate cancer cells were injected s.c. into the flank of nude mice (day 0). KHYG-1 NK cells were labeled with an iron oxide contrast agent and injected s.c., i.v. or i.p. on day 8. Mice were imaged by MRI on days 7, 9 and 12. Tumor sections were examined with fluorescence microscopy and immunohistologic staining for NK cells. NK cells were detected in the tumors by histology after all three administration routes. NK cells and fluorescence from the iron label were co-localized. Signal loss was seen in the areas around the tumors and between the tumor lobes in the s.c. group. We are the first to label this cell line of NK cells with an iron oxide contrast agent. Accumulation of NK cells was visualized by MRI after s.c. injection but not after i.v. and i.p. injection.

Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

PubMed

Zhang, Jialin; Liu, Wenxing; Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao

2016-01-01

From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines.

PubMed

Tanaka, Kyoko; Miyazawa, Masaki; Mikami, Mikio; Aoki, Daisuke; Kiguchi, Kazushige; Iwamori, Masao

2016-10-01

Monoclonal antibody YHD-06 generated by immunization with GM2 reacted with gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, among which GalNAc-GD1a was characterized as an antigen of autoimmune peripheral neuropathies including Guillain-Barré syndrome. When glycolipids were examined by TLC-immunostaining with YHD-06 in seven human cervical carcinoma-derived cell lines, GM2 was found in all cell lines, amounting to 15.5 % to 57.5 % of total gangliosides. Whereas GalNAc-GD1a was present in three cell lines, amounting to 5.4-17.5 % of total gangliosides, and GalNAc-GM1b in four cell lines in amounts of less than 2 %. The elevated amounts of gangliosides with GM2 determinant were closely correlated with the relative intensities of gene expression of GalNAc transferase, this being characteristic of cervical carcinoma-derived cells. However, in tissues from patients with several histological types of cervical carcinomas, GM3 was ubiquitously expressed in amounts of more than 66 % of total gangliosides, GM2 was expressed in only five of 15 tissues, and both GalNAc-GM1b and GalNAc-GD1a were not even detected in trace amounts. Since GM1 was detected in all tissues in amounts of less than 0.06 μg/mg dried tissue, all cervical carcinoma tissues were revealed to exhibit GM2 synthesis, indicating that enhanced synthesis of gangliosides with GM2 determinant is a characteristic of cultivated cells in vitro. Similarly, although I(3)SO3-GalCer was not present in the squamous cell carcinoma (SCC) tissues, SCC-derived cells selectively expressed II(3)SO3-LacCer. Since enhanced synthesis of GM2 has been reported in SV-40 virus-transfected fibroblasts, papilloma virus might be involved in the expression of GM2 in cervical carcinoma-derived cells.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

PubMed

Manca, Antonella; Sini, Maria Cristina; Izzo, Francesco; Ascierto, Paolo A; Tatangelo, Fabiana; Botti, Gerardo; Gentilcore, Giusy; Capone, Marilena; Mozzillo, Nicola; Rozzo, Carla; Cossu, Antonio; Tanda, Francesco; Palmieri, Giuseppe

2011-06-01

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

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

PubMed Central

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

2007-01-01

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

Persistent poliovirus infection of human fetal brain cells.

PubMed

Pavio, N; Buc-Caron, M H; Colbère-Garapin, F

1996-09-01

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis.

E5 can be expressed in anal cancer and leads to epidermal growth factor receptor-induced invasion in a human papillomavirus 16-transformed anal epithelial cell line.

PubMed

Wechsler, Erin Isaacson; Tugizov, Sharof; Herrera, Rossana; Da Costa, Maria; Palefsky, Joel M

2018-05-01

We detected the first human papillomavirus (HPV)-16-immortalized anal epithelial cell line, known as AKC2 cells to establish an in vitro model of HPV-16-induced anal carcinogenesis. Consistent with detection of E6, E7 and E5 expression in anal cancer biopsies, AKC2 cells expressed high levels of all three HPV oncogenes. Also, similar to findings in anal cancer biopsies, epidermal growth factor receptor (EGFR) was overexpressed in AKC2 cells. AKC2 cells exhibited a poorly differentiated and invasive phenotype in three-dimensional raft culture and inhibition of EGFR function abrogated AKC2 invasion. Reducing E5 expression using E5-targeted siRNAs in AKC2 cells led to knockdown of E5 expression, but also HPV-16 E2, E6 and E7 expression. AKC2 cells treated with E5-targeted siRNA had reduced levels of total and phosphorylated EGFR, and reduced invasion. Rescue of E6/E7 expression with simultaneous E5 knockdown confirmed that E5 plays a key role in EGFR overexpression and EGFR-induced invasion.

Microwave frequency sensor for detection of biological cells in microfluidic channels.

PubMed

Nikolic-Jaric, M; Romanuik, S F; Ferrier, G A; Bridges, G E; Butler, M; Sunley, K; Thomson, D J; Freeman, M R

2009-08-12

We present details of an apparatus for capacitive detection of biomaterials in microfluidic channels operating at microwave frequencies where dielectric effects due to interfacial polarization are minimal. A circuit model is presented, which can be used to adapt this detection system for use in other microfluidic applications and to identify ones where it would not be suitable. The detection system is based on a microwave coupled transmission line resonator integrated into an interferometer. At 1.5 GHz the system is capable of detecting changes in capacitance of 650 zF with a 50 Hz bandwidth. This system is well suited to the detection of biomaterials in a variety of suspending fluids, including phosphate-buffered saline. Applications involving both model particles (polystyrene microspheres) and living cells-baker's yeast (Saccharomyces cerevisiae) and Chinese hamster ovary cells-are presented.

Amyloid Precursor-like Protein 2 Association with HLA Class I Molecules

PubMed Central

Tuli, Amit; Sharma, Mahak; Wang, Xiaojian; Simone, Laura C.; Capek, Haley L.; Cate, Steven; Hildebrand, William H.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

2009-01-01

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2Kd and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression. PMID:19184004

A portable cell-based optical detection device for rapid detection of Listeria and Bacillus toxins

NASA Astrophysics Data System (ADS)

Banerjee, Pratik; Banada, Padmapriya P.; Rickus, Jenna L.; Morgan, Mark T.; Bhunia, Arun K.

2005-11-01

A mammalian cell-based optical biosensor was built to detect pathogenic Listeria and Bacillus species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic L. monocytogenes strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from B. cereus strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic L. innocua (F4247) and a B. substilis induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic Listeria and Bacillus species and can potentially be used onsite for food safety or in biosecurity application.

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

PubMed

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

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. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2008-01-01

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

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine.

PubMed

Hayashi, Masamichi; Bernert, Heike; Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O

2014-05-30

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

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

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

Witasp, Erika; Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm; Gustafsson, Ann-Catrin

2005-05-13

Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 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){sub 2}D{sub 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 thismore » model. Moreover, 1,25(OH){sub 2}D{sub 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{sub 3}.« less

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

PubMed

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

2015-06-01

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

Peptidomic analysis of human cell lines

PubMed Central

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

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Optimisation of immunofluorescence methods to determine MCT1 and MCT4 expression in circulating tumour cells.

PubMed

Kershaw, Stephen; Cummings, Jeffrey; Morris, Karen; Tugwood, Jonathan; Dive, Caroline

2015-05-10

The monocarboxylate transporter-1 (MCT1) represents a novel target in rational anticancer drug design while AZD3965 was developed as an inhibitor of this transporter and is undergoing Phase I clinical trials ( http://www.clinicaltrials.gov/show/NCT01791595 ). We describe the optimisation of an immunofluorescence (IF) method for determination of MCT1 and MCT4 in circulating tumour cells (CTC) as potential prognostic and predictive biomarkers of AZD3965 in cancer patients. Antibody selectivity was investigated by western blotting (WB) in K562 and MDAMB231 cell lines acting as positive controls for MCT1 and MCT4 respectively and by flow cytometry also employing the control cell lines. Ability to detect MCT1 and MCT4 in CTC as a 4(th) channel marker utilising the Veridex™ CellSearch system was conducted in both human volunteer blood spiked with control cells and in samples collected from small cell lung cancer (SCLC) patients. Experimental conditions were established which yielded a 10-fold dynamic range (DR) for detection of MCT1 over MCT4 (antibody concentration 6.25 μg/mL; integration time 0.4 seconds) and a 5-fold DR of MCT4 over MCT 1 (8 μg/100 μL and 0.8 seconds). The IF method was sufficiently sensitive to detect both MCT1 and MCT4 in CTCs harvested from cancer patients. The first IF method has been developed and optimised for detection of MCT 1 and MCT4 in cancer patient CTC.

A new indicator cell line established to monitor bovine foamy virus infection.

PubMed

Guo, Hong-Yan; Liang, Zhi-Bin; Li, Yue; Tan, Juan; Chen, Qi-Min; Qiao, Wen-Tao

2011-10-01

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells.

PubMed

Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

2014-03-28

Piwi-interacting RNAs (piRNAs) are 26-31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Fish cell lines as a tool for the ecotoxicity assessment and ranking of engineered nanomaterials.

PubMed

Bermejo-Nogales, A; Fernández-Cruz, M L; Navas, J M

2017-11-01

Risk assessment of engineered nanomaterials (ENMs) is being hindered by the sheer production volume of these materials. In this regard, the grouping and ranking of ENMs appears as a promising strategy. Here we sought to evaluate the usefulness of in vitro systems based on fish cell lines for ranking a set of ENMs on the basis of their cytotoxicity. We used the topminnow (Poeciliopsis lucida) liver cell line (PLHC-1) and the rainbow trout (Oncorhynchus mykiss) fibroblast-like gonadal cell line (RTG-2). ENMs were obtained from the EU Joint Research Centre repository. The size frequency distribution of ENM suspensions in cell culture media was characterized. Cytotoxicity was evaluated after 24 h of exposure. PLHC-1 cells exhibited higher sensitivity to the ENMs than RTG-2 cells. ZnO-NM was found to exert toxicity mainly by altering lysosome function and metabolic activity, while multi-walled carbon nanotubes (MWCNTs) caused plasma membrane disruption at high concentrations. The hazard ranking for toxicity (ZnO-NM > MWCNT ≥ CeO 2 -NM = SiO 2 -NM) was inversely related to the ranking in size detected in culture medium. Our findings reveal the suitability of fish cell lines for establishing hazard rankings of ENMs in the framework of integrated approaches to testing and assessment. Copyright © 2017 Elsevier Inc. All rights reserved.

Acoustical nanometre-scale vibrations of live cells detected by a near-field optical setup

NASA Astrophysics Data System (ADS)

Piga, Rosaria; Micheletto, Ruggero; Kawakami, Yoichi

2007-04-01

The Scanning Near-field Optical Microscope (SNOM) is able to detect tiny vertical movement on the cell membrane in the range of only 1 nanometer or less, about 3 orders of magnitude better than conventional optical microscopes. Here we show intriguing data of cell membrane nanometer-scale dynamics associated to different phenomena of the cell’s The Scanning Near-field Optical Microscope (SNOM) is able to detect tiny vertical movement on the cell membrane in the range of only 1 nanometer or less, about 3 orders of magnitude better than conventional optical microscopes. Here we show intriguing data of cell membrane nanometer-scale dynamics associated to different phenomena of the cell’s life, such as cell cycle and cell death, on rat pheochromocytoma line PC12. Working in culture medium with alive and unperturbed samples, we could detect nanometer-sized movements; Fourier components revealed a clear distinct behavior associated to regulation of neurite outgrowth and changes on morphology after necrotic stimulus.

