JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2006
TRI VUONG
ABSTRACT Serratia vaccinii, a novel bacterium isolated from blueberry microflora, increased the phenolic content of berry juices, and thus increased antioxidant activities. The fermentation capacity of Serratia was investigated with Saskatoon berries, cranberries, strawberries and grapes in both aerobic and anaerobic conditions. It was shown to be compatible with wine yeast in anaerobic fermentations, producing wine with high antioxidant activity. The effects of fermented berry juices were tested on lipopolysaccharide/inferon-gamma-activated macrophages 264.7 NO(,). Data indicated that fermented berry juices strongly inhibited activated-macrophage NO production but induced tumor necrosis factor-alpha production. [source]

MYELIN PROTEIN P-0-SPECIFIC IGM PRODUCING MONOCLONAL B CELL LINES WERE ESTABLISHED FROM POLYNEUROPATHY PATIENTS WITH MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE (MGUS)

JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 4 2002
M Kvarnstrom
Monoclonal expansion of B cells and plasma cells, producing antibodies against ,self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenstroms'macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P-0, using beads coated with P-0. P-0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein-Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P-0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5(+) positive, although the expression declined in vitro over time. The anti-P-0 antibodies were of IgM-lambda type. The antibodies belonged to the V(H)3 gene family with presence of somatic mutations. The IgM reacted with P-0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5(+) clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P-0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation. [source]

Improved In vitro Model Systems for Gastrointestinal Infection by Choice of Cell Line, pH, Microaerobic Conditions, and Optimization of Culture Conditions

HELICOBACTER, Issue 4 2007
Sara K. Lindén
Abstract Background:, Commonly used in vitro infection cultures do not mimic the human gastrointestinal tract with regard to pH and microaerobic conditions. Furthermore, despite the importance of mucin,Helicobacter interactions, the cell lines used have not been selected for appropriate mucin expression. To make in vitro studies more applicable to human disease, we have developed coculture methods taking these factors into account. Materials and methods:, Nine human gastrointestinal epithelial cell lines (MKN1, MKN7, MKN28, MKN45, KATO3, HFE145, PCAA/C11 Caco-2, and LS513) were investigated. Expression and glycosylation of mucins (MUC1, 2, 3, 4, 5AC, 5B, 6, 12, 13, and 16) were determined by immunohistochemistry. We analyzed the effect of microaerobic conditions and acidic pH on cell proliferation, viability, and apoptosis. Results:, Microaerobic culture, which is more physiological for the bacteria, did not adversely affect mammalian cell viability, proliferation, or induce apoptosis The cell lines varied in mucin expression, with MKN7 and MKN45 being most similar to gastric mucosa and Caco-2 and LS513 to intestinal mucosa, although none exactly matched normal mucosa. However, changes in culture conditions did not cause major changes in the mucin expression within cell lines. Conclusions:, Culture conditions mimicking the natural environment and allowing the bacterial cells to thrive had no effect on cell viability or apoptosis, and very little influence on mucin expression of human gastrointestinal cells. Thus, it is feasible, using the simple methods we present here, to substantially improve bacterial,mammalian cell in vitro coculture studies to make them more reflective of human infection. [source]

Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line,,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2008
Hideaki Tomura
Abstract Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E2 (PGE2) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE2 production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca2+ concentration ([Ca2+]i), PGE2 production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca2+]i and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE2 production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for Gq/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/Gq/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE2 production in response to acidic circumstances. [source]

The Dose-Response Effects of Ethanol on the Human Fetal Osteoblastic Cell Line

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2001
A. Maran
Abstract Alcohol is a risk factor for the development of osteoporosis, especially in men. Chronic alcohol abuse decreases bone mass, which contributes to the increased incidence of fractures. To better understand the mechanism of action of ethanol on bone metabolism, we have studied the dose-response effects of ethanol on conditionally immortalized human fetal osteoblasts (hFOB) in culture. Ethanol treatment had no significant effects on osteoblast number after 1 day or 7 days. Ethanol treatment did not reduce type I collagen protein levels at either time point at any dose but slightly reduced alkaline phosphatase activity after 7 days. The messenger RNA (mRNA) levels for alkaline phosphatase, type I collagen, and osteonectin were unaltered by 24 h of ethanol treatment but a high dose (200 mM) reduced mRNA levels for the two bone matrix proteins after 7 days. Ethanol treatment led to dose-dependent increases in transforming growth factor ,1 (TGF-,1) mRNA levels and decreases in TGF-,2 mRNA levels. The concentration of ethanol in the medium decreased with time because of evaporation but there was little degradation caused by metabolism. These results, which show that cultured osteoblasts are less sensitive than osteoblasts in vivo, suggest that the pronounced inhibitory effects of ethanol on bone formation are not caused by direct cell toxicity. [source]

Establishment of Rat Lymphatic Endothelial Cell Line

MICROCIRCULATION, Issue 2 2003
Risuke Mizuno
ABSTRACT Objective: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells. Methods: The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O2, 5% CO2, and 90% N2) or high oxygen (21% O2, 5% CO2, and 74% N2). Results: The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose , and D- N -acetylgalactosamine ,) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3,,3,-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles. Conclusions: The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere (,5%) enabled us to establish rat lymphatic endothelial cell line. [source]

Theoretical Prediction of Antiproliferative Activity against Murine Leukemia Tumor Cell Line (L1210).

MOLECULAR INFORMATICS, Issue 1 2009
3D-Morse Descriptor, its Application in Computational Chemistry
Abstract Cancer is among the top ten causes of death in the world but in spite of the efforts of the pharmaceutical companies and many governmental organizations, new and more effective drugs are urgently needed. Computer-assisted studies have been widely used to predict anticancer activity taking into account different molecular descriptors, statistical techniques, cell lines, and datasets of congeneric and non-congeneric compounds. [source]

Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Maikel E. Giesbers
Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11- cis retinal or all- trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin. [source]

Establishment and Characterization of a Normal Melanocyte Cell Line Derived from Pig Skin

PIGMENT CELL & MELANOMA RESEARCH, Issue 4 2003
Sophia Julé
Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes. [source]

ORIGINAL ARTICLE: Effect of Progesterone on HLA-E Gene Expression in JEG-3 Choriocarcinoma Cell Line

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2009
Zhongying Huang
Problem, Among class Ib human leukocyte antigen (HLA) molecules, HLA-E is known to be a major ligand of CD94/NKG2 receptor on natural killer (NK) cells, and to play a pivotal role in recognition of extravillous trophoblasts (EVTs) by maternal immune cells. However, it is scarcely known how HLA-E expression is regulated in EVTs. Method of study, In this study, we investigated whether progesterone, an essential hormone in maintaining pregnancy, regulated HLA-E expression in EVT-like cell line, JEG-3. HLA-E mRNA amount in cultured JEG-3 cells was assessed by real-time PCR and cell-surface HLA-E protein was analyzed by flowcytometry. Results, Real-time PCR showed 3.5-fold increase 1 hour after the addition of 1000 ng/ml progesterone. This response was dimished by the addition of RU486, an antagonist for progesterone receptor. Flowcytometry indicated that 1000 ng/ml progesterone slightly enhanced HLA-E expression on the surface of JEG-3. Conclusion, These results suggest that progesterone up-regulates HLA-E expression in JEG-3 cells through the pathway mediated by progesterone receptor. Our findings might give a new insight into immunomodulatory function of progesterone at fetomaternal interface. [source]

Effects of Cytokines on VEGF Expression and Secretion by Human First Trimester Trophoblast Cell Line

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 2 2002
SUN JU CHOI
PROBLEM:,The mechanism through which vascular endothelial growth factor (VEGF) regulation occurs at the feto-maternal interface is poorly understood. The aim of this study was to investigate the effects of various cytokines on VEGF expression and secretion by trophoblast cells. METHOD OF STUDY:,We investigated the effects of cytokines on VEGF expression in human first trimester trophoblast cell line by analyzing VEGF messenger RNA (mRNA) by reverse transcription-polymerase chain reaction and VEGF protein secretion by enzyme linked immunosorbent assay. RESULTS:,The trophoblast cells expressed VEGF mRNA constitutively and the main subtypes were identified as VEGF121 and VEGF165. When cultured in the presence of interferon (IFN)-,, interleukin (IL)-1,, tumor necrosis factor (TNF)-,, IL-2, or IL-10, VEGF mRNA expression was found to be significantly increased by IL-1,, IFN-, and TNF-, but to be unaffected by IL-2 and IL-10. Moreover, VEGF secretion was most significantly increased by IFN-, treatment. CONCLUSION:,These results suggest that IL-1,, IFN-,, and TNF-, may regulate the production of VEGF in early gestational trophoblasts. [source]