Diode/magnetic tunnel junction cell for fully scalable matrix-based biochip

NASA Astrophysics Data System (ADS)

Cardoso, F. A.; Ferreira, H. A.; Conde, J. P.; Chu, V.; Freitas, P. P.; Vidal, D.; Germano, J.; Sousa, L.; Piedade, M. S.; Costa, B. A.; Lemos, J. M.

2006-04-01

Magnetoresistive biochips have been recently introduced for the detection of biomolecular recognition. In this work, the detection site incorporates a thin-film diode in series with a magnetic tunnel junction (MTJ), leading to a matrix-based biochip that can be easily scaled up to screen large numbers of different target analytes. The fabricated 16×16 cell matrix integrates hydrogenated amorphous silicon (a-Si:H) diodes with aluminum oxide barrier MTJ. Each detection site also includes a U-shaped current line for magnetically assisted target concentration at probe sites. The biochip is being integrated in a portable, credit card size electronics control platform. Detection of 250 nm diameter magnetic nanoparticles by one of the matrix cells is demonstrated.

Mitigating the risk of Zika virus contamination of raw materials and cell lines in the manufacture of biologicals.

PubMed

Zmurko, Joanna; Vasey, Douglas B; Donald, Claire L; Armstrong, Alison A; McKee, Marian L; Kohl, Alain; Clayton, Reginald F

2018-02-01

Ensuring the virological safety of biologicals is challenging due to the risk of viral contamination of raw materials and cell banks, and exposure during in-process handling to known and/or emerging viral pathogens. Viruses may contaminate raw materials and biologicals intended for human or veterinary use and remain undetected until appropriate testing measures are employed. The outbreak and expansive spread of the mosquito-borne flavivirus Zika virus (ZIKV) poses challenges to screening human- and animal -derived products used in the manufacture of biologicals. Here, we report the results of an in vitro study where detector cell lines were challenged with African and Asian lineages of ZIKV. We demonstrate that this pathogen is robustly detectable by in vitro assay, thereby providing assurance of detection of ZIKV, and in turn underpinning the robustness of in vitro virology assays in safety testing of biologicals.

Inferring genome-wide interplay landscape between DNA methylation and transcriptional regulation.

PubMed

Tang, Binhua; Wang, Xin

2015-01-01

DNA methylation and transcriptional regulation play important roles in cancer cell development and differentiation processes. Based on the currently available cell line profiling information from the ENCODE Consortium, we propose a Bayesian inference model to infer and construct genome-wide interaction landscape between DNA methylation and transcriptional regulation, which sheds light on the underlying complex functional mechanisms important within the human cancer and disease context. For the first time, we select all the currently available cell lines (>=20) and transcription factors (>=80) profiling information from the ENCODE Consortium portal. Through the integration of those genome-wide profiling sources, our genome-wide analysis detects multiple functional loci of interest, and indicates that DNA methylation is cell- and region-specific, due to the interplay mechanisms with transcription regulatory activities. We validate our analysis results with the corresponding RNA-sequencing technique for those detected genomic loci. Our results provide novel and meaningful insights for the interplay mechanisms of transcriptional regulation and gene expression for the human cancer and disease studies.

In situ detection of estrogen receptor dimers in breast carcinoma cells in archival materials using proximity ligation assay (PLA).

PubMed

Iwabuchi, Erina; Miki, Yasuhiro; Ono, Katsuhiko; Onodera, Yoshiaki; Suzuki, Takashi; Hirakawa, Hisashi; Ishida, Takanori; Ohuchi, Noriaki; Sasano, Hironobu

2017-01-01

Estrogen receptor (ER) is required for carcinoma cell proliferation in the great majority of breast cancer and also functions as a dimer. ER dimeric proteins have been largely identified by BRET/FRET analyses but their in situ visualization have not yet been reported. Recently, in situ Proximity Ligation Assay (PLA) has been developed as the methods detecting protein interactions in situ. Therefore, in this study we firstly demonstrated the dimerization of ERα in breast carcinoma cell lines and tissues using PLA. The human breast carcinoma cell lines MCF-7, T-47D and MDA-MB-231 were used in this study. Cells were treated with ER agonist or antagonist and fixed in 4% PFA, and ER dimers were subsequently detected using PLA. The evaluation of ER dimers in breast carcinoma cell lines were quantified by measuring the area of dots localized in the nuclei using image analysis. We also firstly demonstrated the visualization of ER dimer patterns in 10% formalin-fixed paraffin-embedded tissues of breast cancer using PLA technique. Estradiol (E2) administration induced ERα homodimers in the nuclei of MCF-7 and T-47D but not in ER-negative MDA-MB-231. 4-OH tamoxifen also induced ERα homodimers but the subcellular localization of these ERα homodimers was predominant in cytoplasm instead of the nuclei induced by E2 treatment. ICI182,780 treatment did decrease the number of formation of ERα homodimers in MCF-7. In breast cancer patients, ERα PLA score was significantly correlated positively with ERα- or PgR (progesterone receptor) immunohistochemical scores and inversely with Ki-67-labeling index, respectively. We also demonstrated the ERα/β heterodimer as well as ERα homodimers in both breast carcinoma cell lines and surgical pathology specimens. In summary, we did firstly succeed in the visualization of ER dimeric proteins using PLA method. The evaluation of ER dimer patterns could provide pivotal information as to the prediction of response to endocrine therapy of breast cancer patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Increased expression of the PI3K catalytic subunit p110δ underlies elevated S6 phosphorylation and protein synthesis in an individual with autism from a multiplex family.

PubMed

Poopal, Ashwini C; Schroeder, Lindsay M; Horn, Paul S; Bassell, Gary J; Gross, Christina

2016-01-01

Dysfunctions in the PI3K/mTOR pathway have gained a lot of attention in autism research. This was initially based on the discovery of several monogenic autism spectrum disorders with mutations or defects in PI3K/mTOR signaling components. Recent genetic studies corroborate that defective PI3K/mTOR signaling might be a shared pathomechanism in autism disorders of so far unknown etiology, but functional molecular analyses in human cells are rare. The goals of this study were to perform a functional screen of cell lines from patients with idiopathic autism for defects in PI3K/mTOR signaling, to test if further functional analyses are suitable to detect underlying molecular mechanisms, and to evaluate this approach as a biomarker tool to identify therapeutic targets. We performed phospho-S6- and S6-specific ELISA experiments on 21 lymphoblastoid cell lines from the AGRE collection and on 37 lymphoblastoid cell lines from the Simons Simplex Collection and their healthy siblings. Cell lines from one individual with increased S6 phosphorylation and his multiplex family were analyzed in further detail to identify upstream defects in PI3K signaling associated with autism diagnosis. We detected significantly increased S6 phosphorylation in 3 of the 21 lymphoblastoid cell lines from AGRE compared to a healthy control and in 1 of the 37 lymphoblastoid cell lines from the Simons Simplex Collection compared to the healthy sibling. Further analysis of cells from one individual with elevated S6 phosphorylation showed increased expression of the PI3K catalytic subunit p110δ, which was also observed in lymphoblastoid cells from other autistic siblings but not unaffected members in his multiplex family. The p110δ-selective inhibitor IC87114 reduced elevated S6 phosphorylation and protein synthesis in this cell line. Our results suggest that functional analysis of PI3K/mTOR signaling is a biomarker tool to identify disease-associated molecular defects that could serve as therapeutic targets in autism. Using this approach, we discovered impaired signaling and protein synthesis through the PI3K catalytic subunit p110δ as an underlying molecular defect and potential treatment target in select autism spectrum disorders. Increased p110δ activity was recently associated with schizophrenia, and our results suggest that p110δ may also be implicated in autism.

Kefir induces apoptosis and inhibits cell proliferation in human acute erythroleukemia.

PubMed

Jalali, Fatemeh; Sharifi, Mohammadreza; Salehi, Rasoul

2016-01-01

Acute erythroleukemia is an uncommon subtype of acute myeloid leukemia which has been considered to be a subtype of AML with a worse prognosis. Intensive chemotherapy is the first line of treatment. In recent years, the effect of kefir on some malignancies has been experimented. Kefir is a kind of beverage, which obtained by incubation of kefir grains with raw milk. Kefir grains are a symbiotic complex of different kinds of yeasts and bacteria, especially lactic acid bacteria which gather in a mostly carbohydrate matrix, named kefiran. We investigated the effect of kefir on acute erythroleukemia cell line (KG-1) and peripheral blood mononuclear cells (PBMCs). The cell line and PBMCs were treated with different doses of kefir and milk and incubated for three different times. We used Polymixin B to block the lipopolysaccharide and NaOH (1 mol/l) to neutralize the acidic media. Viability was detected by MTT assay. Apoptosis and necrosis were assessed by annexin-propidium iodide staining. Our results showed that kefir induced apoptosis and necrosis in KG-1 cell line. It was revealed that kefir decreased proliferation in erythroleukemia cell line. We did not observe a remarkable effect of kefir on PBMCs. Our study suggested that kefir may have potential to be an effective treatment for erythroleukemia.

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

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

Cell Line Controls for the Genotyping of a Spectrum of Human Single Nucleotide Polymorphisms in the Clinical Laboratory.

PubMed

Kimbacher, Christine; Paar, Christian; Freystetter, Andrea; Berg, Joerg

2018-05-01

Genotyping for clinically important single nucleotide polymorphisms (SNPs) is performed by many clinical routine laboratories. To support testing, quality controls and reference materials are needed. Those may be derived from residual patient samples, left over samples of external quality assurance schemes, plasmid DNA or DNA from cell lines. DNAs from cell lines are commutable and available in large amounts. DNA from 38 cell lines were examined for suitability as controls in 11 SNP assays that are frequently used in a clinical routine laboratory: FV (1691G>A), FII (20210G>A), PAI-1 4G/5G polymorphism, MTHFR (677C>T, 1298A>C), HFE (H63D, S65C, C282Y), APOE (E2, E3, E4), LPH (-13910C>T), UGT1A1 (*28, *36, *37), TPMT (*2, *3A, *3B, *3C), VKORC1 (-1639G>A, 1173C>T), CYP2C9 (*2, *3, *5). Genotyping was performed by real-time PCR with melting curve analysis and confirmed by bi-directional sequencing. We find an almost complete spectrum of genotypic constellations within these 38 cell lines. About 12 cell lines appear sufficient as genotypic controls for the 11 SNP assays by covering almost all of the genotypes. However, hetero- and homozygous genotypes for FII and the alleles TPMT*2, UGT1A1*37 and CYP2C9*5 were not detected in any of the cell lines. DNA from most of the examined cell lines appear suitable as quality controls for these SNP assays in the laboratory routine, as to the implementation of those assays or to prepare samples for quality assurance schemes. Our study may serve as a pilot to further characterize these cell lines to arrive at the status of reference materials.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

PubMed Central

2009-01-01

Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Conclusion Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression. PMID:19545448

Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture.

PubMed

Svobodova, Zuzana; Kucerova, Jana; Autebert, Julien; Horak, Daniel; Bruckova, Lenka; Viovy, Jean-Louis; Bilkova, Zuzana

2014-02-01

In this study, we describe a particular step in developing a microfluidic device for capture and detection of circulating tumor cells-specifically the preparation of an immunosorbent for implementation into the separation chip. We highlight some of the most important specifics connected with superparamegnetic microspheres for microfluidic purposes. Factors such as nonspecific adsorption on microfluidic channels, interactions with model cell lines, and tendency to aggregation were investigated. Poly(glycidyl methacrylate) microspheres with carboxyl groups were employed for this purpose. To address the aforementioned challenges, the microspheres were coated with hydrazide-PEG-hydrazide, and subsequently anti-epithelial cell adhesion molecule (EpCAM) antibody was immobilized. The prepared anti-EpCAM immunosorbent was pretested using model cell lines with differing EpCAM density (MCF7, SKBR3, A549, and Raji) in a batchwise arrangement. Finally, the entire system was implemented and studied in an Ephesia chip and an evaluation was performed by the MCF7 cell line. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Therapeutic potential of endothelin inhibitors in canine hemangiosarcoma.

PubMed

Fukumoto, Shinya; Saida, Kaname; Sakai, Hiroki; Ueno, Hiroshi; Iwano, Hidetomo; Uchide, Tsuyoshi

2016-08-15

Hemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. The preproendothelin-1 (PPET-1), endothelin type A receptor (ETA) and endothelin type B receptor (ETB) mRNA expression levels in HSA cell lines (n=5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n=11) and those in normal splenic tissues (n=6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. The PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. These findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties.

PubMed

Sarrab, Ramadan M; Lennon, Rachel; Ni, Lan; Wherlock, Matthew D; Welsh, Gavin I; Saleem, Moin A

2011-11-01

The aim of this study was to establish an immortalized human mesangial cell line similar to mesangial cells in vivo for use as a tool for understanding glomerular cell function. Mesangial cells were isolated from glomerular outgrowths from a normal human kidney, then retrovirally transfected with a temperature-sensitive SV40T antigen+human telomerase (hTERT). Mesangial cells exhibited features of compact cells with small bodies in a confluent monolayer at 33°C, but the cell shape changed to flat and stellate after 5 days in growth-restrictive conditions (37°C). Western blot and immunofluorescence analysis showed that podocyte markers (nephrin, CD2AP, podocin, Wilms' tumor-1) and an endothelial-specific molecule (VE-cadherin) were not detectable in this cell line, whereas markers characteristic of mesangial cells (α-SMA, fibronectin, and PDGFβ-R) were strongly expressed. In migration assays, a significant reduction in wound surface was observed in podocyte and endothelial cells as soon as 12 h (75 and 62%, respectively) and complete wound closure after 24 h. In contrast, no significant change was observed in mesangial cells after 12 h, and even after 48 h the wounds were not completely closed. Until now, conditionally immortalized podocyte and endothelial cell lines derived from mice and humans have been described, and this has greatly boosted research on glomerular physiology and pathology. We have established the first conditionally immortalized human glomerular mesangial cell line, which will be an important adjunct in studies of representative glomerular cells, as well as in coculture studies. Unexpectedly, mesangial cells' ability to migrate seems to be slower than for other glomerular cells, suggesting this line will demonstrate functional properties distinct from previously available mesangial cell cultures. This conditionally immortalized human mesangial cell line represents a new tool for the study of human mesangial cell biology in vitro.

High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

PubMed

Morimoto, Atsushi; Mogami, Toshifumi; Watanabe, Masaru; Iijima, Kazuki; Akiyama, Yasuyuki; Katayama, Koji; Futami, Toru; Yamamoto, Nobuyuki; Sawada, Takeshi; Koizumi, Fumiaki; Koh, Yasuhiro

2015-01-01

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

PubMed

Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

1998-01-01

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

A Catalog of Genes Homozygously Deleted in Human Lung Cancer and the Candidacy of PTPRD as a Tumor Suppressor Gene

PubMed Central

Kohno, Takashi; Otsuka, Ayaka; Girard, Luc; Sato, Masanori; Iwakawa, Reika; Ogiwara, Hideaki; Sanchez-Cespedes, Montse; Minna, John D.; Yokota, Jun

2010-01-01

A total of 176 genes homozygously deleted in human lung cancer were identified by DNA array-based whole genome scanning of 52 lung cancer cell lines and subsequent genomic PCR in 74 cell lines, including the 52 cell lines scanned. One or more exons of these genes were homozygously deleted in one (1%) to 20 (27%) cell lines. These genes included known tumor suppressor genes, e.g., CDKN2A/p16, RB1, and SMAD4, and candidate tumor suppressor genes whose hemizygous or homozygous deletions were reported in several types of human cancers, such as FHIT, KEAP1, and LRP1B/LRP-DIP. CDKN2A/p16 and p14ARF located in 9p21 were most frequently deleted (20/74, 27%). The PTPRD gene was most frequently deleted (8/74, 11%) among genes mapping to regions other than 9p21. Somatic mutations, including a nonsense mutation, of the PTPRD gene were detected in 8/74 (11%) of cell lines and 4/95 (4%) of surgical specimens of lung cancer. Reduced PTPRD expression was observed in the majority (>80%) of cell lines and surgical specimens of lung cancer. Therefore, PTPRD is a candidate tumor suppressor gene in lung cancer. Microarray-based expression profiling of 19 lung cancer cell lines also indicated that some of the 176 genes, such as KANK and ADAMTS1, are preferentially inactivated by epigenetic alterations. Genetic/epigenetic as well as functional studies of these 176 genes will increase our understanding of molecular mechanisms behind lung carcinogenesis. PMID:20073072

High-throughput microfluidic line scan imaging for cytological characterization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

2015-03-01

Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

Accumulation of True Single Strand Breaks and AP sites in Base Excision Repair Deficient Cells

PubMed Central

Luke, April M.; Chastain, Paul D.; Pachkowski, Brian F.; Afonin, Valeriy; Takeda, Shunichi; Kaufman, David G.; Swenberg, James A.; Nakamura, Jun

2010-01-01

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1 mM MMS compared to control. Furthermore, the Pol β-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays. PMID:20851134

Protein-free culture of the human pancreatic cancer cell line, SUIT-2.

PubMed

Taniguchi, S; Iwamura, T; Kitamura, N; Yamanari, H; Kojima, A; Hidaka, K; Seguchi, K; Setoguchi, T

1994-12-01

A human pancreatic cancer cell line (SUIT-2), usually cultured in serum-supplemented medium (DMEM/FBS), was adapted to protein-free conditions using a 1:1 mixture of DMEM and Ham's F12 medium (DMEM/F12). The cells have been maintained in DMEM/F12 for more than 2 years, with over 50 passages. The SUIT-2 cells grew in DMEM/F12 with a doubling time of 35.7 h, which was similar to that in DMEM/FBS (35.0 h). The cellular morphology was similar in both media. Type IV collagenolytic activity was detected in the conditioned media from cells grown in DMEM/F12. The secretion of CEA and CA19-9 initially decreased in DMEM/F12. CEA was not detected after passage 5 (p5) but the concentration of CA19-9 did not decrease further after the first few serial passages in protein-free medium. Xenografts of SUIT-2 cells cultured in DMEM/F12 remained tumorigenic and could form metastatic tumors in nude mice. In conclusion, SUIT-2 cells grown in protein-free media continued to produce CA19-9 and type IV collagenase in vitro and formed metastatic tumors in vivo.

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-01-01

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

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

PubMed

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

2017-01-05

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

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

PubMed

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

2017-09-16

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

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat.

PubMed

Hu, P F; Guan, W J; Li, X C; Zhang, W X; Li, C L; Ma, Y H

2013-01-01

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.

Cell-Based Sensor System Using L6 Cells for Broad Band Continuous Pollutant Monitoring in Aquatic Environments

PubMed Central

Kubisch, Rebekka; Bohrn, Ulrich; Fleischer, Maximilian; Stütz, Evamaria

2012-01-01

Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni2+ and Cu2+) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity. PMID:22737014

Cell-based sensor system using L6 cells for broad band continuous pollutant monitoring in aquatic environments.

PubMed

Kubisch, Rebekka; Bohrn, Ulrich; Fleischer, Maximilian; Stütz, Evamaria

2012-01-01

Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni(2+) and Cu(2+)) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity.

Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

PubMed Central

Saling, Mark; Duckett, Jordan K; Ackers, Ian; Coschigano, Karen; Jenkinson, Scott; Malgor, Ramiro

2017-01-01

Bladder cancer is the fourth most common cancer in men and the most common malignancy of the urinary tract. Bladder cancers detected at an early stage have a very high five-year survival rate, but when detected after local metastasis the rate is only about 50%. Our group recently reported a positive correlation between the expression of Wnt5a, a member of the Wnt proteins family, and histopathological grade and stage of urothelial carcinoma (UC). The objective of this study was to analyze UC cases reported in Athens, Ohio and investigate the major components of Wnt5a / planar cell polarity (PCP) signaling pathway in UC human tissue samples and UC cell lines. Formalin fixed and paraffin embedded transurethral resection tissues were immunostained for Wnt5a, Ror-2, CTHRC1 and E-cadherin. In addition, in vitro studies using UC cell lines were investigated for Wnt5a/PCP signaling and epithelial mesenchymal transition (EMT) gene expression. The IHC results showed a correlation between the expression of Wnt5a, Ror2 and CTHRC1 with high histological grade of the tumor, while E-cadherin showed an opposite trend of expression. Real time RT-PCR results showed that RNA expression of the Wnt5a/ PCP pathway genes vary in low and high grade UC cell lines and that the high grade cell lines exhibited signs of EMT. These findings support that Wnt5a-Ror2 signaling plays a role in UC, support the potential use of Wnt5a as a prognostic marker and provide evidence that Wnt5a signaling may be used as an effective molecular target for novel therapeutic tools. PMID:28427201

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture

PubMed Central

Prill, Sebastian; Jaeger, Magnus S.; Duschl, Claus

2014-01-01

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro. PMID:24926387

Novel role of MDA-9/syntenin in regulating urothelial cell proliferation by modulating EGFR signaling.

PubMed

Dasgupta, Santanu; Menezes, Mitchell E; Das, Swadesh K; Emdad, Luni; Janjic, Aleksandar; Bhatia, Shilpa; Mukhopadhyay, Nitai D; Shao, Chunbo; Sarkar, Devanand; Fisher, Paul B

2013-09-01

Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knockdown was examined in multiple cells lines and key findings were validated in primary tumors. Significantly higher (P=0.002-0.003) expression of MDA-9/Syntenin was observed in 64% (28 of 44) of primary tumors and an association was evident with stage (P=0.01), grade (P=0.03), and invasion status (P=0.02). MDA-9/Syntenin overexpression in nontumorigenic HUC-1 cells increased proliferation (P=0.0012), invasion (P=0.0001), and EGF receptor (EGFR), AKT, phosphoinositide 3-kinase (PI3K), and c-Src expression. Alteration of β-catenin, E-cadherin, vimentin, claudin-1, ZO-1, and T-cell factor-4 (TCF4) expression was also observed. MDA-9/Syntenin knockdown in three UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and colocalization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin:AKT expressions with stage (P=0.04, EGFR; P=0.01, AKT). A correlation between MDA-9/Syntenin:β-catenin coexpression with stage (P=0.03) and invasion (P=0.04) was also evident. Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring, and therapeutic strategies for managing UCC. ©2013 AACR.

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton, Alexandrium tamarense.

PubMed

Yamasaki, Yasuhiro; Katsuo, Daisuke; Nakayasu, Seiichiro; Salati, Cristina; Duan, JingJing; Zou, Yanan; Matsuyama, Yukihiko; Yamaguchi, Kenichi; Oda, Tatsuya

2008-01-01

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel-filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid-Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N-acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide-based compound. The toxin showed cytotoxic effects on various cell lines in a concentration-dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide-based toxin isolated from red tide phytoplankton can induce apoptotic cell death. (c) 2008 Wiley Periodicals, Inc.

Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.