Epstein-Barr Virus (EBV) Latent Membrane Protein 1 Induces Interleukin-8 through the Nuclear Factor-,B Signaling Pathway in EBV-Infected Nasopharyngeal Carcinoma Cell Line

THE LARYNGOSCOPE, Issue 5 2004
Qingchun Ren MD
Abstract Background/Objectives: Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry. Study Design: In vitro design. Methods: The influence of the EBV genome for IL-8 gene expression was studied using the EBV,genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization. Results: EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)- ,B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role. Conclusions: LMP1 induces IL-8 mainly through the activation of NF-,B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC. [source]

Growing Cholesterol-Dependent NS0 Myeloma Cell Line in the Wave Bioreactor System: Overcoming Cholesterol-Polymer Interaction by Using Pretreated Polymer or Inert Fluorinated Ethylene Propylene

BIOTECHNOLOGY PROGRESS, Issue 4 2005
Judith Kadarusman
Difficulty in growing cholesterol-dependent NS0 cells in the Wave bioreactor using the original low-density polypropylene (LDPE) bags has been encountered. It has been shown that in these bags chemically defined cholesterol is depleted from solution and therefore unavailable for the cells. Our data suggest that the cause of the depletion is not chemical but is due to the physical structure of the polymer. It is proposed that polymer structures with inkbottle pores retain cholesterol, whereas structures with V-shaped pores adsorb cholesterol reversibly. Ultra-low-density polyethylene (ULDPE) bags can support cell growth but need to be pretreated with excess cholesterol. Another material, fluorinated ethylene propylene (FEP) does not need to be pretreated and is found to be superior (negligible cholesterol adsorption) as a result of its inert characteristics. [source]

VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell Line

CANCER SCIENCE, Issue 3 2002
Tatsuya Asai
In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin-containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor-,B, (I,B,), an inhibitor of nuclear factor-,B (NF,B), whether VCP influences NF,B activation or not was examined by using VCP-transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor-, (TNF,), Dunn/VCPs showed constantly activated NF,B, although in the original Dunn cells and control vector transfectant (Dunn/Dunn-c) NF,B activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated I,B, (p-IKB,) in the cytoplasm of confluent Dunn/Dunn-c cells compared to that of Dunn/VCPs. Therefore, decrease of p-IKB, degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn-c cells after TNF, stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP-1 inoculated male C3H mice compared to those in Dunn/Dunn-c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti-apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p-IKB, degrading activity may explain the increased metastatic potential of these cells. [source]

A Murine Osteosarcoma Cell Line with a Potential to Develop Ossification upon Transplantation

CANCER SCIENCE, Issue 6 2001
Tomomi Kusumi
An Osteosarcoma cell line has been established from a soft tissue tumor that occurred spontaneously in a BALB/c mouse. This cell line showed ossification when transplanted into syngeneic mice. To examine the mechanism of bone formation, the expression of mRNAs for osteoblastic and chon-droblastic markers and factors associated with ossification has been investigated. In culture, the cells exhibited a spindle shape in the growth phase, but had a polygonal shape in the stationary phase. Reverse transcription-polymerase chain reaction analysis showed that the cells expressed mRNAs for pro-,(I) chain of type I collagen, alkaline phosphatase, osteopontin, osteocalcin, and core binding factor al, suggesting differentiation into the stage of osteoblasts during the stationary phase. After transplantation, histological examination revealed small foci of pale blue material and basophilic networks that were scattered in the tumor tissues at one week. The former stained positive with alcian blue, suggesting a chondroid matrix. Pro-,(II) chain of type II collagen mRNA was expressed at one week. A large part of tumors at two and three weeks consisted of basophilic networks, which stained positive via von Kossa's method, indicating a calcified woven bone. In situ hybridization analysis showed strong expression of osteopontin and osteocalcin mRNAs in tumor cells surrounding the bone matrix. Bone morphogenetic protein-6 and -7 mRNAs were detected in transplanted tumors, but not in cultured cells. These results suggest that the cell line has the properties of an osteoblastic lineage when cultured in vitro and has an ossifying ability through endochondral bone formation processes when transplanted in vivo. [source]