PubMed Central

Brûlet, P; Babinet, C; Kemler, R; Jacob, F

1980-01-01

Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells. Images PMID:6933460

A search for the prewetting line. [in binary liquid system at vapor-liquid interface

NASA Technical Reports Server (NTRS)

Schmidt, J. W.; Moldover, M. R.

1986-01-01

This paper describes efforts to locate the prewetting line in a binary liquid system (isopropanol-perfluoromethylcyclohexane) at the vapor-liquid interface. Tight upper bounds were placed on the temperature separation (0.2 K) between the prewetting line and the line of bulk liquid phase separation. The prewetting line in systems at equilibrium was not detected. Experimental signatures indicative of the prewetting line occurred only in nonequilibrium situations. Several theories predict that the adsorption of one of the components (the fluorocarbon, in this case) at the liquid-vapor interface should increase abruptly, at a temperature sightly above the temperature at which the mixture separates into two liquid phases. A regular solution calculation indicates that this prewetting line should have been easily detectable with the instruments used in this experiment. Significant features of the experiment are: (1) low-gradient thermostatting, (2) in situ stirring, (3) precision ellipsometry from the vapor-liquid interface, (4) high resolution differential index of refraction measurements using a novel cell design, and (5) computer control.

Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

PubMed

Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

2014-07-04

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

PubMed

Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

2011-01-01

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

Inhibition of urokinase-type plasminogen activator expression by dihydroartemisinin in breast cancer cells

PubMed Central

ZHANG, SHUQUN; MA, YINAN; JIANG, JIANTAO; DAI, ZHIJUN; GAO, XIAOYAN; YIN, XIAORAN; XI, WENTAO; MIN, WEILI

2014-01-01

The aim of the present study was to investigate the inhibitory effects of dihydroartemisinin (DHA) on the primary tumor growth and metastasis of the human breast cancer cell line, MDA-MB-231, in vitro. The expression levels of urokinase-type plasminogen activator (uPA) were detected by immunocytochemistry in two cell lines (MCF-7 and MDA-MB-231). The MDA-MB-231 cell activity was inhibited by various concentration gradients of DHA. The inhibitory rate, cell growth curve and apoptotic morphological observations were obtained using the MTT assay at 0, 24, 48 and 72 h. Cell scratch migration was performed at various time-points to test the cell proliferation and migration capacity. Reverse transcription-polymerase chain reaction was used to analyze the effect of DHA on uPA mRNA expression in breast cancer cells. The human breast cancer cell line, MDA-MB-231, possesses higher metastatic potential and relatively higher expression of uPA when compared with the MCF-7 cell line. DHA was found to inhibit the proliferation and migration capacity of the cell line, MDA-MB-231, in vitro. The growth inhibition occurred in a time- and dose-dependent manner, with IC50 values of 117.76±0.04, 60.26±0.12 and 52.96±0.07 μmol/l following 24, 48 and 72 h, respectively. The inhibition of uPA was observed to decrease breast cancer cell growth and migration. Thus, results of the present study indicate that DHA may be used for further studies with regard to breast cancer therapy. PMID:24765140

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

PubMed

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

2013-04-01

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

Generation and characterization of Atoh1-Cre knock-in mouse line

PubMed Central

Yang, Hua; Xie, Xiaoling; Deng, Min; Chen, Xiaowei; Gan, Lin

2010-01-01

Summary Atoh1 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of the inner ear sensory epithelia, the dorsal spinal cord, brainstem, cerebellum, and intestinal secretory cells. In this study to create a genetic tool for the research on gene function in the ear sensory organs, we generated an Atoh1-Cre knock-in mouse line by replacing the entire Atoh1 coding sequences with the Cre coding sequences. Atoh1Cre/+mice were viable, fertile, and displayed no visible defects whereas the Atoh1Cre/Cremice died perinatally. The spatiotemporal activities of Cre recombinase were examined by crossing Atoh1-Cre mice with the R26R-lacZ conditional reporter mice. Atoh1-Cre activities were detected in the developing inner ear, the hindbrain, the spinal cord, and the intestine. In the inner ear, Atoh1-Cre activities were confined to the sensory organs in which lacZ expression is detected in nearly all of the hair cells and in many supporting cells. Thus, Atoh1-Cre mouse line serves as a useful tool for the functional study of genes in the inner ear. In addition, our results demonstrate that Atoh1 is expressed in the common progenitors destined for both hair and supporting cells. PMID:20533400

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line

PubMed Central

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P.; Parton, Robert G.; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS. PMID:26086601

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

PubMed

Faggi, Fiorella; Codenotti, Silvia; Poliani, Pietro Luigi; Cominelli, Manuela; Chiarelli, Nicola; Colombi, Marina; Vezzoli, Marika; Monti, Eugenio; Bono, Federica; Tulipano, Giovanni; Fiorentini, Chiara; Zanola, Alessandra; Lo, Harriet P; Parton, Robert G; Keller, Charles; Fanzani, Alessandro

2015-01-01

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.

PubMed

Hitzler, Manuel; Bergert, Antje; Luch, Andreas; Peiser, Matthias

2013-09-01

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical's sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

No evidence for involvement of SDHD in neuroblastoma pathogenesis

PubMed Central

De Preter, Katleen; Vandesompele, Jo; Hoebeeck, Jasmien; Vandenbroecke, Caroline; Smet, Jöel; Nuyts, Annick; Laureys, Geneviève; Combaret, Valérie; Van Roy, Nadine; Roels, Frank; Van Coster, Rudy; Praet, Marleen; De Paepe, Anne; Speleman, Frank

2004-01-01

Background Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. Methods SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. Results Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. Conclusions Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis. PMID:15331017

Human pathogenic Mycoplasma species induced cytokine gene expression in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines.

PubMed

Schäffner, E; Opitz, O; Pietsch, K; Bauer, G; Ehlers, S; Jacobs, E

1998-04-01

We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma pneumoniae or M. hominis, with the mycoplasma species (M. fermentans, M. fermentans subsp. incognitus, M. penetrans, M. genitalium) or with mycoplasma species known to be mere commensals of the respiratory tract (M. orale and M. salivarium) would result in expression of mRNAs for IL-2, IL-2R, IL-4 and IL-6 as determined by reverse transcriptase (RT)-PCR after 4 and 24 h of cocultivation. The pattern of cytokine gene expression observed depended on (i) the origin of the transformed cell line, (ii) the pathogenicity of the Mycoplasma species, and (iii) the length of cocultivation. The EBV-immortalized lymphoblastoid cell line HilB-gamma showed mRNA expression for IL-2, IL-2-receptor, IL-4 and IL-6 peaking 24 h after stimulation with M. pneumoniae and all AIDS-related mycoplasma species tested. The Burkitt lymphoma cell line EB-3 showed a distinct and isolated strong II-2/IL-2 R-mRNA expression within 4 h after contact with the pathogenic and all of the AIDS related mycoplasma species. In neither EBV-containing cell line cytokine was gene expression detectable after stimulation with the commensal mycoplasma species, M. orale and M. salivarium, indicating species differences in the ability of mycoplasmas to interact with and stimulate B-cell lines. Our data suggest that some mcyoplasma species may act as immunomodulatory cofactors by eliciting inappropriate cytokine gene expression in B cells latently infected with EBV. Therefore, this cultivation model may prove useful in evaluating the pathogenetic potential of novel isolated mycoplasma species. Copyright 1998 Academic Press Limited.

Transposon Insertion Finder (TIF): a novel program for detection of de novo transpositions of transposable elements.

PubMed

Nakagome, Mariko; Solovieva, Elena; Takahashi, Akira; Yasue, Hiroshi; Hirochika, Hirohiko; Miyao, Akio

2014-03-14

Transposition event detection of transposable element (TE) in the genome using short reads from the next-generation sequence (NGS) was difficult, because the nucleotide sequence of TE itself is repetitive, making it difficult to identify locations of its insertions by alignment programs for NGS. We have developed a program with a new algorithm to detect the transpositions from NGS data. In the process of tool development, we used next-generation sequence (NGS) data of derivative lines (ttm2 and ttm5) of japonica rice cv. Nipponbare, regenerated through cell culture. The new program, called a transposon insertion finder (TIF), was applied to detect the de novo transpositions of Tos17 in the regenerated lines. TIF searched 300 million reads of a line within 20 min, identifying 4 and 12 de novo transposition in ttm2 and ttm5 lines, respectively. All of the transpositions were confirmed by PCR/electrophoresis and sequencing. Using the program, we also detected new transposon insertions of P-element from NGS data of Drosophila melanogaster. TIF operates to find the transposition of any elements provided that target site duplications (TSDs) are generated by their transpositions.

Identification of the long non‑coding RNA LET as a novel tumor suppressor in gastric cancer.

PubMed

Tian, Jingjing; Hu, Xibao; Gao, Wei; Zhang, Jie; Chen, Ming; Zhang, Xinrong; Ma, Junhong; Yuan, Hongxia

2017-04-01

Long non-coding RNAs (lncRNAs) have emerged recently as important factors in regulating fundamental biological processes. Alterations in the expression and function of lncRNAs have been observed to promote tumor formation, progression and metastasis. Although downregulation of the expression levels of LET lncRNA in several tumors has been reported, its role in gastric cancer remains unknown. The aim of the present study was to investigate the expression and function of LET in gastric cancer development. The expression levels of LET in 37 pairs of gastric cancer and adjacent non‑tumor tissues were detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In addition, LET expression in gastric cancer cell lines was analyzed by RT‑qPCR assay analysis. Furthermore, the impact of LET on cell proliferation, migration and apoptosis were detected using the cell counting kit‑8, wound scratch and ELISA assays, respectively. The results demonstrated that the expression level of LET was downregulated in gastric cancer tissues and cell lines (SGC‑7901 and MGC‑803) compared with normal tissues and a normal human gastric epithelial cell line (GES‑1). Restoration of LET expression using a synthesized recombinant overexpression vector transfected into SGC‑7901 and MGC‑803 cells, significantly inhibited cell proliferation and migration, and promoted cell apoptosis in vitro. The present study is the first to demonstrate that LET may function as a tumor suppressor in gastric cancer. The results indicate that LET may be a promising biomarker and/or a therapeutic target for gastric cancer.

MHC class II expression in lung cancer.

PubMed

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

2017-10-01

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

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

PubMed

Shalygin, A V; Vigont, V A; Glushankova, L N; Zimina, O A; Kolesnikov, D O; Skopin, A Yu; Kaznacheeva, E V

2017-07-01

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species.

PubMed

Störmer, Melanie; Vollmer, Tanja; Henrich, Birgit; Kleesiek, Knut; Dreier, Jens

2009-04-01

Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene (encoding elongation factor Tu) were designed. Cell culture supernatants, clinical specimens (vaginal swabs, sputum, cryopreserved heart valve tissues), and reference strains were tested for mollicute contamination as well as to exclude cross-reaction to human nucleic acids and other bacterial species. Nucleic acids were extracted using magnetic separation technology. The coamplification of the human beta2-microglobulin DNA served as an internal control. The PCR assay was highly specific and obtained an analytical sensitivity of one copy per microl sample. The 95% detection limit was calculated to 10 copies per microl sample for Mycoplasma pneumoniae and M. orale. No false-positive results were observed due to cross-reaction of walled bacterial, fungal, and human nucleic acids. To evaluate the PCR, we compared the results to two commercialized test systems. Moreover, in combination with a previously developed broad-range RT-PCR assay for the detection of bacteria in blood products, both mollicute and walled bacterial contamination can be detected simultaneously using multiplex real-time RT-PCR.

Chromosomal Distribution of Endogenous Jaagsiekte Sheep Retrovirus Proviral Sequences in the Sheep Genome

PubMed Central

Carlson, Jonathan; Lyon, Monique; Bishop, Jeanette; Vaiman, Anne; Cribiu, Edmond; Mornex, Jean-François; Brown, Susan; Knudson, Dennis; DeMartini, James; Leroux, Caroline

2003-01-01

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known. PMID:12915578

Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

PubMed Central

Tamm-Rosenstein, Karin; Simm, Jaak; Suhorutshenko, Marina; Salumets, Andres; Metsis, Madis

2013-01-01

Background Estrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively. Methodology/Principal findings Gene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1. Conclusions Significant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage. PMID:23874806

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading

PubMed Central

Dorn, Elizabeth S.; Chastain, Paul D.; Hall, Jonathan R.; Cook, Jeanette Gowen

2009-01-01

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. PMID:19010964

Expression of myeloid differentiation antigens on normal and malignant myeloid cells.

PubMed Central

Griffin, J D; Ritz, J; Nadler, L M; Schlossman, S F

1981-01-01

A series of monoclonal antibodies have been characterized that define four surface antigens (MY3, MY4, MY7, and MY8) of human myeloid cells. They were derived from a fusion of the NS-1 plasmacytoma cell line with splenocytes from a mouse immunized with human acute myelomonocytic leukemia cells. MY3 and MY4 are expressed by normal monocytes and by greater than 90% of patients with acute monocytic leukemia or acute myelomonocytic leukemia, but are detected much less often on other types of myeloid leukemia. MY7 is expressed by granulocytes, monocytes, and 5% of normal bone marrow cells. 79% of all acute myeloblastic leukemia (AML) patients tested (72 patients) express MY7 without preferential expression by any AML subtype. MY8 is expressed by normal monocytes, granulocytes, all peroxidase-positive bone marrow cells, and 50% of AML patients. MY3, MY4, and MY8 define myeloid differentiation antigens in that they are not detected on myeloid precursor cells and appear at discrete stages of differentiation. These antigens are not expressed by lymphocytes, erythrocytes, platelets, or lymphoid malignancies. The monoclonal antisera defining these antigens have been used to study differentiation of normal myeloid cells and malignant cell lines. Images PMID:6945311

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

Bioanalytical system for detection of cancer cells with photoluminescent ZnO nanorods

NASA Astrophysics Data System (ADS)

Viter, R.; Jekabsons, K.; Kalnina, Z.; Poletaev, N.; Hsu, S. H.; Riekstina, U.

2016-11-01

Using photoluminescent ZnO nanorods and carbohydrate marker SSEA-4, a novel cancer cell recognition system was developed. Immobilization of SSEA-4 antibodies (αSSEA-4) on ZnO nanorods was performed in buffer solution (pH = 7.1) over 2 h. The cancer cell line probes were fixed on the glass slide. One hundred microliters of ZnO-αSSEA-4 conjugates were deposited on the cell probe and exposed for 30 min. After washing photoluminescence spectra were recorded. Based on the developed methodology, ZnO-αSSEA-4 probes were tested on patient-derived breast and colorectal carcinoma cells. Our data clearly show that the carbohydrate SSEA-4 molecule is expressed on cancer cell lines and patient-derived cancer cells. Moreover, SSEA-4 targeted ZnO nanorods bind to the patient-derived cancer cells with high selectivity and the photoluminescence signal increased tremendously compared to the signal from the control samples. Furthermore, the photoluminescence intensity increase correlated with the extent of malignancy in the target cell population. A novel portable bioanalytical system, based on optical ZnO nanorods and fiber optic detection system was developed. We propose that carbohydrate SSEA-4 specific ZnO nanorods could be used for the development of cancer diagnostic biosensors and for targeted therapy.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

PubMed

Wright, Joy R; Amisten, Stefan; Goodall, Alison H; Mahaut-Smith, Martyn P

2016-08-01

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

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

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were asmore » capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.« less

Extracting cancer cell line electrochemical parameters at the single cell level using a microfabricated device.

PubMed

Alqabandi, Jassim A; Abdel-Motal, Ussama M; Youcef-Toumi, Kamal

2009-02-01

Cancer cells have distinctive electrochemical properties. This work sheds light on the system design aspects and key challenges that should be considered when experimentally analyzing and extracting the electrical characteristics of a tumor cell line. In this study, we developed a cellularbased functional microfabricated device using lithography technology. This device was used to investigate the electrochemical parameters of cultured cancer cells at the single-cell level. Using impedance spectroscopy analyses, we determined the average specific capacitance and resistance of the membrane of the cancer cell line B16-F10 to be 1.154 +/- 0.29 microF/cm(2), and 3.9 +/- 1.15 KOmega.cm(2) (mean +/- SEM, n =14 cells), respectively. The consistency of our findings via different trails manifests the legitimacy of our experimental procedure. Furthermore, the data were compared with a proposed constructed analytical-circuit model. The results of this work may greatly assist researchers in defining an optimal procedure while extracting electrical properties of cancer cells. Detecting electrical signals at the single cell level could lead to the development of novel approaches for analysis of malignant cells in human tissues and biopsies.

The antiretroviral nucleoside analogue Abacavir reduces cell growth and promotes differentiation of human medulloblastoma cells

PubMed Central

Rossi, Alessandra; Russo, Giuseppe; Puca, Andrew; La Montagna, Raffaele; Caputo, Mariella; Mattioli, Eliseo; Lopez, Massimo; Giordano, Antonio; Pentimalli, Francesca

2009-01-01

Abacavir is one of the most efficacious nucleoside analogues, with a well-characterized inhibitory activity on reverse transcriptase enzymes of retroviral origin, and has been clinically approved for the treatment of AIDS. Recently, Abacavir has been shown to inhibit also the human telomerase activity. Telomerase activity seems to be required in essentially all tumours for the immortalization of a subset of cells, including cancer stem cells. In fact, many cancer cells are dependent on telomerase for their continued replication and therefore telomerase is an attractive target for cancer therapy. Telomerase expression is upregulated in primary primitive neuroectodermal tumours and in the majority of medulloblastomas suggesting that its activation is associated with the development of these diseases. Therefore, we decided to test Abacavir activity on human medulloblastoma cell lines with high telomerase activity. We report that exposure to Abacavir induces a dose-dependent decrease in the proliferation rate of medulloblastoma cells. This is associated with a cell accumulation in the G2/M phase of the cell cycle in the Daoy cell line, and with increased cell death in the D283-MED cell line, and is likely to be dependent on the inhibition of telomerase activity. Interestingly, both cell lines showed features of senescence after Abacavir treatment. Moreover, following Abacavir exposure we detected, by immunofluorescence staining, increased protein expression of the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker synaptophysin (SYN) in both medulloblastoma cell lines. In conclusion, our results suggest that Abacavir reduces proliferation and induces differentiation of human medulloblastoma cells through the downregulation of telomerase activity. Thus, using Abacavir, alone or in combination with current therapies, might be an effective therapeutic strategy for the treatment of medulloblastoma. PMID:19358275

Biochemical signatures of in vitro radiation response in human lung, breast and prostate tumour cells observed with Raman spectroscopy

NASA Astrophysics Data System (ADS)

Matthews, Q.; Jirasek, A.; Lum, J. J.; Brolo, A. G.

2011-11-01

This work applies noninvasive single-cell Raman spectroscopy (RS) and principal component analysis (PCA) to analyze and correlate radiation-induced biochemical changes in a panel of human tumour cell lines that vary by tissue of origin, p53 status and intrinsic radiosensitivity. Six human tumour cell lines, derived from prostate (DU145, PC3 and LNCaP), breast (MDA-MB-231 and MCF7) and lung (H460), were irradiated in vitro with single fractions (15, 30 or 50 Gy) of 6 MV photons. Remaining live cells were harvested for RS analysis at 0, 24, 48 and 72 h post-irradiation, along with unirradiated controls. Single-cell Raman spectra were acquired from 20 cells per sample utilizing a 785 nm excitation laser. All spectra (200 per cell line) were individually post-processed using established methods and the total data set for each cell line was analyzed with PCA using standard algorithms. One radiation-induced PCA component was detected for each cell line by identification of statistically significant changes in the PCA score distributions for irradiated samples, as compared to unirradiated samples, in the first 24-72 h post-irradiation. These RS response signatures arise from radiation-induced changes in cellular concentrations of aromatic amino acids, conformational protein structures and certain nucleic acid and lipid functional groups. Correlation analysis between the radiation-induced PCA components separates the cell lines into three distinct RS response categories: R1 (H460 and MCF7), R2 (MDA-MB-231 and PC3) and R3 (DU145 and LNCaP). These RS categories partially segregate according to radiosensitivity, as the R1 and R2 cell lines are radioresistant (SF2 > 0.6) and the R3 cell lines are radiosensitive (SF2 < 0.5). The R1 and R2 cell lines further segregate according to p53 gene status, corroborated by cell cycle analysis post-irradiation. Potential radiation-induced biochemical response mechanisms underlying our RS observations are proposed, such as (1) the regulated synthesis and degradation of structured proteins and (2) the expression of anti-apoptosis factors or other survival signals. This study demonstrates the utility of RS for noninvasive radiobiological analysis of tumour cell radiation response, and indicates the potential for future RS studies designed to investigate, monitor or predict radiation response.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

PubMed

Hinrichsen, Inga; Ackermann, Anne; Düding, Tonja; Graband, Annika; Filmann, Natalie; Plotz, Guido; Zeuzem, Stefan; Brieger, Angela

2017-07-01

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment. © 2017 Wiley Periodicals, Inc.

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

PubMed Central

Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

2015-01-01

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas. PMID:26368816

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression.

PubMed

Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; Lombardi, Francesca; La Torre, Cristina; Dehcordi, Soheila Raysi; Galzio, Renato; Cimini, Annamaria; Giordano, Antonio; Cifone, Maria Grazia

2017-04-11

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Neurofibromatosis type 2 tumor suppressor protein is expressed in oligodendrocytes and regulates cell proliferation and process formation.

PubMed

Toledo, Andrea; Grieger, Elena; Karram, Khalad; Morrison, Helen; Baader, Stephan L

2018-01-01

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

PubMed

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis

PubMed Central

Chen, Chao; Zhao, Si Cong; Yang, Wen Zheng; Chen, Zong Ping; Yan, Yong

2018-01-01

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC. PMID:29403558

Systematic assessment of the performance of whole-genome amplification for SNP/CNV detection and β-thalassemia genotyping.

PubMed

He, Fei; Zhou, Wanjun; Cai, Ren; Yan, Tizhen; Xu, Xiangmin

2018-04-01

In this study, we aimed to assess the performance of two whole-genome amplification methods, multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycle (MALBAC), for β-thalassemia genotyping and single-nucleotide polymorphism (SNP)/copy-number variant (CNV) detection using two DNA sequencing assays. We collected peripheral blood, cell lines, and discarded embryos, and carried out MALBAC and MDA on single-cell and five-cell samples. We detected and statistically analyzed differences in the amplification efficiency, positive predictive value, sensitivity, allele dropout (ADO) rate, SNPs, and CV values between the two methods. Through Sanger sequencing at the single-cell and five-cell levels, we showed that both the amplification rate and ADO rate of MDA were better than those using MALBAC, and the sensitivity and positive predictive value obtained from MDA were higher than those from MALBAC for β-thalassemia genotyping. Using next-generation sequencing (NGS) at the single-cell level, we confirmed that MDA has better properties than MALBAC for SNP detection. However, MALBAC was more stable and homogeneous than MDA using low-depth NGS at the single-cell level for CNV detection. We conclude that MALBAC is the better option for CNV detection, while MDA is better suited for SNV detection.