Platinum(IV) Complexes of 3- and 4-Picolinic Acids Containing Ammine or Isopropylamine Ligands , Synthesis, Characterization, X-ray Structures, and Evaluation of Their Cytotoxic Activity against Cancer Cell Lines,

EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 30 2008
María J. Macazaga
Abstract The preparation and characterization of the new complexes trans -[PtCl4(NH3)(3-picolinic acid)] (1), trans -[PtCl4{NH2CH(CH3)2}(3-picolinic acid)] (2), trans -[PtCl4(NH3)(4-picolinic acid)] (3), and trans -[PtCl4{NH2CH(CH3)2}(4-picolinic acid)] (4) are described. The main structural feature of these complexes is the presence of ligands capable of multiple hydrogen-bonding interactions. Crystals of 1, 2, 3, and 4 suitable for single-crystal X-ray diffraction were grown, and the molecular structures of these compounds are discussed. In contrast to the inactive parent PtII complexes, the PtIV complexes displayed cytotoxic activity against various cancer cell lines used at the National Cancer Institute (NCI) for in vitro screens. Once more, the isopropylamine derivatives showed the best cytotoxicity values. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source]

Significance of the Grb2 and Son of Sevenless (Sos) Proteins in Human Bladder Cancer Cell Lines

IUBMB LIFE, Issue 4 2000
Takafumi Watanabe
Abstract The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP . However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer. [source]

siRNA-mediated Knockdown of the Heme Synthesis and Degradation Pathways: Modulation of Treatment Effect of 5-Aminolevulinic Acid-based Photodynamic Therapy in Urothelial Cancer Cell Lines

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009
Makito Miyake
Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT)n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT)n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT)n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1. [source]

The Effects of 5-Aminolevulinic Acid Esters on Protoporphyrin IX Production in Human Adenocarcinoma Cell Lines,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2001
H. Brunner
ABSTRACT Photodynamic diagnosis (PDD) and photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA),induced protoporphyrin IX (PPIX) is an interesting approach to detect and treat dysplasia and early cancers in the gastrointestinal tract. Because of low lipophilicity resulting in poor penetration across cell membranes, high doses of ALA should be administered in order to reach clinically relevant levels of PPIX. One way of increasing PPIX accumulation is derivatization of ALA into a more lipophilic molecule. In our in vitro study, different esterifications of ALA were investigated to analyze the effects on PPIX accumulation in human adenocarcinoma cell lines. For systematic analysis of cell type,specific PPIX accumulation, three human adenocarcinoma cell lines (SW480, HT29 and CaCo2) and a fibroblast cell line (CCD18) were tested. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) assays were performed to ensure that the ALA esters showed no cellular dark toxicity. Different concentrations (ranging from 0.012 to 0.6 mmol/L, 3 h) and incubation times (5, 10, 30, 180 min; 0.12 mmol/L) were examined. PPIX accumulation was measured using flow cytometry. ALA esters, especially ALA-hexylester and ALA-benzylester, induced significant higher PPIX levels in adenocarcinoma cell lines when compared with ALA and may be promising candidates for PDT and PDD. [source]

Antiproliferative Effect of Furanocoumarins from the Root of Angelica dahurica on Cultured Human Tumor Cell Lines

PHYTOTHERAPY RESEARCH, Issue 3 2007
Young-Kyoon Kim
Abstract A bioassay-guided fractionation of the root extract of Angelica dahurica (Umbelliferae) led to the isolation of six furanocoumarins as active ingredients responsible for the antitumoral property. The hexane soluble part of the extract demonstrated a signicant inhibition on the proliferation of cultured human tumor cells such as A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) in vitro, whereas the remaining water soluble part exhibited poor inhibition. Intensive investigation of the hexane soluble part of the extract yielded six furanocoumarins, i.e. isoimperatorin, cnidicin, imperatorin, oxypeucedanin, byakangelicol, oxypeucedanin hydrate, all of which exhibited a signicant inhibition on cell proliferation in a dose-dependent manner. Copyright © 2006 John Wiley & Sons, Ltd. [source]

Generation and Characterization of Embryonic Stem-Like Cell Lines Derived from In Vitro Fertilization Buffalo (Bubalus bubalis) Embryos

REPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010
B Huang
Contents In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8,10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent. [source]

ORIGINAL ARTICLE: H3N2 Influenza A Virus Replicates in Immortalized Human First Trimester Trophoblast Cell Lines and Induces Their Rapid Apoptosis

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2009
Quang Duy Trinh
Problem, Epidemiological data suggested that pandemic influenza increased the risks of spontaneous abortion and premature labor, while seasonal influenza also increased the risk of schizophrenia in adolescence. However, their pathogenesis is so far unknown. Method of study, The first trimester trophoblast cell lines, namely, Swan71 and HTR8 cells were challenged with A/Udorn/72 influenza virus (H3N2). At indicated time points, cells were examined for expression of influenza proteins. Viral replication in culture media, apoptosis and the expression of human leukocyte antigen (HLA)-G were also examined. Results Intracellular localization of viral proteins was observed. Twenty-four hours after inoculation, virus was detected in culture media while most cells fell into apoptosis. During apoptosis, expression of HLA-G was unchanged. Conclusion, We revealed replication of low pathogenic influenza virus in the first trimester trophoblast cell lines. Placental damages are likely to be induced by direct cytopathic effects of influenza virus and subsequent apoptosis rather than down regulation of HLA-G expression and subsequent rejection by maternal immune system. [source]

Synthesis of 2-Fluoro N10 -Substituted Acridones and Their Cytotoxicity Studies in Sensitive and Resistant Cancer Cell Lines and Their DNA Intercalation Studies

ARCHIV DER PHARMAZIE, Issue 11 2009
Yergeri C. Mayur
Abstract A series of 2-fluoro N10 -substituted acridone derivatives with varying alkyl side chain length with propyl, butyl substitution, and a tertiary amine group at the terminal end of the alkyl side chain were synthesized and screened against cancer cell lines SW 1573, SW 1573 2R 160 (P-gp substrate) which are non-small lung cancer cell lines, MCF-7, MCF-7/MR (BCRP substrate) are breast cancer cell lines, 2008 WT, 2008MRP1, 2008MRP2, 2008MRP3 are ovarian cancer cell lines, and human embryo kidney cell lines like HEK293, HEK293 MRP4, and HEK293 MRP5i. The propyl-series compounds showed lipophilicity in the range of 1.93 to 4.40 and the butyl series in the range of 2.37 to 4.78. The compounds 4, 7, and 8 showed good cytotoxicity against the 60 human cancer cell line panel of the National Cancer Institute, USA. The compounds 14 and 15 showed a better cytotoxicity in most of the cancer cell lines compared to other compounds tested. The DNA-binding properties of the compounds were evaluated based on their affinity or intercalation with CT-DNA measured with absorption titration. The compound 11 bearing planar tricyclic ring linked with a butyl methylpiperazino side chain showed the highest binding affinity with a binding constant (Ki) of 10.38×10 M,1. Evaluation of the compounds in cell lines with an overexpression of various multidrug resistance-related protein (MRP), P-glycoprotein (P-gp), or Breast Cancer Resistance Protein (BCRP) showed that all compounds are not substrates for any of these transporters. [source]

A New Method to Prepare Multicellular Spheroids in Cancer Cell Lines Using a Thermo-Reversible Gelation Polymer

ARTIFICIAL ORGANS, Issue 7 2003
Satoshi Tsukikawa
Abstract: The purpose of this study is to utilize the thermo-reversible gelation polymer in which the sol-gel transitting phase is reversibly changed by temperature in a three-dimensional culture system. Human cancer cells have been observed to form multicellular spheroids, whereas fibroblasts slowly develop into small spheroids with the culture medium including this polymer. This polymer has some advantages for use as a culture material, as follows: first, cancer cells grow three-dimensionally in the aqueous solution of this polymer; second, it is easy to harvest cells or spheroids in the aqueous solution of this polymer by simply cooling down the temperature; and third, the culture medium including this polymer is so translucent that the cells or spheroids can be observed through a phase-contrast microscope. We thus conclude that this polymer is a very useful material for three-dimensional cultures. [source]