Cancer antibody enhanced real time imaging cell probes--a novel theranostic tool using polymer linked carbon nanotubes and quantum dots.

PubMed

Brakmane, Gerda; Madani, Seyed Yazdan; Seifalian, Alexander

2013-06-01

Cancer is a potentially fatal diagnosis, but due to modern medicine there is a potential cure in many of these cases. The rate of treatment success depends on early disease detection and timely, effective delivery of tumour specific treatment. There are many ongoing researches aimed to improve diagnostics or treatment, but the option to use both modalities concomitantly is deficient. In this project we are using the advances in nanotechnology to develop new theranostic tool using single walled carbon nanotubes (SWCNT) and Quantum dots (QDs) for early cancer cell detection, and option to deliver targeted treatment. SWCNTs were refluxed in HNO3/H2SO4 (1:3) at 120ºC for 120 minutes. Functionalised SWCNT was then covalently attached to octa-ammonium polyhedral oligomeric silsesquioxane (POSS), QDs and conjugated with antibodies for targeted cell detection. Fourier transforms infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), UV/NIR analysis, Raman and UV-VIS spectroscopy were used in order to prove the successful conjugation. Toxicology study using alamar blue analysis and DNA assay was conducted in order to choose the best concentration of SWCNT, octa-ammonium-POSS and QDs before commencing the conjugation process. Human colorectal cancer cell line HT29, pancreas cancer cell line PANC-1 and mouse fibroblasts 3T3 were then treated with or without antibody conjugated SWCNT-POSS-QDs (CPQ) compound solution. The cell response was observed under the microscope after 24, 48 and 72 hours. FTIR and Raman spectroscopies confirmed covalent binding of the SWCNTs to Octa-Ammonium-POSS. This was supported by TEM images and photos obtained, which showed well dispersed SWCNTs following its treatment with Octa-Ammonium-POSS compared to pristine SWCNT samples. UV-VIS graphs determined the presence of antibody within the compound. UV/NIR demonstrated QD fluorescence even after attachment of SWCNT-POSS. The cellular behaviour revealed high CPQ-antibody complex affinity towards cancer cells when compared to healthy cell line which internalised the complex only on day three. The pancreas cancer cell line had appearance of lysed pulp after 72 hours of incubation. Colonic cancer cells seemed to regain ability to populate from day three signifying that higher treatment payload is necessary. We have successfully manufactured novel compound consisting of Octa-Ammonium-POSS linked SWCNTs, QDs, and tumour specific antibodies. The complex has proven its potential as cell probing tool, and the attachment of antibodies has shown high affinity to cancer cells rendering this an attractive model for further theranostic developments.

A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.

PubMed

Ugolotti, Elisabetta; Vanni, Irene

2014-01-01

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

Cytokine-mediated FOXO3a phosphorylation suppresses FasL expression in hemopoietic cell lines: investigations of the role of Fas in apoptosis due to cytokine starvation.

PubMed

Behzad, Hayedeh; Jamil, Sarwat; Denny, Trisha A; Duronio, Vincent

2007-05-01

We have investigated phosphatidylinositol 3-kinase (PI3K)-dependent survival signalling pathways using several cytokines in three different hemopoietic cell lines, MC/9, FDC-P1, and TF-1. Cytokines caused PI3K- and PKB-dependent phosphorylation of FOXO3a (previously known as FKHRL1) at three distinct sites. Following cytokine withdrawal or PI3K inhibition, both of which are known to lead to apoptosis, there was a loss of FOXO3a phosphorylation, and a resulting increase in forkhead transcriptional activity, along with increased expression of Fas Ligand (FasL), which could be detected at the cell surface. Concurrently, an increase in cell surface expression of Fas was also detected. Despite the presence of both FasL and Fas, there was no detectable evidence that activation of Fas-mediated apoptotic events was contributing to apoptosis resulting from cytokine starvation or inhibition of PI3K activity. Thus, inhibition of FOXO3a activity is mediated by the PI3K-PKB pathway, but regulation of FasL is not the primary means by which cell survival is regulated in cytokine-dependent hemopoietic cells. We were also able to confirm increased expression of known FOXO3a targets, Bim and p27kip1. Together, these results support the conclusion that mitochondrial-mediated signals play the major role in apoptosis of hemopoietic cells due to loss of cytokine signalling.

Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

PubMed

Ohno, S; Luka, J; Falk, L; Klein, G

1977-12-15

In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.

Automatic microscopy for mitotic cell location.

NASA Technical Reports Server (NTRS)

Herron, J.; Ranshaw, R.; Castle, J.; Wald, N.

1972-01-01

Advances are reported in the development of an automatic microscope with which to locate hematologic or other cells in mitosis for subsequent chromosome analysis. The system under development is designed to perform the functions of: slide scanning to locate metaphase cells; conversion of images of selected cells into binary form; and on-line computer analysis of the digitized image for significant cytogenetic data. Cell detection criteria are evaluated using a test sample of 100 mitotic cells and 100 artifacts.

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

PubMed

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

2012-05-01

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

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

PubMed Central

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

2012-01-01

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

Novel Role of MDA-9/Syntenin in Regulating Urothelial Cell Proliferation by Modulating EGFR Signaling

PubMed Central

Dasgupta, Santanu; Menezes, Mitchell E.; Das, Swadesh K.; Emdad, Luni; Janjic, Aleksandar; Bhatia, Shilpa; Mukhopadhyay, Nitai D; Shao, Chunbo; Sarkar, Devanand; Fisher, Paul B.

2013-01-01

Purpose Urothelial cell carcinoma (UCC) rapidly progresses from superficial to muscle-invasive tumors. The key molecules involved in metastatic progression and its early detection require clarification. The present study defines a seminal role of the metastasis-associated gene MDA-9/Syntenin in UCC progression. Experimental Design Expression pattern of MDA-9/Syntenin was examined in 44 primary UCC and the impact of its overexpression and knock down was examined in multiple cells lines and key findings were validated in primary tumors. Results Significantly higher (p= 0.002–0.003) expression of MDA-9/Syntenin was observed in 64% (28/44) of primary tumors and an association was evident with stage (p=0.01), grade (p=0.03) and invasion status (p=0.02). MDA-9/Syntenin overexpression in non-tumorigenic HUC-1 cells increased proliferation (p=0.0012), invasion (p=0.0001) and EGFR, AKT, PI3K and c-Src expression. Alteration of Beta-catenin, E-Cadherin, Vimentin, Claudin-1, ZO-1 and TCF4 expression were also observed. MDA-9/Syntenin knock down in 3 UCC cell lines reversed phenotypic and molecular changes observed in the HUC-1 cells and reduced in vivo metastasis. Key molecular changes observed in the cell lines were confirmed in primary tumors. A physical interaction and co-localization of MDA-9/Syntenin and EGFR was evident in UCC cell lines and primary tumors. A logistic regression model analysis revealed a significant correlation between MDA-9/Syntenin:EGFR and MDA-9/Syntenin: AKT expressions with stage (p=0.04, EGFR), (p=0.01, AKT). A correlation between MDA-9/Syntenin: β-catenin co-expression with stage (p=0.03) and invasion (p=0.04) was also evident. Conclusions Our findings indicate that MDA-9/Syntenin might provide an attractive target for developing detection, monitoring and therapeutic strategies for managing UCC. PMID:23873690

Comparison of test methods to screen for residual chemical contamination on medical device surfaces.

PubMed

Kulkarni, Prachi; Shoff, Megan; Lucas, Anne

2012-01-01

Reprocessing medical devices involves several steps including cleaning and disinfection or sterilization. Chemical residuals can occur at various stages of reprocessing. These residues could interfere with device function and potentially harm patients. These solutions are composed of a combination of various chemicals and their residues are highly diluted post rinsing, therefore, it is difficult to find a sensitive and rapid method to detect toxicity due to chemical residues. This study focused on (1) finding the levels of residues that are cytotoxic using two mammalian cell lines and Daphnia magna, and (2) evaluating two test methods, Total Organic Carbon (TOC) and the Luminescent Bacteria Test (LBT), to measure residual chemicals levels. The two mammalian cell lines were equal in their cytotoxicity responses. However, Daphnia were more sensitive to some chemical residue than the two mammalian cell lines. TOC and LBT were able to detect the presence of residue well below the levels that were determined to cause mammalian cytotoxicity. LBT was more sensitive for some chemicals and TOC for others, both in solution and in simulated cleaning and rinsing for the limited number of solutions tested in this study.

Role of Hunk and Punc in Breast Cancer and Mammary Development

DTIC Science & Technology

2001-08-01

of the 4 small cell lung carcinoma cell lines, but is detected in only one of ten other lung cancer cell Lung < branchio adeno Kids ml Pell CNS PNCK 3...polarity in Schizosaccharomyces Schnurch, H., and Risau, W. (1993). Expression of tie-2, a member of porn be. Proc. Natl. Acad. Sci. USA 87: 8272-8276

Telomere elongation in immortal human cells without detectable telomerase activity.

PubMed Central

Bryan, T M; Englezou, A; Gupta, J; Bacchetti, S; Reddel, R R

1995-01-01

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism. Images PMID:7556065

Telomere elongation in immortal human cells without detectable telomerase activity.

PubMed

Bryan, T M; Englezou, A; Gupta, J; Bacchetti, S; Reddel, R R

1995-09-01

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

PubMed

Yoda, N; Konno, R; Nagashima, S

2001-01-01

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar-Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Sakamoto, Atsushi; Matsumaru, Takehisa; Yamamura, Norio; Suzuki, Shinobu; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2015-09-01

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Human herpes simplex viruses in benign and malignant thyroid tumours.

PubMed

Jensen, Kirk; Patel, Aneeta; Larin, Alexander; Hoperia, Victoria; Saji, Motoyasu; Bauer, Andrew; Yim, Kevin; Hemming, Val; Vasko, Vasyl

2010-06-01

To test the hypothesis that herpes viruses may have a role in thyroid neoplasia, we analysed thyroid tissues from patients with benign (44) and malignant (65) lesions for HSV1 and HSV2 DNA. Confirmatory studies included direct sequencing, analysis of viral gene expression, and activation of viral-inducible signalling pathways. Expression of viral entry receptor nectin-1 was examined in human samples and in cancer cell lines. In vitro experiments were performed to explore the molecular mechanisms underlying thyroid cancer cell susceptibility to HSV. HSV DNA was detected in 43/109 (39.4%) examined samples. HSV capsid protein expression correlated with HSV DNA status. HSV-positive tumours were characterized by activation of virus-inducible signalling such as interferon-beta expression and nuclear NFkappaB expression. Lymphocyte infiltration and oncocytic cellular features were common in HSV-positive tumours. HSV1 was detected with the same frequency in benign and malignant thyroid tumours. HSV2 was significantly associated with papillary thyroid cancer and the presence of lymph node metastases. The expression of HSV entry receptor nectin-1 was increased in thyroid tumours compared to normal thyroid tissue and further increased in papillary thyroid cancer. Nectin-1 expression was detected in all examined thyroid cancer cell lines. Nectin-1 expression in cancer cells correlated with their susceptibility to HSV. Inhibition of PI3K/AKT or MAPK/ERK signalling did not affect the level of nectin-1 expression but decreased thyroid cancer cell susceptibility to HSV. These findings showed that HSV is frequently detected in thyroid cancer. During tumour progression, thyroid cells acquire increased susceptibility to HSV due to increased expression of viral entry mediator nectin-1 and activation of mitogenic signalling in cancer cells.

Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma

PubMed Central

Gerhard, M C; Schmid, R M; Häcker, G

2002-01-01

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome c-induced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis. British Journal of Cancer (2002) 86, 893–898. DOI: 10.1038/sj/bjc/6600171 www.bjcancer.com © 2002 Cancer Research UK PMID:11953820

Screening strategy to avoid toxicological hazards of inhaled nanoparticles for drug delivery: The use of a-quartz and nano zinc oxide particles as benchmark

NASA Astrophysics Data System (ADS)

Beyerle, Andrea; Schulz, Holger; Kissel, Thomas; Stoeger, Tobias

2009-02-01

Nanotechnology is a broad, revolutionary field with promising advantages for new medicine. In this context the rapid development and improvement of so called nanocarriers is of high pharmaceutical interest and some devices are already on the market. In our project we aim to develop well characterized nanoscaled drug delivery systems for an inhalative application. To this end, we focus on the most adverse side-effects within the lung, the cytotoxic and the proinflammatory responses to these nanoparticles (NPs). Before performing any animal experiments, we start with an in vitro screening for analyzing the cytotoxic and proinflammatory effects of the investigated particles on two murine lung target cell lines, the alveolar epithelial like typ II cell line (LA4) and the alveolar macrophage cell line (MH-S). Three different endpoints were estimated, (i) cellular metabolic activity, determined by the WST-1 assay, (ii) membrane integrity, by detection of LDH release and hemolytic activity, and (iii) secretion of inflammatory mediators. To analyze the relative particle toxicity we choose two reference particles as benchmarks, (i) fine a-quartz, and (ii) ultrafine ZnO particles. The investigation of dose-response and kinetics of proinflammatory and toxic effects caused to the named cell lines provide an insight to a close evaluation of our cell based screening strategy. oc-quartz is well known for its inflammatory and toxic potential caused by inhalation, and nanosized ZnO particles - used in a broad field of nanotechnology like electronics, but also cosmetics and pharmaceuticals - is to a high degree cytotoxic and proinflammatory in vitro. Preliminary experiments indicated not only particle and cell specific inflammatory responses, but also different susceptibilities of the cell types being exposed to our benchmark particles regarding their size and surface activities. Exposure to the μm-sized a-quartz particles affected the viability of epithelia cells less than that of macrophages, pointing to the impact of particle uptake by phagocytosis. In contrast, the nanosized ZnO particles caused much stronger decrease in cell viability and higher levels of LDH in the macrophage cell line compared to epithelial cells, even though the hemolytic activity was much higher for the a-quartz particles than for the nanosized ZnO. For the proinflammatory effects, we observed a clear dose-dependent release of acute phase cytokines (TNF-α, IL-6, G-CSF> CXCL10>CCL2) for both alveolar cell lines after Min-U-Sil exposure. After ZnO treatment the cytokine responses were negligible compare to control cells. In conclusion, our data attach value to the use of different cell types to detect different pathways of toxicity generated by different particle properties. Therefore, we will establish both lung target cell lines for an in vitro screening to analyze proinflammatory and cytotoxicity effects of nanocarriers. The implementation of the two reference particles facilitate the validated classification of the cytotoxic responses caused by the NPs investigated.

Correlation between cell death and induction of non-rejoining PCC breaks by carbon-ion beams.

PubMed

Suzuki, M; Kase, Y; Kanai, T; Ando, K

1998-01-01

We have shown a correlation between cell death and induction of non-rejoining chromatin breaks in two normal human cells and three human tumor cell lines irradiated by carbon-ion beams and X rays. Non-rejoining chromatin breaks were measured by counting the number of remaining chromatin fragments detected by the premature chromosome condensation (PCC) technique. Carbon-ion beams were accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC). The cells were irradiated by two different mono-LET beams (LET = 13 keV/micrometer and 77 keV/micrometer ) and 200 kV X rays. The RBE values of cell death for carbon-ion beams relative to X rays were 1.1 to 1.4 for 13 keV/micrometer beams and 2.5 to 2.9 for 77 keV/micrometer beams. The induction rate of non-rejoining PCC breaks per cell per Gy was found to be highest for the 77 keV/micrometer beams for all of the cell lines. The results found in this study show that there is a good correlation between cell death and induction of non-rejoining PCC breaks for these human cell lines.

Integration of high-risk human papillomavirus into cellular cancer-related genes in head and neck cancer cell lines.

PubMed

Walline, Heather M; Goudsmit, Christine M; McHugh, Jonathan B; Tang, Alice L; Owen, John H; Teh, Bin T; McKean, Erin; Glover, Thomas W; Graham, Martin P; Prince, Mark E; Chepeha, Douglas B; Chinn, Steven B; Ferris, Robert L; Gollin, Susanne M; Hoffmann, Thomas K; Bier, Henning; Brakenhoff, Ruud; Bradford, Carol R; Carey, Thomas E

2017-05-01

Human papillomavirus (HPV)-positive oropharyngeal cancer is generally associated with excellent response to therapy, but some HPV-positive tumors progress despite aggressive therapy. The purpose of this study was to evaluate viral oncogene expression and viral integration sites in HPV16- and HPV18-positive squamous cell carcinoma lines. E6/E7 alternate transcripts were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Detection of integrated papillomavirus sequences (DIPS-PCR) and sequencing identified viral insertion sites and affected host genes. Cellular gene expression was assessed across viral integration sites. All HPV-positive cell lines expressed alternate HPVE6/E7 splicing indicative of active viral oncogenesis. HPV integration occurred within cancer-related genes TP63, DCC, JAK1, TERT, ATR, ETV6, PGR, PTPRN2, and TMEM237 in 8 head and neck squamous cell carcinoma (HNSCC) lines but UM-SCC-105 and UM-GCC-1 had only intergenic integration. HPV integration into cancer-related genes occurred in 7 of 9 HPV-positive cell lines and of these 6 were from tumors that progressed. HPV integration into cancer-related genes may be a secondary carcinogenic driver in HPV-driven tumors. © 2017 Wiley Periodicals, Inc. Head Neck 39: 840-852, 2017. © 2017 Wiley Periodicals, Inc.

A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

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

Chang, Junghwa; Jung, Hye Jin; Jeong, Seung Hun

2014-12-12

Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identificationmore » and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.« less

Pro-neurotensin/neuromedin N expression and processing in human colon cancer cell lines.

PubMed

Rovère, C; Barbero, P; Maoret, J J; Laburthe, M; Kitabgi, P

1998-05-08

The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.

Sensitive and specific detection of EML4-ALK rearrangements in non-small cell lung cancer (NSCLC) specimens by multiplex amplicon RNA massive parallel sequencing.

PubMed

Moskalev, Evgeny A; Frohnauer, Judith; Merkelbach-Bruse, Sabine; Schildhaus, Hans-Ulrich; Dimmler, Arno; Schubert, Thomas; Boltze, Carsten; König, Helmut; Fuchs, Florian; Sirbu, Horia; Rieker, Ralf J; Agaimy, Abbas; Hartmann, Arndt; Haller, Florian

2014-06-01

Recurrent gene fusions of anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) have been recently identified in ∼5% of non-small cell lung cancers (NSCLCs) and are targets for selective tyrosine kinase inhibitors. While fluorescent in situ hybridization (FISH) is the current gold standard for detection of EML4-ALK rearrangements, several limitations exist including high costs, time-consuming evaluation and somewhat equivocal interpretation of results. In contrast, targeted massive parallel sequencing has been introduced as a powerful method for simultaneous and sensitive detection of multiple somatic mutations even in limited biopsies, and is currently evolving as the method of choice for molecular diagnostic work-up of NSCLCs. We developed a novel approach for indirect detection of EML4-ALK rearrangements based on 454 massive parallel sequencing after reverse transcription and subsequent multiplex amplification (multiplex ALK RNA-seq) which takes advantage of unbalanced expression of the 5' and 3' ALK mRNA regions. Two lung cancer cell lines and a selected series of 32 NSCLC samples including 11 cases with EML4-ALK rearrangement were analyzed with this novel approach in comparison to ALK FISH, ALK qRT-PCR and EML4-ALK RT-PCR. The H2228 cell line with known EML4-ALK rearrangement showed 171 and 729 reads for 5' and 3' ALK regions, respectively, demonstrating a clearly unbalanced expression pattern. In contrast, the H1299 cell line with ALK wildtype status displayed no reads for both ALK regions. Considering a threshold of 100 reads for 3' ALK region as indirect indicator of EML4-ALK rearrangement, there was 100% concordance between the novel multiplex ALK RNA-seq approach and ALK FISH among all 32 NSCLC samples. Multiplex ALK RNA-seq is a sensitive and specific method for indirect detection of EML4-ALK rearrangements, and can be easily implemented in panel based molecular diagnostic work-up of NSCLCs by massive parallel sequencing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

DTIC Science & Technology

2014-10-01

Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation, Senescence. 16. SECURITY CLASSIFICATION OF: 17...Tyrosine Kinase, Her/ErbB2 signaling, Rab Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation...lines included RCP condition to internalization and detection of E-cadherin, a well-known adherent junction and epithelial mesenchymal transition

The sensitivity of glioma cells to pyropheophorbide-αmethyl ester-mediated photodynamic therapy is enhanced by inhibiting ABCG2.

PubMed

Pan, Li; Lin, Haidan; Tian, Si; Bai, Dingqun; Kong, Yuhan; Yu, Lehua

2017-09-01

To study the mechanisms of human glioblastoma cell resistance to methyl ester pyropheophorbide-a-mediated photodynamic therapy (MPPa-PDT) and the relationship between the cells and adenosine triphosphate-binding cassette superfamily G member 2 (ABCG2). The sensitivity of four human glioma cell lines (U87, A172, SHG-44, and U251) to MPPa-PDT was detected with a CCK-8 assay. Cell apoptosis, intracellular MPPa, and singlet oxygen were tested with flow cytometry. The mRNA and protein expression of ATP-binding cassette transporters (ABCG2, MRP1, and MDR1) were detected by PCR and Western blot, respectively. Both the sensitivity to MPPa-PDT and intracellular MPPa in A172 were the lowest among the four cell lines, while expression of ABCG2 mRNA and protein in A172 were the highest. The intracellular MPPa and ROS in A172 receiving MPPa-PDT significantly increased after using the ABCG2 inhibitor fumitremorgin C (FTC). Both cell viability and apoptosis in A172 cells undergoing MPPa-PDT were significantly improved with FTC. ABCG2 plays a significant role in the resistance of A172 to MPPa-PDT. Lasers Surg. Med. 49:719-726, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

PubMed

Dragan, Y; Valdés, R; Gomez-Angelats, M; Felipe, A; Javier Casado, F; Pitot, H; Pastor-Anglada, M

2000-08-01

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

PubMed Central

Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O.

2014-01-01

Background: To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. Methods: We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Results: Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Conclusion: Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes. PMID:24830820

Volatile metabolomic signature of human breast cancer cell lines

PubMed Central

Silva, Catarina L.; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S.

2017-01-01

Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC–MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways. PMID:28256598

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

PubMed

Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C

2012-09-01

What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest. © 2012 BJU INTERNATIONAL.

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

PubMed

Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

Combined M-FISH and CGH analysis allows comprehensive description of genetic alterations in neuroblastoma cell lines.