Hepatocyte Cell Lines as the Biological Component of Liver Support

ARTIFICIAL ORGANS, Issue 7 2001
Hugo O. Jauregui M.D. Guest Editor
First page of article [source]

Improved Fermentation Processes for NS0 Cell Lines Expressing Human Antibodies and Glutamine Synthetase

BIOTECHNOLOGY PROGRESS, Issue 1 2003
Jonathan Dempsey
To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed-batch process was developed for GS-NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine- and serum-free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5-fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin-containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum-and protein-free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2-fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10-fold over the starting concentrations. [source]

Comparison of Growth and Recombinant Protein Expression in Two Different Insect Cell Lines in Attached and Suspension Culture

BIOTECHNOLOGY PROGRESS, Issue 4 2001
R. A. Taticek
Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPL,-Sf21-AE) and Trichoplusia ni (Tn 5,-1,4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150,160 rpm were necessary for maximum growth of suspended Tn 5,-1,4 cells compared to 125,150 rpm for Sf-21 cells. An inoculum size of 5 × 105 cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for ,-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5,-1,4 cells are 2,4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of ,-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5,-1,4 cells produced 2.6,4.4 and 2.7,3 times more ,-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the ,-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of ,-galactosidase by Tn 5,-1,4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production. [source]

RASSF1A, BLU, NORE1A, PTEN and MGMT Expression and Promoter Methylation in Gliomas and Glioma Cell Lines and Evidence of Deregulated Expression of de novo DNMTs

BRAIN PATHOLOGY, Issue 2 2009
Aiala Lorente
Abstract Methylation of CpG islands in gene promoters can lead to gene silencing. Together with deletion or mutation, it may cause a loss of function of tumor suppressor genes. RASSF1A (3p21.3), NORE1A (1q32.1) and BLU (3p21.3) have been shown to be downregulated by methylation in cancer, and PTEN (10q23.3) and MGMT (10q26.1) are located in areas commonly deleted in astrocytomas. MGMT methylation predicts a better response and a longer overall survival in patients with glioblastomas treated with temozolomide. We analyzed 53 astrocytoma samples and 10 high-grade glioma cell lines. Gene expression was assessed by RT-PCR. Bisulfite sequencing, MSP and a melting curve analysis-based real-time PCR were performed to detect promoter methylation. Treatments with 5,-aza-2,-deoxicitidine were applied to restore gene expression in cell lines. Ninety-two percent of tumor samples were methylated for RASSF1A, 30%,57% for BLU and 47% for MGMT, suggesting promoter methylation of these genes to be a common event in glioma tumorigenesis. Only 4% of the tumors revealed a methylated promoter for NORE1A. No association between methylation and loss of expression could be established for PTEN. We identified de novo DNMTs overexpression in a subset of tumors which may explain the methylation phenotype of individual gliomas. [source]

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

CANCER SCIENCE, Issue 8 2002
Teiichiro Honda
The TSLC1 (tumor suppressor in lung cancer,1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers. [source]

X-Irradiation Induces Up-regulation of ATM Gene Expression in Wild-type Lymphoblastoid Cell Lines, but Not in Their Heterozygous or Homozygous Ataxia-telangiectasia Counterparts

CANCER SCIENCE, Issue 6 2001
Yuko Hirai
Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradi-ated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM+/+), AT heterozygotes (ATM+/,), or AT homozy-gotes (ATM,/,), respectively. In ATM+/+ LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this tune. The results obtained with the ATM+/, LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM+/, LCL. Twelve of the mutations in the ATM,/, LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM,/, LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way. [source]

Cell Lines (cell + line)

Selected Abstracts

ANTIOXIDANT ACTIVITY OF FERMENTED BERRY JUICES AND THEIR EFFECTS ON NITRIC OXIDE AND TUMOR NECROSIS FACTOR-ALPHA PRODUCTION IN MACROPHAGES 264.7 GAMMA NO(,) CELL LINE