PubMed

Van Roy, N; Van Limbergen, H; Vandesompele, J; Van Gele, M; Poppe, B; Salwen, H; Laureys, G; Manoel, N; De Paepe, A; Speleman, F

2001-10-01

Cancer cell lines are essential gene discovery tools and have often served as models in genetic and functional studies of particular tumor types. One of the future challenges is comparison and interpretation of gene expression data with the available knowledge on the genomic abnormalities in these cell lines. In this context, accurate description of these genomic abnormalities is required. Here, we show that a combination of M-FISH with banding analysis, standard FISH, and CGH allowed a detailed description of the genetic alterations in 16 neuroblastoma cell lines. In total, 14 cryptic chromosome rearrangements were detected, including a balanced t(2;4)(p24.3;q34.3) translocation in cell line NBL-S, with the 2p24 breakpoint located at about 40 kb from MYCN. The chromosomal origin of 22 marker chromosomes and 41 cytogenetically undefined translocated segments was determined. Chromosome arm 2 short arm translocations were observed in six cell lines (38%) with and five (31%) without MYCN amplification, leading to partial chromosome arm 2p gain in all but one cell line and loss of material in the various partner chromosomes, including 1p and 11q. These 2p gains were often masked in the GGH profiles due to MYCN amplification. The commonly overrepresented region was chromosome segment 2pter-2p22, which contains the MYCN gene, and five out of eleven 2p breakpoints clustered to the interface of chromosome bands 2p16 and 2p21. In neuroblastoma cell line SJNB-12, with double minutes (dmins) but no MYCN amplification, the dmins were shown to be derived from 16q22-q23 sequences. The ATBF1 gene, an AT-binding transcription factor involved in normal neurogenesis and located at 16q22.2, was shown to be present in the amplicon. This is the first report describing the possible implication of ATBF1 in neuroblastoma cells. We conclude that a combined approach of M-FISH, cytogenetics, and CGH allowed a more complete and accurate description of the genetic alterations occurring in the investigated cell lines. Copyright 2001 Wiley-Liss, Inc.

BCORL1 is an independent prognostic marker and contributes to cell migration and invasion in human hepatocellular carcinoma.

PubMed

Yin, Guozhi; Liu, Zhikui; Wang, Yufeng; Dou, Changwei; Li, Chao; Yang, Wei; Yao, Yingmin; Liu, Qingguang; Tu, Kangsheng

2016-02-15

The deregulation of E-cadherin has been considered as a leading cause of hepatocellular carcinoma (HCC) metastasis. BCL6 corepressor-like 1 (BCORL1) is a transcriptional corepressor and contributes to the repression of E-cadherin. However, the clinical significance of BCORL1 and its role in the metastasis of HCC remain unknown. Differentially expressed BCORL1 between HCC and matched tumor-adjacent tissues, HCC cell lines and normal hepatic cell line were detected by Western blot. The expression of BCORL1 was altered by siRNAs or lentivirus-mediated vectors. Transwell assays were performed to determine HCC cell invasion and migration. Increased expression of BCORL1 protein was detected in HCC specimens and cell lines. Clinical association analysis showed that BCORL1 protein was expressed at significant higher levels in HCC patients with multiple tumor nodes, venous infiltration and advanced TNM tumor stage. Survival analysis indicated that high expression of BCORL1 protein conferred shorter overall survival (OS) and recurrence-free survival (RFS) of HCC patients. Multivariate Cox regression analysis disclosed that BCORL1 expression was an independent prognostic marker for predicting survival of HCC patients. Our in vitro studies demonstrated that BCORL1 prominently promoted HCC cell migration and invasion. Otherwise, an inverse correlation between BCORL1 and E-cadherin expression was observed in HCC tissues. BCORL1 inversely regulated E-cadherin abundance and subsequently facilitated epithelial-mesenchymal transition (EMT) in HCC cells. Notably, the effect of BCORL1 knockdown on HCC cells was abrogated by E-cadherin silencing. BCORL1 may be a novel prognostic factor and promotes cell migration and invasion through E-cadherin repression-induced EMT in HCC.

Sulforaphane Preconditioning Sensitizes Human Colon Cancer Cells towards the Bioreductive Anticancer Prodrug PR-104A.

PubMed

Erzinger, Melanie M; Bovet, Cédric; Hecht, Katrin M; Senger, Sabine; Winiker, Pascale; Sobotzki, Nadine; Cristea, Simona; Beerenwinkel, Niko; Shay, Jerry W; Marra, Giancarlo; Wollscheid, Bernd; Sturla, Shana J

2016-01-01

The chemoprotective properties of sulforaphane (SF), derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3), bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC50 of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

PubMed

Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells

PubMed Central

2014-01-01

Background The cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, its efficacy is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Programmed necrosis is an alternative, molecularly distinct mode of programmed cell death that is elicited by TRAIL under conditions when the classical apoptosis machinery fails or is actively inhibited. The potential of TRAIL-induced programmed necrosis in tumor therapy is, however, almost completely uncharacterized. We therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin. Methods Cell death/viability was measured by flow cytometry/determination of intracellular ATP levels/crystal violet staining. Cell surface expression of TRAIL receptors was detected by flow cytometry, expression of proteins by Western blot. Ceramide levels were quantified by high-performance thin layer chromatography and densitometric analysis, clonogenic survival of cells was determined by crystal violet staining or by soft agarose cloning. Results TRAIL-induced programmed necrosis killed eight out of 14 tumor cell lines. Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested. TRAIL synergized with chemotherapeutics in killing tumor cell lines by programmed necrosis, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. Susceptibility/resistance of the investigated tumor cell lines to programmed necrosis seems to primarily depend on expression of the pro-necrotic kinase RIPK3 rather than the related kinase RIPK1 or cell surface expression of TRAIL receptors. Furthermore, interference with production of the lipid ceramide protected all tested tumor cell lines. Conclusions Our study provides evidence that TRAIL-induced programmed necrosis represents a feasible approach for the elimination of tumor cells, and that this treatment may represent a promising new option for the future development of combination therapies. Our data also suggest that RIPK3 expression may serve as a potential predictive marker for the sensitivity of tumor cells to programmed necrosis and extend the previously established role of ceramide as a key mediator of death receptor-induced programmed necrosis (and thus as a potential target for future therapies) also to the tumor cell lines examined here. PMID:24507727

Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5

PubMed Central

Yoshida, S; Arakawa, F; Higuchi, F; Ishibashi, Y; Goto, M; Sugita, Y; Nomura, Y; Niino, D; Shimizu, K; Aoki, R; Hashikawa, K; Kimura, Y; Yasuda, K; Tashiro, K; Kuhara, S; Nagata, K; Ohshima, K

2012-01-01

Objectives The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. Methods Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. Results The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. Conclusions Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology. PMID:22401175

Assessment of the Tumorigenic Potential of Spontaneously Immortalized and hTERT-Immortalized Cultured Dental Pulp Stem Cells

PubMed Central

Wilson, Ryan; Urraca, Nora; Skobowiat, Cezary; Hope, Kevin A.; Miravalle, Leticia; Chamberlin, Reed; Donaldson, Martin; Seagroves, Tiffany N.

2015-01-01

Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases. Significance This study demonstrated that immortalized dental pulp stem cells (DPSCs) do not form tumors in animals and that immortalized DPSCs can be differentiated into neurons in culture. These results lend support to the use of primary and immortalized DPSCs for future therapeutic approaches to treatment of neurobiological diseases. PMID:26032749

Overexpression of PBK/TOPK relates to tumour malignant potential and poor outcome of gastric carcinoma

PubMed Central

Ohashi, Takuma; Komatsu, Shuhei; Ichikawa, Daisuke; Miyamae, Mahito; Okajima, Wataru; Imamura, Taisuke; Kiuchi, Jun; Kosuga, Toshiyuki; Konishi, Hirotaka; Shiozaki, Atsushi; Fujiwara, Hitoshi; Okamoto, Kazuma; Tsuda, Hitoshi; Otsuji, Eigo

2017-01-01

Background: PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) is a serine–threonine kinase and overexpressed in various types of cancer by inhibiting the transactivation activities of p53 and PTEN. We tested whether PBK/TOPK acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Methods: We analysed five GC cell lines and 144 primary tumours, which were curatively resected in our hospital between 2001 and 2003. Results: Overexpression of the PBK/TOPK protein was frequently detected in GC cell lines (4 out of 5 lines, 80.0%) was detected in primary tumour samples of GC (24 out of 144 cases, 16.6%) and was significantly correlated with venous invasion, tumour depth and recurrence rate. PDZ-binding kinase/T-LAK cell-originated protein kinase-overexpressing tumours had a worse survival rate than those with non-expressing tumours (P=0.0009, log-rank test). PDZ-binding kinase/T-LAK cell-originated protein kinase positivity was independently associated with a worse outcome in multivariate analysis (P<0.0001, hazard ratio 6.40 (2.71–14.49)). In PBK/TOPK-overexpressing GC cells, knockdown of PBK/TOPK inhibited the cell proliferation through the p53 activation in a TP53 mutation-dependent manner and inhibited the migration/invasion through the PTEN upregulation in a TP53 mutation-independent manner. Conclusions: These findings suggest PBK/TOPK plays a crucial role in tumour malignant potential through its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in GC. PMID:27898655

The interaction of bee products with temozolomide in human diffuse astrocytoma, glioblastoma multiforme and astroglia cell lines.

PubMed

Borawska, Maria H; Markiewicz-Żukowska, Renata; Naliwajko, Sylwia K; Moskwa, Justyna; Bartosiuk, Emilia; Socha, Katarzyna; Surażyński, Arkadiusz; Kochanowicz, Jan; Mariak, Zenon

2014-01-01

In the present study, we investigated the influence of extracts from Salix spp. honey (ESH), beebread (EBB), and royal jelly (ERJ) with and without temozolomide (TMZ) on cell lines derived from a patient with diffuse astrocytoma (DASC), human glioblastoma multiforme (U87MG), and normal human astroglia (SVGp12). DASC was identified by immunocytochemistry. TMZ (20 μM) in combination with ESH (30 μg/mL), EBB (50 μg/mL), and ERJ (30 μg/mL) has stronger cytotoxic activity on U87MG cells after 72 h (20.0, 26.5, and 29.3% of control, respectively) than TMZ alone (about 6% of control). An increase of the cytotoxic effect and inhibition of DNA synthesis in SVGp12 were detected after administering TMZ with the studied extracts. NF-κB p50 subunit was reduced in U87MG cells after treatment with ESH (70.9%) and ESH + TMZ (74.7%). A significant decline of MMP-9 and MMP-2 secretion in cultured U87MG was detected after incubation with EBB (42.9% and 73.0%, respectively) and EBB + TMZ (38.4% and 68.5%, respectively). In conclusion, the use of bee products may increase the cytotoxic effect of TMZ in U87MG but also in SVGp12 cell line. It is important to note that the U87MG cells were sensitive to natural bee products, although there was no influence of natural bee products on the DASC cells.

Residual chromatin breaks as biodosimetry for cell killing by carbon ions.

PubMed

Suzuki, M; Kase, Y; Nakano, T; Kanai, T; Ando, K

1998-01-01

We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET = 13 keV/micrometer, 76 keV/micrometer) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour post-irradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/micrometer beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.

Residual chromatin breaks as biodosimetry for cell killing by carbon ions

NASA Astrophysics Data System (ADS)

Suzuki, M.; Kase, Y.; Nakano, T.; Kanai, T.; Ando, K.

1998-11-01

We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET = 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour post-irradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

PubMed Central

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

PubMed

English, Diana P; Bellone, Stefania; Schwab, Carlton L; Roque, Dana M; Lopez, Salvatore; Bortolomai, Ileana; Cocco, Emiliano; Bonazzoli, Elena; Chatterjee, Sudeshna; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Santin, Alessandro D

2015-02-01

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM. © 2014 American Cancer Society.

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells.

PubMed

Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai-Kei; Zhang, Wei

2015-07-26

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells). MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells. Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.